Signal Transduction: Pathways, Mechanisms and Diseases [1 ed.] 3642021115, 9783642021114, 9783642021121

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Signal Transduction: Pathways, Mechanisms and Diseases

Ari Sitaramayya Editor

Signal Transduction: Pathways, Mechanisms and Diseases

Editor Ari Sitaramayya Professor of Biomedical Sciences 423 DHE Eye Research Institute Oakland University Rochester, MI 48309 USA [email protected]

ISBN 978-3-642-02111-4 e-ISBN 978-3-642-02112-1 DOI 10.1007/978-3-642-02112-1 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2009928304 # Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover illustration: Cellular and subcellular detection of M 2 receptors in striatal neurons; see Fig. 2.1 in Chap. 2 ‘‘Regulation of Intraneuronal Trafficking of G-Protein-Coupled Receptors by Neurotransmitters In Vivo’’ by Ve´ronique Bernard Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

For Usha, Vani, and Aruna

Preface

Teaching a graduate course on signal transduction in the fall of 2007 was very enjoyable. Each week, my students and I discussed a recently published research paper in some area of signaling, argued about the appropriateness of the methodology used in the research, the design of the experiments, and whether the conclusions drawn were supported by the results presented. Finally, each student was asked to list what was good and what was deficient in the paper and whether she/he would have accepted or rejected it had she/he reviewed it for publication. For a good number of recent papers, a consistent complaint of the students was that the background to the research was not adequately described. It can of course be argued that students should research the background. Either way, the need for a good source of background material in order to appreciate the research presented in a paper became apparent to me and was the inspiration for developing this book. The goal in bringing this book was to provide students with a review of recent developments in specific areas of current interest in signal transduction, sufficiently in depth to make recent research publications accessible. However, given the wide range of research topics being investigated today in signaling, a choice had to be made to focus on a select few. This choice was made not only keeping the current research interest in mind, but also with the anticipation that these areas will continue to be of interest over the next several years. In choosing papers for discussion in my class, I look for health-relatedness of the research, and that is reflected in the list of the broad areas covered in this book – G protein coupled receptors, growth factors, nuclear receptors, reactive oxygen and nitrogen species, cell cycle and cancer; the emphasis has been on areas of signaling research with immediate clinical significance. I believe this book will serve well as supplemental reading material for undergraduate and graduate students with similar interest. Another area covered in this book, one not often highlighted in signal transduction books, is that of signaling platforms, which is emerging as a significant area of research relevant to cellular metabolism, cell proliferation, differentiation, apoptosis, neurodegenerative diseases, and cancer. The authors contributing to this book

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Preface

are experts and active contributors to research in their specific areas of signaling. I would like to thank them for their contributions and their suggestions in the development of the book. I would like to acknowledge the help of Frank Tedeschi, a student in my department, who assisted in editing the manuscripts and in preparing the index. I would also like to thank Dr. Sabine Schwarz, Life Sciences Editor at Springer Verlag, and Dr. Jutta Lindenborn who have been very helpful at every stage of production of this book. Ari Sitaramayya

Contents

Part I G Proteins and G Protein–Coupled Receptors 1

Traditional GPCR Pharmacology and Beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Annette Gilchrist and Maria R. Mazzoni

2

Regulation of Intraneuronal Trafficking of G-Protein-Coupled Receptors by Neurotransmitters In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Ve´ronique Bernard

3

Small GTPases and Their Role in Regulating G Protein-Coupled Receptor Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Fabiola M. Ribeiro and Stephen S.G. Ferguson

4

Regulation of G Protein Receptor Coupling, Mood Disorders and Mechanism of Action of Antidepressants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Moran Golan, Gabriel Schreiber, and Sofia Avissar

5

Dysregulation of G Protein-Coupled Receptor Signaling in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 JoAnn Trejo

Part II Growth Factors 6

Insulin Signaling in Normal and Diabetic Conditions . . . . . . . . . . . . . . . . . 101 Patrice E Fort, Hisanori Imai, Raju Rajala, and Thomas W. Gardner

7

Epidermal Growth Factor (EGF) Receptor Signaling and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Elizabeth S. Henson and Spencer B. Gibson ix

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Contents

8

Leptin Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Hiroyuki Shimizu

9

Signaling in Normal and Pathological Angiogenesis . . . . . . . . . . . . . . . . . . 159 Michael R. Mancuso and Calvin J. Kuo

Part III Signaling Platforms 10

Spatial and Temporal Control of Cell Signaling by A-Kinase Anchoring Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 F. Donelson Smith, Lorene K. Langeberg, and John D. Scott

11

Mitochondria, a Platform for Diverse Signaling Pathways . . . . . . . . . . 199 Astrid C. Schauss and Heidi M. McBridee

12

Mitogen-Activated Protein Kinases and Their Scaffolding Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Danny N. Dhanasekaran and E. Premkumar Reddy

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Molecular and Functional Determinants of Ca2þ Signaling Microdomains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 Indu S. Ambudkar, Hwei L. Ong, and Brij B. Singh

Part IV Nuclear Receptors / Transcription 14

Eukaryotic Gene Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Jennifer H. Gromek and Arik Dvir

15

Estrogen Signaling Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Dapeng Zhang and Vance L. Trudeau

16

Signal Transduction Pathways Involved in Glucocorticoid Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Peter J. Barnes

Part V Reactive Signaling Molecules 17

Cellular Signaling by Reactive Oxygen Species: Biochemical Basis and Physiological Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Michel B. Toledano, Simon Fourquet, and Benoıˆt D’Autre´aux

18

Soluble Guanylyl Cyclase: The Nitric Oxide Receptor . . . . . . . . . . . . . . . 337 Doris Koesling and Ari Sitaramayya

Contents

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Part VI Cell Cycle, Cell Death and Cancer 19

Distinct Roles of the Pocket Proteins in the Control of Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 Paraskevi Vogiatzi and Pier Paolo Claudio

20

Activation of the p53 Tumor Suppressor and its Multiple Roles in Cell Cycle and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Luciana E. Giono and James J. Manfredi

21

Aging and Cancer: Caretakers and Gatekeepers . . . . . . . . . . . . . . . . . . . . . 397 Diana van Heemst

22

Signal Transduction in Embryonic Stem Cells and the Rise of iPS Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Solene Jamet, Ruairi Friel, and P. Joseph Mee

Index

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427

Part I

G Proteins and G Protein–Coupled Receptors

Chapter 1

Traditional GPCR Pharmacology and Beyond Annette Gilchrist and Maria R. Mazzoni

Abbreviations AGS AR CRE DAG ERK FRET G proteins GEF GIP GLP GPCR GRK HTS IP3 PIP2 RGS TM

1.1

Activator of G protein signaling Adrenergic receptor cAMP response element 1,2 diacylglycerol Extracellular signal related kinase Fluorescence resonance energy transfer Guanine nucleotide binding proteins Guanine exchange factor G protein coupled receptor interacting protein Glucagon-like peptide G protein coupled receptor G protein coupled receptor kinase High-throughput screening Inositol 1,4,5-trisphosphate Phosphatidylinositol 4,5-bisphosphate Regulator of G protein signaling Transmembrane

Introduction

Many biologically active molecules convey their signals via G protein coupled receptors (GPCRs) which are coupled to heterotrimeric guanine nucleotide binding proteins (G proteins). GPCRs respond to a large variety of molecules from small A. Gilchrist (*) Midwestern University, 555 31st Street, Downers Grove, IL 40515, USA e-mail: [email protected] M.R. Mazzoni University of Pisa, Via Banano 6, Pisa 56126, Italy A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_1, © Springer-Verlag Berlin Heidelberg 2010

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molecules such as acetylcholine, norepinepherine, serotonin, dopamine, and histamine to large peptides and glycoprotein hormones. There are over 800 GPCRs in the human genome, and the expansive list has resulted in GPCRs frequently being the focus for those searching for new therapeutics. But GPCRs are not unique to mammals as seven transmembrane (7TM)-containing proteins have now been identified in five of the six kingdoms of life (Bacteria, Protozoa, Plantae, Fungi and Animalia). Genetic variation in GPCRs, leading to inactive, overactive, or constitutively active receptors, has been shown to be present in a wide variety of human diseases (Thompson et al. 2008a). Interventions aimed at correcting genetic GPCR dysfunction include calcimimetics used to compensate for some calcium sensing receptor mutations, compounds that revert the gonadotropin releasing hormone receptor loss from the cell surface which is found in idiopathic hypogonadotropic hypogonadism, and novel drugs to rescue the purinergic P2Y12 receptor which results in a rare bleeding disorder. By excluding the sensory receptors such as the olfactory, bitter taste, vomeronasal, sweet/umami taste and opsin/rhodopin related receptors, it has been suggested that, of the over 800 GPCRs, some 369 are “physiologically relevant” and could serve as drug targets (Lagerström and Schiöth 2008). Of these, the activating ligand has not been identified for approximately 150 of these receptors, and they remain “orphans.” A continually updated list of GPCRs is available online at http://www. iupharbb.org/receptorList/results.php (Foord et al. 2005). Members of the GPCR superfamily are diverse in their primary structure, and this has been used for the phylogenetic classification of the family members such as that provided by Kolakowski in 1994 with an A–F classification system (Kolakowski 1994). A similar but extended nomenclature system was introduced in 1999, with the receptors being divided into five families on the basis of structural and ligandbinding criteria (Bockaert and Pin 1999). Using a draft of the human genome which became available in 2001, Fredriksson and colleagues divided 802 (known and predicted) human GPCRs into five families on the basis of phylogenetic criteria, termed Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin (GRAFS) (Fredriksson et al. 2003). One can also use BIAS-PROFS (http://igrid-ext.cryst.bbk. ac.uk/gpcrtree/), an internet site that classifies GPCRs at the class, subfamily and sub-subfamily level when a protein sequence is entered (Davies et al. 2008) Although for years the underlying premise of GPCR signaling centered on their ability to couple to G proteins, this fundamental paradigm of receptor biology has been expanded as research showing GPCRs participating in protein–protein interactions independent of G proteins has emerged. Some of the GPCR–protein interactions appear to serve as scaffolds, to tether the receptor to particular subcellular locations or to effector enzymes and regulatory proteins. Several of these proteins control receptor trafficking, while others modulate the kinetics of GPCRmediated signaling transduction (Wang and Limbird 2007). Many GPCR-associated proteins bind other signaling molecules suggesting that they serve to provide a means to signal via non-G protein-regulated effectors (Bockaert et al. 2004). In fact, several GPCRs have been shown to couple (directly or through adapter proteins) to enzymatic effectors, including guanine exchange factors (GEFs) for small G proteins, non-receptor tyrosine kinases, and components of the extracellular

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signal related kinase (ERK) pathway, indicating that GPCR signal transduction can occur independent of heterotrimeric G proteins. An example of GPCR/G proteinindependent signaling is through b-arrestins which bind to activated, phosphorylated receptors and control internalization of the receptors, G protein coupling, and secondary signaling that is independent of G protein signaling. The events appear to be differentially regulated by GPCR kinase (GRK) phosphorylation, ubiquitination and b-arrestin oligomerization, which may be both receptor- and cell-typedependent (Dromey and Pfleger 2008). There is a bias to the GPCRs that have been successfully targeted, with compounds being identified for > 39 Rhodopsin (class A) receptors, four Secretin (class B) receptors, and three Glutamate (class C) receptors. Of those GPCRs that have proved to be good therapeutic targets, 17% (23/133) bind peptides or proteins (including enzymes); 29% (12/41) bind biogenic amines; 20% (7/35) bind lipidlike-molecules; 17% (2/12) bind amino acids, and 6% (1/16) bind purines. Perhaps most surprising is that even with such a large number of candidates, only 46 GPCRs (~12%) have been successfully targeted by drugs (Lagerström and Schiöth 2008). Yet, nearly half of all prescription drugs were targeted towards GPCRs in 2000 in the United States (Drews 2000). No doubt this success provides the financial basis for the ongoing pursuit by pharmaceutical companies in exploring both new and old targets from this important receptor family. The common structural feature of GPCRs is 7TM a-helical domains, each composed of 25–35 amino acid residues. The highly hydrophobic helices are connected by three extracellular and three intracellular loops, with the amino (N-) terminus being extracellular and the carboxyl (C-) terminus being intracellular (Fig. 1.1). This structure gave rise to an alternative name for these molecules, 7TM receptors. One of the greatest advances in GPCR knowledge came with the high-resolution crystal structure of inactive rhodopsin (Palczewski et al. 2000), which was eventually followed with the crystal structures for rhodopsin with a deprotonated retinylidene Schiff base linkage (Salom et al. 2006), b2-adrenergic receptor (AR) (Cherezov et al. 2007; Rasmussen et al. 2007), b1-AR (Warne et al. 2008), squid rhodopsin (Murakami and Kouyama 2008; Shimamura et al. 2008), and active rhodopsin (Park et al. 2008). Moreover, the surface movement of rhodopsin upon photoactivation was mapped (Altenbach et al. 2008) using an electron pair spin resonance technology called “double electron–electron resonance” spectroscopy, which interrogates pairs of nitroxide spin labels to provides a solid foundation for the so-called “helix movement model” of receptor activation. Stimulation of heterotrimeric G proteins via GPCRs results in the regulation of a variety of intracellular signaling pathways through activation of downstream effectors such as enzymes, ion channels, and nuclear transcription factors. Heterotrimeric G proteins are comprised of a Ga subunit that binds and hydrolyzes GTP, and a Gbg dimer that serves as a functional monomer. The Gabg holoprotein acts as a molecular switch that can be turned “on” and “off” via the GTPase cycle (Fig. 1.2). Numerous members comprise the heterotrimer G protein family, including genes for 16 Ga subunits, five Gb subunits, and 14 Gg subunits (Milligan and Kostenis 2006). The Ga subunits are a family of 39–52 kDa proteins commonly divided into four families based on their sequence similarity: Gi/o, Gs, Gq/11, and

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Ag

EC2 EC1 EC3

N-

extracellular space IV

I

III

II

V

IC1

P

VII

cytosol

VI IC2

P IC3

C-

P

P γ Heterotrimeric G-protein

α β

Fig. 1.1 Topology of a typical GPCR. The receptor’s N-terminus is extracellular, and its C-terminus is intracellular. The receptor traverses the plane of the cell membrane seven times. The hydrophobic transmembrane segments (light color) are designated by roman numerals (I–VII). The agonist (Ag) approaches the receptor from the extracellular side, while G proteins interact with cytoplasmic regions of the receptor, especially portions of the third cytoplasmic loop between transmembrane regions V and VI. The receptor’s cytoplasmic terminal tail contains numerous serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated, and this phosphorylation is often associated with receptor internalization

G12/13. Several of the Ga subunits are expressed ubiquitously, while others have temporal and/or differential expression (Eglen et al. 2008). Intensive studies of Ga proteins have helped elucidate distinct regions involved in receptor recognition, GTP binding and hydrolysis, guanine nucleotide-induced conformational changes, and effector interaction. Structurally, each Ga subunit consists of two domains, a GTPase domain and an a helical domain. In between these two domains is a cleft where the guanine nucleotide binds (Tanabe et al. 1985). Lipid modification of a Cys residue near the N-terminus of most Ga subunits allows for binding to membrane (Linder et al. 1993) while the C-terminus of all Ga subunits appears to be important for interaction with the receptor. Gbg is an absolute requirement for the binding of the Ga subunit to the receptor, for the formation of high-affinity agonist binding, and for receptor catalyzed activation of G protein (Fung 1983; Blumer and

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‘7TM’ - receptor

extracellular space I

II

VI

VII

Ligand

V

cytosol γ β

α

Pi

GDP

V IV

I

Inactive G-protein

+

III II II VII VI

α

RGS

GDP

I

VII

VI

α

V

γ β

V

γ β

GTP

GDP-GTP Exchange

GTP

Active Gα

Active Gβγ

Downstream Effectors

Fig. 1.2 Activation of G protein signaling via ligand binding to a GPCR. Following ligand-initiated stimulation, the 7TM receptor becomes activated and changes its conformation leading to an interaction between Gabg and the GPCR. This interaction induces a conformation change in Ga that decreases its affinity for GDP whereby it dissociates and is replaced with GTP. Once GTP is bound, the Ga subunit assumes an activated conformation and rearrange/dissociates from receptor and Gbg. Both Ga-GTP and Gbg initiate downstream signaling events. The activated state lasts until the GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the Ga subunit, resulting in association of the Ga-GDP with Gbg subunits to form inactive Gabg, and this process may be influenced by RGS proteins

Thorner 1990). Gbg is also tethered to the membrane by a lipid modification of the Gg subunit and together the Gbg dimer acts as a scaffolding protein. Following receptor activation, both Ga and Gbg act as signaling molecules (Fig. 1.2). Although much has been learned about how ligand-stimulated GPCRs mediate G protein activation, the exact mechanisms along the way, such as the existence of precoupled G proteins, the role of GEFs in nucleotide exchange on Ga, and dissociation of Ga and Gbg from the receptor, are now being considered in greater detail. A number of studies have shown that G proteins may not physically dissociate from the receptors (Frank et al. 2005; Digby et al. 2006; Yuan et al. 2007). In addition, there appear to be several downstream effectors that can be activated by the GPCR independent of nucleotide exchange (Sun et al. 2007). There has also been recognition that other proteins such as GRKs, activator of G protein signaling (AGS) proteins, regulator of G protein signaling (RGS) proteins,

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and GPCR interacting proteins (GIPs) may play an important role in the activation and deactivation processes (Bockaert et al. 2004; Jean-Baptiste et al. 2006; Blumer et al. 2007; Ribas et al. 2007). Adding to the complexity is the substantial evidence indicating that posttranscriptional modifications, receptor oligomerization, and the presence (or absence) of specific G proteins can influence GPCR activation and signaling (Gilchrist 2007). Activation of a GPCR results in the release of GDP from Ga. Until GTP binds, a high-affinity complex is formed between the receptor and “empty pocket” G protein. Current structural models of the receptor with the G protein suggest that the nucleotide-binding pocket of Ga is at a distance from the closest receptor contact site (~30 Å) lending to the “action at a distance” hypothesis (Oldham and Hamm 2008). The exchange of GDP for GTP leads to dissociation of the Gbg dimer from the Ga subunit, and both initiate unique intracellular signaling responses often referred to as second messengers (Sprang et al. 2007). The second messengers go on to activate or inhibit other components of the cellular machinery. For example, activation of Gq may lead to activation of phospholipase C, an enzyme that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 interacts with receptors on intracellular calcium stores resulting in cytosolic release of calcium while DAG can activate protein kinase C isoforms. Other G proteins modulate a large number of second messenger systems including cyclic AMP, cyclic GMP, calmodulin, and kinases. Attempts to understand how a receptor catalyzes G protein activation has led to the proposition of several models including the “lever-arm,” “gear-shift,” and “latch” models as mechanisms by which the GDP is released (Johnston and Siderovski 2007). In such models, the interactions between Ga and Gbg have a crucial role in G protein activation. With the lever-arm model, the GPCR uses the N-terminal helix of Ga as a lever arm to pull Gbg away from Ga, which then forces open the nucleotide-binding pocket resulting in GDP release (Rondard et al. 2001). In the gear-shift model the GPCR uses the aN-helix to force Gbg into Ga, permitting the Ga N-terminus to engage the helical domain, resulting in an interdomain gap between the helical and GTPase domains of the Ga, thus leading to GDP release (Cherfils and Chabre 2003). In the latch model (Nanoff et al. 2006) it is proposed that the GPCR can engage the C-terminus of Ga to destabilize nucleotide binding from the “back side” of the nucleotide binding pocket. Although the studies emphasize the uncertainty that still surrounds the exact mechanisms of GPCR–G protein activation, structural studies have provided detailed information on the three-dimensional structures (Wall et al. 1995; Lambright et al. 1996; Sondek et al. 1996), including localization of the nucleotidebinding pocket of Ga which is approximately 30 Å away from the nearest receptor contact site. Thus, the activated receptor must induce conformational changes such that upon interaction with the G protein the signal is transmitted over this distance resulting in GDP release (Ciarkowski et al. 2005). Recently, site-directed spin-labeling studies have shown that several regions in the Ga subunit undergo changes in mobility upon receptor binding (Oldham et al. 2006). Besides several C-terminal residues, other distant residues show changes in mobility consistent with

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rotation/translation of the a5 helix towards the b6 strand and rearrangement of switch I and switch II regions at the Gb-binding surface. Moreover, the a5 helix is in close proximity to the receptor-binding C-terminus of Ga and the guanine-ringbinding TCAT motif is in the b6–a5 loop connecting the b6 strand to the a5 helix. In order for GDP to be released, some of the contacts between the GTPase and the helical domains of Ga may be broken. Although experimental data do not support a role of interdomain interactions in G protein activation, inspection of the crystal structure seems to suggest the requirement of a movement of these two domains relative to each other to release GDP. Indeed, both molecular dynamic simulation (Ceruso et al. 2004) and site-directed mutagenesis in the interdomain linkers (Majumdar et al. 2004) suggest an interdomain reorientation. The G protein heterotrimer is reformed by GTPase activity of the Ga subunit, forming Ga-GDP and by doing so, allowing Ga and Gbg to reassociate/recombine (Fig. 1.2). This terminates the action of the Ga and Gbg subunits on their effectors. Agonist activation increases the rate of guanine nucleotide exchange and, therefore, the amount of active Ga-GTP and free Gbg. RGS proteins increase the rate of hydrolysis of GTP and thus shorten the lifetime of Ga-GTP and therefore of free Gbg. Consequently, the G proteins regulate both the specificity and duration of the initiating signal. In recent years, additional methods of regulating signaling through heterotrimeric G proteins have been identified, thus widening the perspective on the role of a G protein as a “signaling switch.” Besides RGS proteins, other accessory proteins can interact with G proteins and some of these interactions may be independent of receptor activation. In this regard, receptor-independent AGS proteins have emerged (Cismowski 2006). These proteins are classified into different groups depending on their role in signal transduction. Some AGS proteins such as AGS1 increase GTPgS binding to both Gai subunits and heterotrimeric Gi proteins acting as putative GEFs (Cismowski et al. 2000), while others such as AGS8 bind to Gbg and activate it in the heterotrimeric complex without causing subunit dissociation (Yuan et al. 2007). Indeed, some experimental evidence indicates that subunit dissociation is not necessarily a required step in the G protein activation cycle. A fluorescence resonance energy transfer (FRET) study has shown that Gi1 protein activation in cells involves subunit rearrangement rather than dissociation (Bünemann et al. 2003). Other investigations using fluorescence recovery after photobleaching have shown that some G protein subunits (Gi3 and GoA) physically dissociate in living cells while other heterotrimers (Gs) do not (Digby et al. 2006). It seems from the experimental work thus far that more extensive studies will be required to obtain a complete knowledge of G protein activation and signaling in living cells. Some GPCRs may assume active conformations in the absence of agonists and thus, constitutively or spontaneously, activate G proteins. An example is the histamine H3 receptor which shows a high level of constitutive activity both in vitro and in vivo. Constitutive activity has been observed for many other wild-type GPCRs from humans and commonly used laboratory animals. However, there are documented differences in the extent of constitutive activity among GPCRs, even between subtypes of the same receptor family (Milligan 2003). Even for those

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receptors that have little or no constitutive activity, such as rhodopsin, constitutively activating mutations (CAMs) can increase the level of spontaneous activity (Parnot et al. 2002). There are several indications that constitutive activity may reflect the inherent flexibility of a GPCR and its tendency to exist in more than one conformation in the absence of ligands (Kobilka and Deupi 2007). For a variety of GPCRs, point mutations have been clearly linked to human disease. For example, there are approximately 50 different CAMs found in the thyroid-stimulating hormone receptor which are associated with hyperfunctioning thyroid adenomas. Additionally, GPCRs encoded and expressed in infected cells by herpesviruses such as Epstein– Barr virus, human cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus (KSHV) are interesting examples of the pathophysiological importance of constitutive GPCR activity. The GPCR encoded by KSHV acts as an oncogene and angiogenesis activator, suggesting a role of somatic CAMs in cancer development. In view of the potential role of herpesvirus-encoded GPCRs in viral dissemination and redirection of intracellular signaling pathways, they are exploited as targets for drug discovery efforts to identify compounds that can inhibit their constitutive signaling (inverse agonists). There are a number of regions on G protein a subunits that could serve to block receptor–G protein interactions. For example, with all G proteins studied the GTPand Mg2+-binding sites are tightly coupled. Dominant-negative constructs of the Ga subunit have been made in which mutations are introduced at residues known to contact the magnesium ion. For the Ga subunit, this includes mutations of the Gly residue within the invariant sequence (G203T; G204A) (Hermouet et al. 1991; Murray-Whelan et al. 1995), as well as mutations of a Ser residue in the effector loop, switch I region (S47C) (Slepak et al. 1993). Although this approach was quite successful with p21ras and other small G proteins, dominant-negative Ga subunits using this method were less effective, perhaps due to the degree to which Mg2+ is necessary to support GDP binding (Barren and Artemyev 2007). p21ras forms a tight and฀ nearly฀ irreversible฀ GDP•Mg2+ complex, while Ga subunits bind Mg2+ in the GDP•Mg2+฀complex฀with฀lower฀affinity฀than฀in฀the฀GTP•Mg2+ complex. An alternative approach to make an inhibitor of G protein coupling is to use the C-terminus of Ga which is essential for receptor contact (Gilchrist et al. 2002). Antibodies, peptides, and minigene vectors expressing C-terminal peptides can be used as inhibitors of receptor–G protein interactions resulting in competitive blockade of downstream events. This interaction has been shown to be quite specific with single amino acid changes in the C-terminal peptide resulting in functional differences (Gilchrist et al. 1998). A similar approach has been used by other investigators to look at a variety of responses related to G proteins including (1) binding of pleckstrin homology (PH) domains to Gbg, (2) inhibiting GPCRs by expressing the C-terminus of b2-adrenergic receptor kinase, and (3) identifying intracellular domains of GPCRs critical for G protein coupling. In addition, researchers have made transgenic mice targeting the Ga C-terminus in specific tissues such as the myocardium (DeGeorge et al. 2008) and smooth muscle (McGraw et al. 2007) and the results indicate a marked inhibition of GPCR-mediated responses.

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GPCR signaling is also substantially influenced by receptor expression, dimerization, desensitization, and internalization, as well as in response to binding by different ligands and within various cellular contexts. For example, receptor internalization reduces the pool of receptors available for binding thus diminishing signaling when downstream effector pathways are measured.

1.2

Traditional GPCR Pharmacology

1.2.1

Basic Concepts and Language

1.2.1.1

Agonism, Antagonism, Efficacy versus Affinity, Dose–Response Curves, and Drug Receptor Theory

In the most general definition, a drug is any substance that brings about a change in the biologic function of a cell. An agonist binds to and activates the receptor, often mimicking the action of an endogenous ligand (such as a neurotransmitter) that binds to the same receptor. Agonists come in several versions such as full agonists which bind and activate a receptor with complete efficacy. An example of a full agonist is isoproterenol which mimics the endogenous ligand epinepherine and acts on b-ARs. Partial agonists are drugs that bind to receptors and activate them but do not evoke as great a response as the full agonists. It is worth mentioning that if both a full agonist (such as the endogenous ligand) and partial agonist are present, the partial agonist will actually serve as an antagonist, competing with the full agonist for receptor occupancy and thus producing a net decrease in the receptor activation relative to that observed with the full agonist alone. Thus, pindolol, which is a partial agonist for b-AR, may act as either a partial agonist (if no full agonist is present) or an antagonist (if a full agonist such as epinephrine is present). Other drugs act as pharmacological antagonists; i.e., they bind to receptors but do not lead to any signaling by the cell; rather, they interfere with the ability of an agonist to activate the receptor. The effect of a so-called “pure” or “neutral” antagonist on a cell or in a patient depends entirely on its preventing the binding of agonist molecules and blocking their biologic actions. An endogenous ligand such as acetylcholine or serotonin may bind and stimulate receptors that couple to different subsets of G proteins. The apparent promiscuity allows a ligand to elicit a variety of G protein-dependent responses in different cell types. For instance, the heart may respond to catecholamines (norepinephrine and epinepherine) by increasing heart rate by acting on Gs-coupled b2-ARs while in skin such ligands induce constriction of blood vessels through Gq-coupled ARs. It is becoming apparent that the relative efficacy of compounds may depend on the read-out chosen for the assay or the conditions used in the experiment. There

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are, in fact, many examples of differences in the pharmacological profile for one receptor when different assay read-outs or conditions are used (Kenakin 2005; Gilchrist 2008).

1.3 1.3.1

And Beyond Recognition of Inverse Agonists

Because of their underlying nature, inverse agonists (which decrease basal signaling) are easier to identify with functional screens versus classical binding assays. In 2004, Terry Kenakin wrote a landmark paper that included a survey of antagonist–receptor pairings on some 73 GPCR targets. Using results from over a hundred articles he concluded that of the 380 antagonist-receptor pairings, 322 were inverse agonists (85%), while 58 (15%) were neutral antagonists (Kenakin 2004). Somewhat tellingly, all clinically prescribed antagonists were noted to be inverse agonists in the survey. These and other findings have moved inverse agonism to the forefront of receptor theory. However, as was pointed out by Parra and Bond (Parra and Bond 2007), the reclassification does not address the clinical importance of inverse agonism versus neutral antagonism. Constitutive activity was first shown using recombinant systems, and the validity of this finding was questioned due to the use of a heterologous expression system. The use of recombinant systems has been quite beneficial for drug discovery efforts; however, they have highlighted that GPCRs are not merely on/off switches, but are rather more complex micro-processors. For example, the relative potency profiles for agonists of calcitonin receptor vary depending on whether the receptor is transfected into Chinese hamster ovary (CHO), human embryonic kidney (HEK), or African green monkey kidney (COS) cells. Such variations are probably the result of a long list of phenotypic differences between host cells, including rate of gene transcription, posttranslational transfer, glycosylation, phosphorylation, compartmentalization, as well as expression levels of intracellular partner components such as G proteins, scaffolding proteins, and accessory proteins (Kenakin 2003). The a1b-AR was the first GPCR in which specific point mutations were shown to lead to constitutive receptor activation (Kjelsberg et al. 1992). The discovery of CAMs in the AR family triggered other researchers to look for physiological settings in which GPCRs were constitutively active. Many diseases have been shown to be the result of constitutive GPCR activity caused by mutations leading to increased G protein signaling, or upregulated GPCR expression levels (Thompson et al. 2008a). Serotonin 5-HT receptors are a large family, containing some 14 members. One of these, 5-HT1A, is a key target for the treatment of mood disorders such as anxiety and depression. Recently, researchers showed that native 5-HT1A receptors constitutively activate Gao in rat brain tissue homogenates (Martel et al. 2007). Moreover,

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it has been suggested that alterations in the constitutive activity at the 5-HT2A and 5-HT2C receptors are involved in anxiety and depression (Berg et al. 2008). Histamine receptors are another family that contain constitutively active members. High-throughput screening (HTS) using a radioligand binding assay with the rat H3 receptor, and a secondary screening with a guinea pig ileum assay, suggested species differences in compounds identified as antagonists (Hancock 2006). Repeating the screening with human histamine H3 receptor led to a number of new compounds being identified, although the author points out that there are 20 potential variant isoforms with numerous signal transduction pathways, unique distribution patterns, and perhaps differential responses to pharmalogical agents. When a receptor lacks constitutive activity, an inverse agonist behaves as a neutral competitive antagonist. This point was emphasized by a recent report in which what were thought to be neutral antagonists of 5HT7a were shown to be inverse agonists (Rauly-Lestienne et al. 2007). Thus, the classification of neutral antagonist should be used cautiously until the constitutive activity of the GPCR has been characterized. What seems clear after reviewing numerous papers is a lack of literature in which inverse agonists and neutral antagonists are compared in a clinical setting. Without this direct information, it is difficult to determine if the priority, going forward, should be to screen for neutral antagonists or inverse agonists. This is a critical question given that programs to identify neutral antagonists are often more technically demanding in terms of both screening and medicinal chemistry.

1.3.2

Coming of Age for Allosteric Modulation

GPCRs are naturally allosteric, in that they possess at least two binding sites: one for the endogenous ligand; and one for the G protein. Early observations with GPCRs showed multiple ligand binding states in the absence of GTP, while agonist binding was mostly of a low-affinity form with GTP. The necessity for a distinction between the endogenous ligand binding site on the GPCR and other topographically distinct binding sites led to the terms orthosteric and allosteric, respectively (May and Christopoulos 2003). Drugs that act via the orthosteric site rely on the drug’s affinity, while allosteric modulators are characterized by cooperative binding. Traditionally, modulators of GPCRs (both allosteric and orthosteric) were studied using radioligand binding assays (Tränkle et al. 1999). However, this approach is limited in that it may limit detection of allosteric ligands to those that modulate the affinity of the orthosteric ligand (Kenakin 2006). The advent of functional screening using methods such as changes in calcium transients, activation of inositol phosphates and ERK has opened the floodgates for the identification of small molecules that can modulate GPCR function, while having no effect (or even an opposing effect) on agonist binding. Recently two groups reported identification of small-molecule allosteric agonists for the glucagon-like peptide (GLP) receptor (Chen et al. 2007; Knudsen et al. 2007). The work is noteworthy given that no small-molecule agonists had

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been previously discovered for any member of the family B GPCR group. In the case of Knudsen et al. none of the 500,000 small molecules served as agonists when assessed by competitive binding assays. The discovery of a substituted quinoxaline with an IC50 of 1.4 mM emerged when a cAMP assay was used to screen 250,000 compounds. Additional studies showed the compound increased binding of the radiolabeled peptide ligand, suggesting it might have been identified in competitive binding assays if the screen had initially been set up to look for increased ligand binding. Chen et al. also utilized a cAMP assay to screen GLP receptor, but utilized a cAMP response element (CRE)-driven luciferase reporter plasmid when screening their library of 48,160 compounds. Their assay resulted in the identification of five initial hits of which two compounds were confirmed to increase cAMP levels and displace ligand binding. The use of cAMP and CRE assays is interesting given that some compounds may not necessarily stimulate cAMP responses, but their responses become substantial at the level of CRE-gene transcription (Baker et al. 2004). The ability to identify allosteric modulators for GPCRs has only emphasized the need to characterize the pharmacology of the ligands in as many assays as possible, given that a lack of effect in one assay does not necessarily translate to the same effect in all systems. Rather, the “efficacy” of a compound be it allosteric or orthosteric is dependent on the assay system in which it is tested (Niedernberg et al. 2003).

1.3.3

Advent of Functional Selectivity

In classical pharmacological terms, intrinsic efficacy is a system-independent parameter that is constant for each ligand at each receptor. Unfortunately for GPCR researchers, this premise is usually incorrect. Recognition that some ligands have diverse functional consequences when they bind led to the hypothesis that each ligand could induce changes in the GPCR resulting in unique conformations. This led to the theory that certain drugs have the ability to preferentially activate one intracellular signaling pathway over another. The phenomenon is known by a number of different terms including “functional selectivity”, “agonist-directed trafficking of receptor stimulus”, “biased agonism”, “protean agonism”, “differential engagement”, and “stimulus trafficking”. During the last year researchers have added substantially to the growing body of evidence that functional selectivity is not just a theoretical possibility, but a robust biological phenomenon. Working with recombinant Gai1 and Gao2, researchers revealed significant differences in the activation patterns of four out of 10 m-opioid receptor agonists (Saidak et al. 2006). While other scientists (Maillet et al. 2007) provided an example of a compound (LPI805) showing functional selectivity identified in a screen for the tachykinin NK2 receptor. The orthosteric agonist neurokinin A stabilizes the receptor in at least two different active conformations, and LPI805 modulates the relative proportion of the two active conformation states. A first-rate illustration of

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R

α

γ β

G protein dependent signaling

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R

R

ARR2

ARR1

ARR2

GIP

Endocytosis

G protein independent signaling

Endocytosis

G protein independent signaling

α

γ β

G protein dependent signaling

Fig. 1.3 Functional selectivity can occur with GPCRs. The conformation of GPCRs appears to be ligand-dependent, and different agonists can produce selective G protein dependent or independent signaling. Certain ligands may have functional selectivity towards responses not mediated through G proteins, but rather signal via arrestins or other GPCR-interacting proteins (GIPs). Which messenger proteins first become activated as a result of GPCR/ligand interaction influence the downstream effectors employed. Thus, signaling via G proteins, arrestins, or GIPs are critical in determining cellular responses

functional selectivity was shown in the study of the cortical 5-HT2A receptor response to hallucinogens (González-Maeso et al. 2007). Another case in point for the extent of functional selectivity used cells that express either b1-AR or b2-AR measuring two distinct signaling pathways (adenylyl cyclase and ERK1/2) to profile several clinically relevant ligands (Galandrin et al. 2008). The antibody-capture [35S]GTPgS scintillation proximity assay provided evidence of functional selectivity for several GPCRs including the dopamine D1 receptor (Gazi et al. 2003), and 5-HT1A receptor (Mannoury la Cour et al. 2006). Taken together, these data are consistent with the idea that different ligands can stabilize distinct receptor conformations, and these conformations promote an affinity for one G protein over another, which subsequently govern downstream signaling events (Fig. 1.3).

1.3.4

Where will Dimerization take Us?

GPCRs were initially thought to be monomeric entities, coupling to the G proteins with a 1:1 stoichiometery. Over a decade ago, researchers showed that the GABAB1 receptor needed to couple to the GABAB2 receptor to traffic to the cell surface (Kaupmann et al. 1998; White et al. 1998). Since that time, using a number of methods of detection including co-immunoprecipitation, FRET and bioluminescence resonance energy transfer (BRET), an increasing number of GPCRs have

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been shown to be expressed at the plasma membranes as homodimers, heterodimers, or higher order oligomers (Gurevich and Gurevich 2008). These “horizontal molecular networks” (Agnati et al. 2005) may also contain other interacting proteins. While the functional significance of receptor dimerization is still not completely understood, and in some cases even the applied methodological approaches are debated, it is now generally accepted that oligomerization of GPCRs not only exists, but that it is relevant to receptor expression and function, including agonist binding, potency, and efficacy (Szidonya et al. 2008). An open question still remains on whether there is a requirement for the receptor to be dimeric for G protein activation (White et al. 2007; Damian et al. 2008). As a heterodimer, the receptors may display a different conformation, and thus may have a pharmalogical profile (ligand binding and/or G protein coupling) that is distinct from the receptor monomer or homodimer. For example the D2 receptor normally couples to Gi/o proteins; however, in the D1–D2 heterodimer it switches to Gq/11 when D1 is co-activated (Lee et al. 2004; Rashid et al. 2007). New equations to fit the binding data for receptor homodimers have been introduced (Giraldo 2008). In addition to providing equilibrium dissociation constants for binding of the first and second molecule (low- and high-affinity, respectively) to the dimer, they provide a measure of the dimer cooperativity index. No doubt similar equations for fitting binding data for heterodimer formations will soon be determined as well. It is important to point out that in vivo, receptors form heteromers that differ depending on the cell type, and pharmacological differences in antagonist activity/ affinity depending on the partner protein have been noted. This adds a new level of complexity to drug development, but reveals a new arena in which drugs can be designed to bind preferentially to one dimer formation over another. Another novel therapeutic approach for dimers is the design of dimeric compounds that act on both receptors in the heterodimer complex (Waldhoer et al. 2005; Rashid et al. 2007).

1.3.5

Quantifying Drug Activity

One of the most well known models for GPCRs is the two-state model of receptor activation, which postulates that there are at least two conformational forms that are in equilibrium: R and R* (Leff 1995). In this model R* is capable of signaling, and it is considered the “active” molecule, while R is considered to be the inactive state of the receptor. More complex models which include allosteric sites such as the “ternary complex model” proposed by De Lean et al. (De Lean et al. 1980) include an allosteric or regulatory site where G proteins can bind. Samama and colleagues (Samama et al. 1993) expanded this model and developed the “extended ternary complex model,” which includes different affinity states for the receptor uncoupled and coupled to the G protein, R and R*, respectively. From this the “allosteric twostate model” was developed in which allosteric modulation was not restricted to

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G proteins (Hall 2000). More recently, the “quaternary complex model” of allosteric interactions have been proposed (Christopoulos and Kenakin 2002). The use of radiolabeled ligands and binding assays to determine binding constants (association, KA, or dissociation, KD = 1/KA) has proven to be an invaluable tool for screening GPCRs for potential drugs. Curve fitting of experimental data points by mathematical models is a common way to extract relevant information from biological systems being regulated by GPCRs. Mathematical models are classified as mechanistic or empirical. Mechanistic models are the ideal formulations for the analysis of experimental curve data points, and can lead to new knowledge of the underlying biological mechanisms. Unfortunately, they often have a high number of parameters precluding their use by classical curve-fitting procedures such as gradient nonlinear regression. Empirical models employ the minimum number of parameters for the determination of the shape of the curve and, accordingly, do not present difficulties for standard curve fitting. In general, empirical models lack physical basis and are limited to obtaining the common geometric descriptors (midpoint location and slope, asymptotes, etc.) of the curves. Over the years the pharmaceutical industry has shifted away from ligand binding assays to functional assays for their large primary screens of GPCRs. The reasons for the change include a desire to decrease the use of radioactivity, reduce the cost of screening their large (hundreds of thousands) compound libraries, and identify allosteric modulators targeting sites independent from the orthosteric ligand binding site. The initial functional assays employed were focused on well-validated second messenger signaling pathways such as calcium, and cAMP. New assays that look to alternatives in the downstream signaling cascade have been introduced, including IP3 (Trinquet et al. 2006) and ERK (Osmond et al. 2005; Leroy et al. 2007). ERK1/2 phosphorylation assay offers an advantage in that it is a convergent signaling point, and its activation can be both G protein dependent and independent. More recent methods of screening for small-molecule modulators of GPCRs include cellular dielectric spectroscopy, flow cytometry, capillary electrophoresis, electronic biosensors, and the use of genetic selection in yeast (Gilchrist 2008). There has also been a trend towards adapting high content screening (HCS) approaches for GPCRs (Henriksen et al. 2008; Ross et al. 2008). Another interesting GPCR screening trend was the advent of b-arrestin screening approaches (Ghosh et al. 2005; Hammer et al. 2007). Arrestins are a family of four GPCR-binding proteins that play a key role in homologous desensitization and endocytosis (Defea 2008). They were believed to play a role only in limiting GPCR signaling by binding the GPCR and physically interfering with the binding of the G protein. However, scientists have now demonstrated that b-arrestin signaling extends beyond the regulation of G protein pathways and receptor trafficking and suggest that G proteins and arrestins link GPCRs to distinct sets of downstream effectors capable of eliciting both redundant and unique cellular responses (DeWire et al. 2007). In fact, recent evidence indicates that certain ligands can selectively activate b-arrestin signaling independent of G protein signaling (Violin and Lefkowitz 2007). For example, the parathyroid hormone (PTH) analog (D-Trp12,

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Tyr34) PTH(7–34) acts as an inverse agonist for PTH1 receptor–Gs coupling while promoting arrestin-dependent sequestration (Gesty-Palmer et al. 2006). Similarly, ICI118551, propranolol, and carvedilol, b2-AR ligands that act as inverse agonists with respect to Gs activation, function as partial agonists for the ERK1/2 pathway by engaging the b-arrestin (Azzi et al. 2003; Wisler et al. 2007). The opposite is also possible, such that Trp1-PTHrP(1–36), another PTH analog, promotes Gs coupling without inducing b-arrestin recruitment or desensitization (Gesty-Palmer et al. 2006). Such findings support the idea that GPCRs are capable of assuming multiple conformations, but the clinical utility of functionally selective compounds will ultimately depend on whether inducing only a subset of the full response profile confers therapeutic benefits. Work in the field of functional selectivity has made it clear that a compound’s efficacy is ultimately defined in terms of the assay used to measure it, as compounds that were disregarded on the basis of apparently poor efficacy for one response (i.e., calcium) may be found to be effective if other assays are employed (i.e., ERK1/2). Researchers also now recognize the importance of the “environment” for the GPCRs during the drug discovery process as changes in the GPCR expression level, G proteins present, G protein:GPCR ratio, as well as the availability of accessory proteins all influence the efficacy of the drugs being tested (Eglen et al. 2008; Gilchrist 2008). The transduction of many diverse signals from the extracellular to intracellular environments requires the actions of GPCRs, and at the same time, mutations in GPCRs underlie a variety of human diseases (Thompson et al. 2008b). However, in spite of the advances made by researchers in past years, the answers to many fundamental questions remain unanswered, including how GPCR activation through ligand binding promotes GDP–GTP exchange on Ga, and defining the critical role that Gbg plays in this process. It will also be important to discern how binding events at the orthosteric and allosteric binding sites mechanistically lead to their downstream events. Many of the answers will require a better understanding of the how, when, and why specific heterotrimeric G proteins or alternative GPCR signaling partners get activated.

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Chapter 2

Regulation of Intraneuronal Trafficking of G-Protein-Coupled Receptors by Neurotransmitters In Vivo Véronique Bernard

2.1

Introduction

Most neurotransmitters and related drugs modulate neuronal activity through G-protein-coupled receptors (GPCRs), which are synthesized in the endoplasmic reticulum and mature in the Golgi complex. Immunohistochemical studies at the electron microscopic level demonstrate that GPCRs are targeted to the plasma membrane to interact with neurotransmitters. This membrane targeting of GPCRs leads to their presynaptic or postsynaptic distribution and their synaptic or extrasynaptic localization. This determines the sites of neurotransmitter action and thus the putative role of GPCRs in regulation of neuronal activities. Activation of these receptors triggers a cascade of intracellular events that involves a wide variety of effector systems and leads to the modulation of neuronal postsynaptic activity (Koenig and Edwardson 1997; Krueger et al. 1997; Yoburn et al. 2004). In vitro studies have widely demonstrated that the abundance and availability of these receptors at the cell surface is regulated by the neuronal environment and is the result of complex intraneuronal trafficking. The amplitude of neuronal response to the neurochemical environment variations depends on, besides the quantity of released neurotransmitter, the number of postsynaptic receptors (Oakley et al. 1999; Anborgh et al. 2000). This control of the abundance and availability of GPCRs at the neuronal membrane probably contributes to modulation of how neurons respond to their endogenous or exogenous ligands under physiological, pathological or therapeutic conditions. It is possible that the modulation of receptor availability at the plasma membrane is a key event of

V. Bernard Biologie des Jonctions Neuromusculaires Normales et Pathologiques, INSERM U686 Université Paris Descartes, 45 Rue des Saints-Pères, Paris, France e-mail: [email protected] A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_2, © Springer-Verlag Berlin Heidelberg 2010

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neuronal activity regulation in vivo, other facets of which include neurotransmitter release and neuronal excitability. However, this regulation is still poorly understood in vivo. Because the mechanisms of GPCR trafficking might differ in vivo and in vitro, as has been shown for the muscarinic ACh receptor M2, specific analyses are required in vivo (Roseberry and Hosey 2001; van Koppen 2001; Bernard et al. 2003). In vivo analyses of the mechanisms that regulate the availability of GPCRs are thus important for improving our understanding of drug effects in pathology and therapy. We discuss the role of neurochemical environment in the intraneuronal trafficking of GPCRs in vivo and we present data demonstrating that the abundance of GPCRs at the cell membrane and their intracellular trafficking are modulated by levels of neurotransmitters. We also show that subcellular distribution of GPCRs is determined by different criteria, such as the type of stimulation (acute vs. chronic) and the neuronal compartment (somatodendritic vs. axonal).

2.2

Experimental Approaches used to Study Trafficking of G-Protein-Coupled Receptors In Vivo

Different strategies in which the neurochemical environment is impaired have been used to study GPCR trafficking in animals in vivo. Two such approaches are pharmacological treatment (using direct or indirect agonists or, more rarely, antagonists), and knockout mice for molecules involved in neurotransmitter level. GPCRs are detected in brain sections using antibodies or fluorescent ligands (Yoburn et al. 2004). Alternatively, viral-mediated gene transfer and epitopetagged GPCRs can be used (Haberstock-Debic et al. 2003). Receptors are usually visualized at the light-microscopic level; immunohistochemistry at this level enables the distinction between membrane and intracytoplasmic localization of the receptors to be made efficiently. Identification of cytoplasmic organelles or compartments containing GPCRs is necessary for understanding the dynamic of trafficking in neurons and for identifying events such as endocytosis, synthesis and degradation. Some organelles can be identified easily on the basis of their ultrastructure: these include the endoplasmic reticulum, Golgi complex and multivesicular bodies. Cytoplasmic trafficking also involves vesicular compartments such as endosomes; the endosomal compartment is identified by its vesicular aspect at the light-microscopic level (Mantyh et al. 1995) or its ultrastructural characteristics (Bernard et al. 1998; Dumartin et al. 1998; Bernard et al. 1999, 2003; Csaba et al. 2001; Liste et al. 2002). Subcellular compartments can be identified at the light-microscopic level by co-detection of GPCRs with molecular markers of cytoplasmic compartments, such as transferrin or the transferrin receptor (Faure et al. 1995; Keith et al. 1998; Csaba et al. 2001; Bernard et al. 2003). Counting immunoparticles at the ultrastructural level is also important for comparing the abundance of receptor in each compartment in basal and

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experimental conditions (Bernard et al. 1998, 1999, 2003; Dumartin et al. 1998; Csaba et al. 2001; Liste et al. 2002).

2.3

Regulation of the Intraneuronal Distribution of GPCRs Under Physiological Conditions: Constitutive Endocytosis

In vitro, many studies have demonstrated that some GPCRs display constitutive endocytosis, indicating that a proportion of the receptor population spontaneously undergoes internalization in the absence of stimulation with an agonist (Leterrier et al. 2004; Xu et al. 2007). The neuron is a highly polarized cell that comprises two large domains: the somatodendritic compartment and the axonal compartment. The somatodendritic compartment receives and transduces external signals, and the axon and axonal terminal transmit a relevant response. Regulation of receptor distribution on the cell surface, including axonal polarization may be the result of constitutive internalization under physiological conditions. This polarization is achieved and maintained through a specific sorting signal that selectively targets the neuronal membrane proteins to somatodendritic or to axonal compartments (Higgins et al. 1997; Burack et al. 2000). A recent in vitro study has shown that constitutive somatodendritic endocytosis is required for the proper axonal targeting of the type 1 cannabinoid receptor (CB1R). Blockade of constitutive somatodendritic endocytosis abolished CB1R targeting to the axonal plasma membrane (Leterrier et al. 2006). In vivo, constitutive endocytosis may be a means to adapt receptor density to the intensity of stimulation and thus to regulate neuronal activity e.g., neurotransmitter release for axonal receptors, neuronal excitability for somatodendritic receptors. Indeed, there seems to be a correlation between the intensity of the stimulation by the endogenous neurotransmitter and the availability of the receptors at the plasma membrane. For example, two striatal neuronal subpopulations have been identified according to the density of M4 muscarinic ACh receptors in their plasma membranes (Bernard et al. 1999). These subpopulations are localized in two different striatal territories (striosomes and matrix) that display different ACh levels (Graybiel and Ragsdale 1978; Graybiel 1986; Hirsch et al. 1989; Lowenstein et al. 1989). The higher the ACh-mediated activity, the fewer ACh receptors are present at the membrane. In addition, an inverse relationship exists in rat brain between the density of somatostatin-containing afferents and the density of somatostatin sst2A receptors at the plasma membrane (Dournaud et al. 1998). Similarly, µ-opiate receptor trafficking has been shown to be regulated by afferent inputs in dorsal horn neurons (Morinville et al. 2004).

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Regulation of GPCR Distribution in the Somatodendritic Field After Acute and Chronic Stimulation

2.4.1

Decreased Number of GPCRs at the Plasma Membrane

In vivo data suggest that the mechanisms of regulation and adaptation of GPCR compartmentalization are different after acute and chronic stimulation (Bernard et al. 1998; Dumartin et al. 1998, 2000; Riad et al. 2001; Bernard et al. 2003; Riad et al. 2008). Several processes seem to control the abundance of GPCRs at the plasma membrane, including endocytosis, recycling, degradation and neosynthesis. In both acute and chronic conditions, the stimulation of GPCRs is associated with a decrease in receptor density at the plasma membrane of cell bodies and dendrites (Figs. 2.1 and 2.2). Acetylcholinesterase (AChE) and dopamine-transporter knockout mice, which are models of constitutive chronic receptor stimulation by ACh and dopamine, display a decreased density or even disappearance from the plasma membrane of M2 receptors and dopamine D1 receptors, respectively (Bernard et al. 1998, 2003; Dumartin et al. 1998, 2000). However, although the abundance of these receptors is reduced at the plasma membrane following both acute and chronic stimulation, their intracytoplasmic fate and trafficking are different. Anatomical studies have demonstrated that different GPCRs are differentially redistributed in different intraneuronal compartments.

2.4.2

Redistribution of GPCRs in the Cytoplasm After Acute Stimulation

2.4.2.1

Endocytosis

In vitro, acute stimulation induces endocytosis of GPCRs, which occurs mainly through the formation of clathrin-coated pits (von Zastrow and Kobilka 1992; Koenig and Edwardson 1997; Lamb et al. 2001; Yoburn et al. 2004). After binding of the agonist, the GPCR is phosphorylated and binds b-arrestin, which is responsible for receptor uncoupling from its G protein. Clathrin is then recruited to the plasma membrane to form pits; detachment of these pits from the membrane is induced by dynamin. Alternatively, endocytosis of some GPCRs can involve the formation of caveolae and not of clathrin-coated pits (Lamb et al. 2001; Sabourin et al. 2002). In vivo, light- and electronmicroscopic analyses show that acute stimulation reduces the abundance at the plasma membrane of GPCRs, including muscarinic, dopamine, opioid, substance P, serotonin-1A and somatostatin sst2A receptors (Mantyh et al. 1995; Bernard et al. 1998, 1999; Dumartin et al. 1998; Abbadie and Pasternak 2001; Csaba et al. 2001; Riad et al. 2001; He et al. 2002; Liste et al. 2002; Decossas et al. 2003;

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Fig. 2.1 Effect of acute and chronic modifications of ACh levels on the cellular and subcellular distribution of M2 receptors in neurons of the striatum in vivo. Cellular and subcellular detection of M2 receptors in striatal neurons. Images were collected under epifluorescence (a–e) and electron microscopy (f–h) using fluorescent immunohistochemistry and a pre-embedding immunogold method. (a): In control mice, M2 receptor immunoreactivity is mostly detected at the plasma membrane. (b): After acute treatment with oxotremorine (“Oxo”; 0.5 mg/kg subcutaneously for 1 h), M2 receptor immunoreactivity is seen in the cytoplasm. (c): After chronic stimulation of ACh receptors in acetylcholinesterase knockout mice (AChE–/–), no staining is observed at the membrane, whereas strong immunoreactivity is detected in the cytoplasm. (d,e): Organotypic cultures of striatum were co-incubated for 1 h with NaCl (9 g/L) (d), or with oxotremorine (25 µM) and transferrin (Tf; 50 µg/ml) (e), a constitutively endocytosed molecule used as a marker of endocytosis. In control animals (d), M2 receptors are localized at the membrane (red) whereas transferrin is endocytosed (green). After oxotremorine treatment (e), M2 receptors (red) are partially cointernalized with transferrin (yellow; the boxed area is enlarged in the inset). This suggests that the stimulation of muscarinic receptors induces the endocytosis of M2 receptors through clathrincoated pits. (f): In a control mouse, immunoparticles are associated mostly with the internal side of the plasma membrane (arrowheads). Some immunoparticles are associated with the endoplasmic reticulum (“er,” small arrow) and the Golgi apparatus (g). (g,h) In AChE–/– mice, few immunoparticles are detected in association with the plasma membrane (arrowheads). By contrast, numerous particles are seen in the cytoplasm associated with the endoplasmic reticulum and Golgi apparatus. This suggests that when ACh receptors are chronically stimulated, targeting of m M2 receptors is blocked in the intraneuronal compartments of synthesis and maturation, and thus they are no longer targeted to the membrane. Additional abbreviation: n, nucleus. Scale bars, 10 µm in (a–e); 500 nm (f,g); 50 nm (h). Reproduced, with permission, from Bernard et al. 2003

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V. Bernard A. Control

B. Acute stimulation

m2R ACh Clathrin-coated endosome MVB Lysosome neosynthesis

degradation

Endoplasmic reticulum Golgi apparatus

internalization

recycling

C. Chronic stimulation

neosynthesis

degradation

Fig. 2.2 A model of the regulation of the neuronal trafficking of GPCR in vivo by the neurochemical environment based on data obtained for the M2 receptors in cholinergic neurons of the basalo-cortical or striatal pathways. (a): In control mice, most GPCRs are located at the plasma membrane of the somatodendritic compartment and at axonal varicosities. GPCRs are also present in intraneuronal compartments involved in synthesis (endoplamsic reticulum and outer nuclear membrane) and maturation (Golgi apparatus). (b): After acute stimulation, GPCRs are internalized from the plasma membrane to the cytoplasm by endocytosis through clathrin-coated pits. After endocytosis, receptors can be either recycled to the plasma membrane or sent to the degradation pathway via multivesicular bodies (MVBs) and lysosomes. Recycling may occur directly after endocytosis or after going through the Golgi apparatus. The receptors continue to be synthesized. (c): After chronic stimulation, GPCRs are trapped in the cytoplasm in association with the Golgi apparatus and endoplasmic reticulum, and few of them are targeted to the plasma membrane; mostly to the membrane of the axon terminal. After blockade in compartments of synthesis and maturation, M2 receptors are degraded in lysosomes

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Haberstock-Debic et al. 2003; Morinville et al. 2004; Trafton and Basbaum 2004). GPCRs have also been detected in association with small labeled vesicles in the cytoplasm, without a loss in the total number of receptors (Bernard et al. 1998, 1999, 2003; Dumartin et al. 1998; Csaba et al. 2001; Liste et al. 2002; Decossas et al. 2003). These compartments co-internalize transferrin, a constitutively endocytosed molecule, or co-express transferrin receptors (Csaba et al. 2001; Bernard et al. 2003) (Fig. 2.1). These data demonstrate that acute stimulation induces in vivo, as it does in vitro, internalization of GPCRs with the bound ligand from the cell surface into intraneuronal compartments. This is achieved in the same way as in vitro, by the classical endocytosis of GPCRs through formation of clathrin-coated pits. In vivo, no proof exists for a non-classical clathrinindependent endocytotic pathway involving caveolae (as has been demonstrated for GPCRs in vitro (Feron et al. 1997)). The molecular mechanisms following ligand binding and endocytosis (phosphorylation of GPCRs, interaction with a-arrestin and uncoupling of the receptor from its G protein) have been described in vitro, but are still poorly understood in vivo. However, phosphorylation of µ-opioid receptors after agonist stimulation and the absence of such phosphorylation in GPCR kinase (GRK3) knockout mice, suggest a role for GPCR phosphorylation in the process of endocytosis of GPCRs in vivo (McLaughlin et al. 2004). Endocytosis can occur throughout the membrane of the somatodendritic tree or can be compartment specific. Endocytosis of a same receptor may also be brain region specific (Riad, 2001). For example, after acute stimulation, muscarinic, D1 or sst2A receptors are endocytosed in both the soma and dendrites, whereas µ-opioid receptors are endocytosed only in dendrites (Bernard et al. 1998, 1999, 2003; Dumartin et al. 1998; Csaba et al. 2001; Liste et al. 2002; Decossas et al. 2003; Haberstock-Debic et al. 2003). This might be due to different subneuronal comparmentalization of cytoplasmic regulatory factors involved in the endocytotic pathway (e.g., arrestin and/or dynamin). Such subcellular compartmentalization occurs in retinal photoreceptors for arrestin, which binds to rhodopsin, a GPCR (Elias et al. 2004). Because neuronal functions depend on the integration of neurochemical signals transmitted by different parts of the neuron, these data suggest that endocytosis might occur selectively in different neuronal compartments and thus contribute to the modulation of the membrane receptor availability and, hence, to the neuronal response. Endocytosis mechanisms in vivo seem to depend on the type of agonist. For example, it has been demonstrated that µ-opioid receptors are internalized after treatment with D-ala2,me-phe4,gly(ol)5-enkephalin (DAMGO), but not after morphine treatment (Abbadie and Pasternak 2001).

2.4.2.2

Fate of GPCRs After Endocytosis

In vitro, degradation and recycling of GPCRs are thought to be important events after endocytosis. They participate in regulation of plasma membrane receptor

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abundance and thus modulate the receptivity of neurons to further stimulation (Alvarez et al. 2002). Degradation and recycling have also been shown to occur in vivo. Anatomical data demonstrate that acute stimulation induces an increase in the number of muscarinic receptors associated with multi-vesicular bodies (MVBs) and lysosomes – two subcellular compartments that are involved in the degradation pathway (Bernard et al. 1998, 1999; Liste et al. 2002; Decossas et al. 2003) (Fig. 2.2). MVBs are considered to be intermediary compartments between endosomes and lysosomes where proteins are degraded. This suggests that some of these receptors are degraded after endocytosis. Alternatively, other endocytosed M2 receptors might be recycled to the plasma membrane hours after acute stimulation (Bernard et al. 1998, 2003). It is possible that some GPCRs follow exclusively one or the other post-endocytotic pathways, as has been shown in vitro. For example, substance P NK1 receptors and µ-opioid receptors are mostly recycled to the plasma membrane, µ-opioid receptors are not, and instead are almost all sent to lysosomes and degraded (Grady et al. 1995; Wang et al. 2003). In vitro, GPCRs might also be degraded in the proteasome, as demonstrated for µ-opioid receptors, but no such data are available in vivo (Li et al. 2000). Ex vivo studies on organotypic cultures of striatum suggest that the re-expression of GPCRs at the membrane might occur without neosynthesis. For example, the blockade of neosynthesis of the M2 receptor by cycloheximide has no effect on its reappearance at the plasma membrane (organotypic sections incubated first with 1 µM oxotremorine and 100 µM cycloheximide for 20 min, and then with 100 µM cycloheximide for 2 h; V. Bernard, unpublished). Moreover, no increase in levels of M2 receptor mRNA was observed in neurons of the nucleus basalis magnocellularis (NBM) after acute stimulation, despite the internalization of M2 receptors (Decossas et al. 2003). This suggests that activation of gene expression might not contribute to the synthesis of new M2 receptors for recycling to the plasma membrane. It is usually accepted that reclycling occurs directly after endocytosis. Alternatively, it has been recently shown in vivo that after endocytosis, the somatostatin type 2 receptor (sst2) may be retrogradely transported through a microtubule-dependent mechanism to a trans-Golgi network, before recycling (Csaba et al. 2007).

2.4.2.3

Functional Role of Endocytosis

In vitro, the function of endocytosis is still under debate, but it might be involved in processes of desensitization, resensitization and/or signaling (Ferguson, 2001; Alvarez et al. 2002). Desensitization is a reversible reduction in neuronal response during sustained agonist stimulation. Some authors consider desensitization of GPCRs to be a consequence of endocytosis (Ferguson, 2001). Alternatively, densitization might not be linked to endocytosis, because the blockade of 5-HT2A receptor endocytosis has no effect on agonist-induced desensitization (Gray et al. 2001). In vivo, the links between changes in subcellular compartmentalization of a GPCR and the functions regulated by the same receptor are also unclear. Is densensitization a consequence of endocytosis, or are these processes independent?

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Recently, Scherrer et al. (2006) studied the consequences of delta opioid receptor (DOR) sequestration on receptor function in vivo (locomotion) in knockin mice expressing fluorescent DOR. Groups of animals were pretreated with an agonist of DOR. After 2 h, when fluorescent DOR has internalized as a function of agonist concentration , a second dose was injected to all groups. Animals pretreated with either vehicle or the low agonist dose, whose receptors remain on the surface, showed a significant hyperlocomotor response. On the contrary, animals pretreated with doses producing both the locomotor response and receptor redistribution showed no significant increase of locomotor activity in response to the second injection. Mice with endocytosed receptors, therefore, are insensitive to the agonist. This last experiment strongly suggests that DOR internalization prevents further DOR signaling. Thus, that receptor internalization represents a main mechanism for receptor desensitization in vivo. The relationship between opioid-induced endocytosis and anti-nociceptive tolerance has been investigated but the conclusions were conflicting. In b-arrestin 2 knockout mice, in which endocytosis is blocked, agonist-induced desensitization of µ-opioid receptors is strongly impaired and mice exhibit increased sensitivity to the acute anti-nociceptive effects of morphine (Bohn et al. 1999; von Zastrow 2004). These results suggest that arrestin-mediated endocytosis of opioid receptors is induced by morphine in vivo and contributes directly to the development of physiological tolerance to opioids (Bohn et al. 2000). However, opioid tolerance-related changes in signaling after stimulation of µ-opioid receptors do not correlate with the endocytosis of these receptors in vivo (Trafton and Basbaum 2004).

2.4.3

Redistribution of GPCRs in the Cytoplasm After Chronic Stimulation

2.4.3.1

Downregulation

Downregulation of GPCRs is characterized by a decrease in the total number of receptors in neurons and a decrease in the number of receptors at the membrane. Downregulation can be distinguished from internalization, which is defined by redistribution of receptors from the plasma membrane to the cytoplasm without modification of total receptor number. In vitro, the number of receptors present in cells can be regulated at the level of receptor gene expression and biosynthesis, in addition to the level of receptor degradation (von Zastrow 2001). In the case of b2 adrenoceptors, proteolysis is believed to be the predominant mechanism of downregulation (Heck and Bylund 1998). In vivo, the decrease in M2- and M4-receptor abundance in dendrites (i.e., in the larger compartment of the neuron), and the decrease in the number of membrane-bound M2 receptors after chronic cholinergic neuron stimulation, show that these receptors are downregulated (Liste et al. 2002; Decossas et al. 2003). Different mechanisms might induce downregulation, including modulation of gene expression. The decrease in receptor M2 mRNA in NBM or

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striatum neurons of mice in which levels of ACh are chronically high (chronic hypercholinergic mice) might partially explain the loss of M4 receptors in dendrites. A decrease in D1 receptor mRNA has also been demonstrated in mice with chronically high levels of dopamine (chronic hyperdopaminergic mice) (Giros et al. 1996; Dumartin et al. 2000). Alternatively, downregulation might result from increased proteolysis of GPCRs. The mechanism by which plasma-membrane abundance of GPCRs decreases after repetitive and/or long-lasting stimulation in vivo seems to involve at least two phenomena: (1) limited delivery of the receptors to the plasma membrane because of their sequestration in protein synthesis and maturation compartments; and (2) degradation in lysosomes.

2.4.3.2

Intraneuronal Sequestration of GPCRs

Electron-microscopic analyses after immunohistochemistry demonstrate that, in constitutive chronic hyperdopaminergic or hypercholinergic mice, D1, M2 and M4 receptors are trapped in the cytoplasmic compartments of synthesis and maturation (i.e., the endoplasmic reticulum and Golgi apparatus) (Dumartin et al. 2000; Liste et al. 2002; Bernard et al. 2003; Decossas et al. 2003) (Figs. 2.1 and 2.2). In hypercholinergic mice, M2 receptors are almost absent at the plasma membrane (Fig. 2.1). This suggests that, once synthesized, GPCRs are trapped in endoplasmic reticulum and Golgi apparatus, and not targeted to the plasma membrane of the somatodendritic compartment. The decrease in total M2 receptor number might also be explained by decrease in receptor neosynthesis, because M2 receptor mRNA expression is decreased in neurons of AChE knockout mice (Decossas et al. 2003). The molecular mechanisms that prevent the newly synthesized proteins reaching the plasma membrane are still poorly understood. However, a membrane protein associated with the endoplasmic reticulum, dopamine-receptor-interacting protein 78 (DRIP78), has been linked to the transport of GPCRs, including D1 and M2, from the endoplasmic reticulum to the cell membrane (Bermak et al. 2001). Neurons from DRIP78 knockout mice do indeed accumulate D1 and M2 receptors in the endoplasmic reticulum. We therefore suspect that such a mechanism is impaired during chronic stimulation. Intraneuronal sequestration of GPCRs is a reversible process, because reduction of hyperstimulation enables the receptors to return to the membrane, as has been shown for M2 and D1 (Dumartin et al. 2000; Bernard et al. 2003).

2.4.3.3

Fate of Receptors After Sequestration

Under normal conditions, the majority of GPCRs are targeted from the Golgi apparatus to the plasma membrane, and only a few of them are degraded. In vitro, long-lasting stimulation activates degradation of GPCRs in lysosomes, as has been shown for b2 adrenoceptors (Kallal et al. 1998). In vivo, GPCRs are mainly sent

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from the Golgi apparatus to the degradation lysosomal compartment, as observed in the NBM and striatal neurons of hypercholinergic mice (Bernard et al. 2003) (V. Bernard, unpublished).

2.4.3.4

Function of Downregulation

There is probably a functional link between the decrease in number of membrane GPCRs after chronic stimulation and the changes in functions that are regulated by this receptor. For example, in vitro data demonstrate that downregulation of M3 receptors after chronic stimulation induces desensitization (Detjen et al. 1995). In vivo, AChE knockout mice are resistant to M2-agonist-induced salivation and hypothermia (Li et al. 2003). This is due to the absence of M2 receptor stimulation, because the same response has been demonstrated in M2 receptor knockout mice (Gomeza et al. 1999; Bymaster et al. 2001). Similarly, recycling of the µ-opioid receptor to the plasma membrane correlates with the increase in µ-receptormediated anti-nociception (Cahill et al. 2001, 2003).

2.4.4

Relationships Between Endocytosis and Downregulation

The relationship between endocytosis and downregulation of GPCRs is still being debated. In vitro data suggest that they are independent phenomena. Deletion of a part of the third intracytoplasmic loop of the human M2 receptor inhibits internalization after agonist stimulation, but partially inhibits M2 receptor downregulation (Tsuga et al. 1998). Alternatively, the mutation of one specific amino acid of the M2 receptor decreases its ability to display downregulation, without affecting its internalization properties (Goldman and Nathanson 1994). Phosphorylation of some residues of the histamine H1 receptor is required for receptor transport from endosomes to lysosomes, and thus downregulation has no effect on internalization (Horio et al. 2004). Ex vivo experiments on organotypic cultures of chronic AChstimulated striatum (Bernard et al. 2003) suggest that endocytosis does not contribute to the decrease in the abundance of M2 receptors in the plasma membrane, because M2 receptors are not co-incorporated with transferrin, which characterizes an endocytotic process. However, other in vivo and in vitro data suggest a link between endocytosis and downregulation (Cahill et al. 2001; Liste et al. 2002). In vivo, internalization and intracytoplasmic sequestration of M2 receptors might contribute to the decrease in the membrane-bound M2 receptors. Indeed, subchronic stimulation of muscarinic receptors leads to increased numbers of M2 receptors in both endosomes and endoplasmic reticulum (Liste et al. 2002). The molecular mechanisms leading to the downregulation are still unclear. However, if internalization and downregulation are linked, we can hypothesize that these two processes share common molecular mechanisms at least in the first step, such as phosphorylation of GPCRs and endocytosis in clathrin-coated pits,

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which has been demonstrated for b2 adrenoceptors in vitro (Gagnon et al. 1998). In addition, downregulation of the µ-opioid receptor in vitro involves molecules activated during endocytosis, such as GPCR kinase (GRK), arrestin 2, dynamin, rab5 and rab7 (Li et al. 2000).

2.5

Regulation of GPCR Distribution in the Axonal Field After Acute and Chronic Stimulation

Regulation of GPCR compartmentalization at neuronal terminals by the neurochemical environment might contribute to modulation of functional responses, including neurotransmitter release. Few studies have addressed this question in vitro or in vivo. In vitro, the metabotropic glutamate mGlu5 receptor, the neurotensin NTS1 receptor and the dopamine D1 receptor (three GPCRs) display endocytosis in axons and/or terminals after acute stimulation by their respective agonists (Martin-Negrier et al. 2000; Nguyen et al. 2002; Fourgeaud et al. 2003). In vivo, no modification of M2 receptor density at varicosities was shown after acute stimulation. Conversely, chronic stimulation of ACh receptors induces an increase in M2 receptor density at cortical cholinergic varicosities (Decossas et al. 2003) (Fig. 2.2). The mechanisms underlying these different effects remain unidentified. However, we hypothesize that different regulation of the sorting signals by chronic stimulation might direct M2 receptors from the Golgi apparatus to the terminals, and so lead to accumulation of the receptors in varicosities.

2.6

Concluding Remarks

The results obtained for different GPCRs in the brain suggest a model of trafficking of GPCRs in vivo under acute and chronic stimulation conditions (Fig. 2.2). Acute stimulation induces endocytosis of GPCRs through clathrin-coated pits. These receptors might then be either degraded directly in lysosomes or recycled to the plasma membrane. Chronic stimulation inhibits the delivery of receptors to the plasma membrane from synthesis and maturation compartments (the endoplasmic reticulum and Golgi apparatus). The receptors that are no longer targeted to the membrane are thus directly degraded in lysosomes, leading to the downregulation of GPCRs. Chronic high levels of ACh had opposite effects on the regulation of M2 receptor density at the plasma membrane in postsynaptic somatodendritic and presynaptic axonal compartments of the same neuron in AChE knockout mice (Decossas et al. 2003) (Fig. 2.2). In addition to the intraneuronal redistribution observed in the somatodendritic field, M2 receptors were redistributed along the plasma membrane of the soma, dendrites and axon: the M2 receptor density decreased at the

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plasma membrane of the somatodendritic field and increased at the membrane of terminals. This suggests that the mechanism regulating the GPCR membrane targeting by the neurochemical environment differs at the plasma membrane depending on the subcellular compartment. The molecular mechanisms that underlie the targeting of M2 receptors to varicosities are unclear (Trimmer 1999).The different effects at somatodendritic and axonal membranes might also result from subcellular compartmentalization of cytoplasmic regulatory factors involved in trafficking of GPCRs. The sorting signals that direct M2 receptors from the Golgi apparatus to the nerve terminals might be regulated, which could lead to accumulation of the receptor in varicosities, as suggested by the increase in the total receptor numbers at the terminals of basalocortical cholinergic neurons. The regional differences might also result from the differences in receptor membrane recycling and degradation efficiencies between the somatodendritic and axonal fields, as has been demonstrated for the neurotensin receptor NT1 (Nguyen et al. 2002). More of this receptor might be recycled, and less of it degraded, in axon terminals than in the soma and dendrites. The opposing regulation of the abundance of receptors at presynaptic and postsynaptic sites suggests differences in the functions transmitted by these GPCRs at these sites. This has been observed for the adenosine A1 receptor, which differentially desensitizes the neuronal response depending on its presynaptic or postsynaptic localization (Wetherington and Lambert 2002). We have reviewed the trafficking of GPCRs after stimulation; however, inhibition of receptors by antagonists also induces changes in receptor distribution that shed additional light on multiple mechanisms for trafficking of GPCRs (Gray and Roth 2001). Further investigations will be required for a better understanding of the link between intraneuronal trafficking of GPCRs and neuronal responses induced by GPCR activation. This might enable the development of new strategies for treating neurological diseases associated with altered GPCR signaling, such as Parkinson’s and Alzheimer’s diseases (Levey 1996; Muriel et al. 1999; von Zastrow 2001).

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Chapter 3

Small GTPases and Their Role in Regulating G Protein-Coupled Receptor Signal Transduction Fabiola M. Ribeiro and Stephen S.G. Ferguson

3.1

Introduction

Heterotrimeric guanine nucleotide (G) protein-coupled receptors (GPCRs) are integral membrane receptor proteins that are coupled to a diversity of signal transduction mechanisms. The receptor family includes more than 800 distinct genes and alternative splicing of these genes results in the expression of as many as 1,000–2,000 distinct receptor proteins (Lander et al. 2001; Venter et al. 2001; Gesty-Palmer and Luttrell 2008). GPCRs function to modulate many important physiological processes including vision, olfaction, inflammation, immunity, cognition, pain perception, cardiac function, and neurotransmission (Gainetdinov et al. 2004; Vroon et al. 2006; Harris et al. 2008; Insel et al. 2007; Pan et al. 2008). Thus, the diverse array of GPCRs available for pharmaceutical manipulation makes them ideal targets for the treatment of human disease. As a consequence, GPCRs are the primary target for more than 40% of pharmaceutical agents in clinical use for the treatment of diseases and are also a primary target for drugs of abuse (Flower 1999; GestyPalmer and Luttrell 2008). The traditional view of GPCR signaling is that they transduce the information provided by a diverse assortment of stimuli into intracellular second messengers by functioning as ligand-regulated guanine nucleotide exchange factors for the family of heterotrimeric GTP-binding proteins (G proteins) (Neer 1995). In this model, agonist binding within the ligand binding domain of the GPCR stabilizes a receptor conformation that allows intracellular GPCR domains to facilitate the exchange of GDP for GTP on the G protein Ga-subunit, leading to the dissociation of the Ga- and Gbg-subunits. The activated proteins Ga and Gbg can positively or S.S.G. Ferguson (*) and F.M. Ribeiro J. Allyn Taylor Centre for Cell Biology, Molecular Brain Research Group, Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada e-mail: [email protected] A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_3, © Springer-Verlag Berlin Heidelberg 2010

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negatively regulate various downstream effectors such as phospholipases, adenylyl cyclases, and ion channels. However, recent work in this field has demonstrated that some ligands that normally function as agonists to activate one given signaling pathway may also function as antagonists, or even inverse agonists, on a different signaling pathways in the same cell (Galandrin et al. 2007; Kenakin 2007). This so-called “biased agonism” is particularly important with respect to b-arrestin signaling and may underlie the therapeutic utility of a number of clinically utilized drugs to target GPCRs (Wisler et al. 2007). In addition, GPCRs are now recognized to play an important role in the organization of cell signaling complexes that may function independently from heterotrimeric G protein activation. This includes the recruitment of modular scaffold proteins via their carboxyl-terminal PDZ domain-binding motifs (Bockaert and Pin 1999; Hall et al. 1999; Hall and Lefkowitz 2002). In this chapter, we summarize GPCR signaling mediated by small G proteins of the Arf, Ras, Rho, and Rab families of GTPases that are activated either downstream of heterotrimeric G proteins or through their direct interaction with GPCRs and their regulatory proteins (Bhattacharya et al. 2004a). In particular, we emphasize how the association of small GTPases with both GPCRs and GPCR regulatory proteins may influence GPCR signaling and intracellular trafficking.

3.2

The Family of Small GTPases

The small GTPases are monomeric G proteins with molecular masses ranging from 20 to 30 kDa (Exton 1998). The small GTP-binding protein superfamily contains over 100 members that are generally classified by structural similarity into five subfamilies: Ras family GTPases (e.g., Ras, Rap and Ral), Rho family GTPases (Rho, Rac and cdc42), Arf family GTPases (Arf 1–6, Arl 1–7 and Sar), Rab family GTPases (>60 members, e.g., Rab5), and Ran family GTPases. These small GTPases play major roles in the regulation of growth, morphogenesis, cell motility, axonal guidance, cytokinesis, and trafficking through the Golgi, nucleus, and endosomes (Bos 1998; Sah et al. 2000; Takai et al. 2001; Zerial and McBride 2001; Wettschureck and Offermanns 2005; Lundquist 2006). In general, Ras family GTPases regulate cell signaling events that lead to alterations in gene transcription. The Rho family GTPases function as regulators of the actin cytoskeleton and can also influence gene transcription. The Rab and Arf family GTPases control the formation, fusion, and movement of vesicular traffic between different membrane compartments of the cell, and Ran GTPases regulate both microtubule organization and nucleocytoplasmic protein transport. All of these small GTPases function as molecular switches that control eukaryotic cell function by cycling between two interconvertible, GDPbound “inactive” and GTP-bound “active” forms (Bos 1998; Takai et al. 2001). The rate-limiting step of the GDP for GTP exchange, which is the dissociation of GDP, is promoted by the association of guanine nucleotide exchange factors (GEFs) (Rossman et al. 2005). The activity of GEFs for small GTPases may be regulated by

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upstream signaling events such as the activation of a heterotrimeric G protein. The binding of GTP eventually leads to a conformational change in the effector-binding domain of the small G protein such that it interacts with downstream effector(s) to modulate the activity of a variety of signaling pathways. The inactivation of small GTPases is facilitated by the association of GTPase-activating proteins (GAP) (Ross 2008). GAPs function to stimulate the intrinsic GTPase activity of small GTPases converting them back to the inactive GDP-bound state.

3.2.1

GPCR Signaling via Ras Family GTPases

The first Ras family members (H-Ras and K-Ras) were identified as oncogenes of sarcoma viruses (Shih et al. 1978). Shortly after, Ras was found to be a family of small GTPases mainly functioning to regulate gene expression (Takai et al. 2001). Ras family GTPases include the Ras proto-oncogene H-Ras, K-Ras, M-Ras, N-Ras, R-Ras, RalA, RalB, the Rap proteins Rap1 and Rap2, and Tc21 (Feig 2003). Rasmediated pathways can regulate cell proliferation, differentiation, morphology, and apoptosis (Noda et al. 1985; Feramisco et al. 1984; Kauffmann-Zeh et al. 1997). The best studied Ras pathways are mediated by (1) Raf kinase, (2) phosphatidylinositol-3 lipid kinase (PI3K); and (3) Ral guanine nucleotide exchange factors (RalGEFs) which activate the Ras family member Ral.

3.2.1.1

Ras-Dependent GPCR Signaling

Ras-dependent activation of Raf protein kinase (C-Raf-1, A-Raf, and B-Raf) is induced by a number of GPCRs and subsequently leads to the activation of MEK (mitogen-activated protein kinase (MAPK) kinase), the phosphorylation of extracellular regulated kinase (ERK or MAPK), and the translocation of ERK into the nucleus to activate gene transcription (Dent et al. 1992; Huang et al. 1993). The activation of Ras/MAPK by GPCRs can occur through two main mechanisms: (1) via activation of classical heterotrimeric G protein effectors, such as increased intracellular Ca2+, leading to the activation of second messenger-dependent protein kinases, and (2) via the transactivation of receptor tyrosine kinases (RTKs) (Luttrell 2002). Gq/11-coupled receptor-mediated activation of Ca2+-dependent protein kinase C (PKC) can result in the phosphorylation of Raf-1, leading to the subsequent activation of ERK1/2 (Kolch et al. 1993). In neurons, increases in cytoplasmic Ca2+ leads to the activation of the Ca2+/calmodulin-dependent GEF, p140 RasGRF, which activates Ras (Farnsworth et al. 1995). Furthermore, the Ca2+-dependent activation of the FAK family kinase, Pyk2, also leads to the Ras-dependent activation of Raf (Lev et al. 1995). In the case of Gs-coupled GPCRs, the activation of the MAPK cascade involves either the cAMP-dependent activation of the Rap1 GEF, EPAC, or the phosphorylation of Rap1 by cAMP-dependent protein kinase (Vossler et al. 1997).

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GPCR-mediated transactivation of RTKs is an additional mechanism by which Go/i- and Gq/11-coupled GPCRs can activate the Ras/MAPK signal transduction cascade. For Gi-coupled GPCRs, Gbg-subunits stimulate the Src-dependent activation of matrix metalloproteinases which release substances such as heparin-binding epidermal growth factor (EGF) from the cell surface. These substances in turn stimulate either the autocrine or paracrine activation of cell surface EGF receptors resulting in Ras-dependent activation of MAPK (Prenzel et al. 1999). For receptors coupled to Gq/11, the activation of the matrix metalloproteinases is mediated by Gaq/11 and results in the Ras-dependent ERK activation (Seo et al. 2000). 3.2.1.2

Ral-Dependent GPCR Signaling

The Ral subfamily consists of two members, RalA and RalB, which are implicated in the regulation of vesicle trafficking, cell morphology, gene transcription, and oncogenesis (Chardin and Tavitian 1989; Feig 2003; Chardin and Tavitian 1986). Several RalGEFs have been identified including RalGDS, Rgl, RIf, and Rgr (Feig et al. 1996). Several groups have reported that Ral proteins and RalGEFs constitute a distinct downstream pathway from Ras and induce cellular transformation in parallel with activation of the Raf/MAPK cascade (Hofer et al. 1994; Herrmann et al. 1996; Urano et al. 1996; White et al. 1996). Consistent with this observation, stimulation of insulin and EGF receptors activates Ral, and this activation is inhibited by a dominant-negative mutant of Ras (Wolthuis et al. 1998). In the case of the N-formylmethionyl-leucylphenylalanine (fMLP) receptor, Ral activation can be accomplished by both Ras-dependent and Ras-independent mechanisms (M’Rabet et al. 1999). The Ras-independent mechanism for GPCR-mediated Ral activation appears to involve the b-arrestin-dependent regulation of RalGDS (Bhattacharya et al. 2002). In the absence of fMLP receptor activation, RalGDS exists as a complex with b-arrestin in the cytosol (Bhattacharya et al. 2002) (Fig. 3.1b). However, in response to fMLP receptor activation, this complex translocates to the cell surface and RalGDS is displaced from b-arrestin, upon b-arrestin binding to the receptor, allowing membrane localized RalGDS to activate Ral. The activation of Ral leads to the subsequent reorganization of the actin cytoskeleton. This molecular mechanism may explain, in part, the impairment in chemotaxis observed for lymphocytes obtained from b-arrestin2 null mice (Fong et al. 2002). For Gq/11-coupled receptors, Ral may be activated by the direct binding of Ca2+-calmodulin to the carboxyl-terminus of RalA (Wang et al. 1997). RalGDS activity is also regulated by Gs-coupled receptors through the activation of cAMP-dependent protein kinase (Kikuchi and Williams 1996).

3.2.2

GPCR Signaling via Rho Family GTPases

The Rho family of GTPases is composed of at least 20 members (RhoA, RhoB, RhoC, RhoD, Rif, Rnd1, Rnd2, Rnd3/RhoE, RhoH/TTF, Rac1, Rac2, Rac3, RhoG,

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Cdc42, TC10 (RhoQ), TCL (RhoJ), Wrch1 (RhoV), Chp/Wrch2 (RhoU), RhoBTB1, RhoBTB2) and the best studied members are RhoA, Rac, and Cdc42 (Boureux et al. 2007). Rho family members share significant amino acid homology with each other, ranging from 50 to 90% homology. Rho pathways regulate multiple cellular functions, such as cytoskeletal reorganization in response to extracellular signals, regulation of Ca2+ sensitivity of smooth muscle contraction, and gene expression (Hirata et al. 1992; Ridley and Hall 1992, 1994; Coso et al. 1995; Hill et al. 1995). RhoA can be activated by multiple Ga12/13-coupled GPCRs. GPCR-mediated activation of RhoA modulates the activity of several downstream effector proteins including Rho kinase (ROCK), protein kinase N (PKN), jun kinase (JNK), citron kinase, phospholipase D (PLD), LIM kinase (LIMK), diaphanous (Dia)1, rhophilin, and rhotekin (Bishop and Hall 2000; Jaffe and Hall 2005). Furthermore, GPCRmediated Jak/STAT activation also appears to require the activation of Rho GTPases (Pelletier et al. 2003). The physiological consequences of Ga12/13-coupled GPCRmediated activation of Rho include: (1) thrombin, thromboxane, sphingosine 1-phosphate, endothelin, angiotensin II, and vasopressin receptor-mediated vasoconstriction (Gohla et al. 2000; Seasholtz and Brown 2004; Watterson et al. 2005), (2) LPA receptor-mediated neurite retraction (Sayas et al. 1999), and (3) thrombin or lysophosphatidic acid (LPA) receptor-mediated cell proliferation and metastasis (Sahai and Marshall 2002; Kelly et al. 2007). Ga12/13 proteins mediate RhoA activation via their ability to interact with and stimulate a variety of RhoGEFs in response to GPCR activation. These GEFs include: PDZ-RhoGEF, leukemia-associated RhoGEF (LARG) which is also activated by Gaq, and Lbc-RhoGEF (Dutt et al. 2004). Moreover, Ga13, but not Ga12, can stimulate p115RhoGEF activity (Kozasa et al. 1998; Hart et al. 1998). Gaq may also activate Rho activation via a Gaq-specific RhoGEF, p63RhoGEF (Lutz et al. 2005).

3.2.2.1

Rho-Dependent GPCR Cytoskeletal Signaling

Rho GTPases play a crucial role in the regulation of cytoskeletal rearrangement and adhesive functions underlying cell migration (Ridley et al. 2003). While Rac and Cdc42 are involved in the formation of cell protrusions and the formation of adhesions, RhoA plays an important role in the retraction of cell protrusions. In neurons, LPA-mediated Rho GTPase modulation is very important for neuronal positioning, migration, and development (Moers et al. 2008). LPA-induced neurite retraction is mainly driven by actinomyosin-based contractile forces initiated by Ga12/13 activation of RhoA and its downstream effector ROCK (Jalink et al. 1994). Glycogen synthase kinase-3 (GSK-3) can also be activated by LPA through RhoA and the tyrosine kinase Pyk2 (Sayas et al. 2002, 2006). GSK3 appears to contribute to optimal neurite retraction by phosphorylating microtubule-binding proteins leading to microtubule destabilization (Sayas et al. 2006).

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Rho-dependent GPCR Proliferative Signaling

The activation of GPCRs for acetylcholine, LPA, thrombin, and endothelin can promote proliferative signaling pathways (Sah et al. 2000). Mutations in the thyroid-stimulating hormone have been found in 30% of hyperfunctioning thyroid adenomas (Parma et al. 1993). Furthermore, mutation in Ga-subunits has also revealed the growth-promoting activity of Ga12, Ga13, Gai2, Gaq, and Gao (Dhanasekaran et al. 1998). The Ga12/13 family has exhibited the greatest transforming potency. The mechanism employed by GPCRs to promote cell growth and proliferation appears to be related to the regulation of many signal transduction pathways, such as the activation of Ras and Rho. The Rho family of proteins can promote the disordered proliferative, invasive, and metastastic properties of tumor cells by activating many transcription factors, such as the c-Fos and c-Jun oncoproteins (reviewed by Benitah et al. 2004). For example, thrombin receptor activation leads to RhoA-dependent expression of the cysteine-rich 61 protein (Cyr61), which, upon secretion, promotes cell proliferation via integrin activation (Walsh et al. 2008).

3.2.2.3

Rho- and GPCR-Dependent Cardiovascular Remodeling

Rho family GTPases also appear to play important roles in mediating cardiovascular remodeling, inducing hypertrophy, hyperplasia, and migration of vascular smooth muscle cells (VSMCs) induced by a number of GPCRs. Several Rho family members, including Rac, are activated in response to angiotensin II receptor (AT1AR) signaling. AT1AR activation has been reported to result in reactive oxygen species formation in VSMCs as well as the JNK-mediated VSMC hypertrophy (Gregg et al. 2003; Woolfolk et al. 2005; Ohtsu et al. 2006a, 2006b). Rho-dependent ROCK has also been implicated in cardiovascular remodeling associated with hypertension and other cardiovascular diseases (Ohtsu et al. 2006b; Higuchi et al. 2007). AT1AR stimulation can also activate RhoA and stimulate the formation of stress fibers via a mechanism that depends upon both b-arrestin1 and Gaq (Barnes et al. 2005). Interestingly, AT1AR-stimulated b-arrestin-mediated ERK signaling is independent of G protein activation (Wei et al. 2003). AT1AR activation also leads to the formation of a complex composed of ARF6 and Rac1 (Cotton et al. 2007). Further studies have demonstrated that AP-2 and clathrin are also part of an ARF6 complex, and that depletion of ARF6 leads to the inhibition of receptor endocytosis (Poupart et al. 2007).

3.2.3

Arf Family GTPases and GPCRs

Arf GTPases play an essential role in regulating secretory vesicle budding, Golgi transport, and membrane trafficking events involved in endocytosis (D’Souza-Schorey

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and Chavrier 2006). Based on amino acid sequence homology, the Arf family of GTPases can be subdivided into three subclasses: Class I Arf proteins (Arf 1, Arf 2 and Arf3) are considered to play a role in the regulation of the assembly of coat proteins on budding vesicles within the secretory pathway (Bonifacino and Glick 2004). The function of Class II Arf proteins (Arf4 and Arf5) is less well described, although there is evidence that they may contribute to the regulation of trans-Golgi coat protein assembly (Claude et al. 1999). Arf6 is the sole member of the Class III Arf proteins and is recognized as playing an important role in regulating vesicular membrane trafficking from the cell surface to endosomes (D’Souza-Schorey et al. 1995). The Arf proteins are ubiquitously expressed and are remarkably well conserved from yeast to humans. Of these Arf family proteins, there is evidence that Arf1, Arf3 and Arf6 may contribute to the regulation of GPCR signaling either via direct receptor association or by regulating the vesicular pathways utilized by GPCRs as they traffic through the intracellular compartment.

3.2.3.1

Regulation of GPCRs by Class I Arf proteins

There are several reports in the literature suggesting that Arf1 and Arf3 may directly interact with a variety of GPCRs and facilitate the coupling of GPCRs to the activation of phospholipase D (PLD) (Mitchell et al. 1998, 2003; McCulloch et al. 2001; Ronaldson et al. 2002; Koch et al. 2003; Robertson et al. 2003; Johnson et al. 2006). Michell and colleagues (1998) were the first to report that both Arf1 and Arf3 can be co-immunoprecipitated with the M3 muscarinic acetylcholine receptor (mAChR) and AT1AR, and that agonist activation of the M3 mAChR results in membrane translocation of Arf1, Arf3, and Rho. Moreover, the study suggests that GPCRs that activate PLD in an Arf1/3-dependent manner contain an NPxxY motif in their seventh transmembrane spanning domain. Subsequent investigation demonstrated that the third intracellular loop domain (il3) of the M3 mAChR associates with Arf1 and is essential for the selective activation of PLD1 (Fig. 3.1a) (Mitchell et al. 2003). Arf1 has also been shown to interact with the il3 and carboxyl-terminal tail of the 5-HT2A receptor and that the highly conserved NPxxY motif is required for 5HT2A receptor-mediated activation of PLD (Robertson et al. 2003; Johnson et al. 2006). Furthermore, the insertion of the hop1 cassette localized within the il3 of a PAC1 receptor splice variant is required for brefeldin A-sensitive (presumably Arf1/3-dependent) activation of PLD (McCulloch et al. 2001; Ronaldson et al. 2002). Thus Arf1/3 appear to interact with intracellular GPCR domains that have previously been implicated in heterotrimeric G protein coupling. Arf1 activity also plays a key role in regulating the PLD-dependent endocytosis of the m-opioid receptor (Fig. 3.1a) (Koch et al. 2003). PLD2 has been shown to specifically interact with the carboxyl-terminal tail of the m-opioid receptor by yeast two-hybrid and both Arf and PLD activity are required for m-opioid receptor endocytosis. In addition, to the m-opioid receptor, the endocytosis of the AT1AR, d-opioid receptor, cannabinoid receptor isoform 1 and metabotropic glutamate

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Fig. 3.1 Interaction of GPCRs with small G proteins. (a) Agonist binding to a GPCR promotes the translocation of Arf1 and Arf3 to the third intracellular loop of GPCRs and allows for both receptor activation of PLD and the subsequent endocytosis of the receptor. Arf6 interacts with GPCRs both directly and in a complex with ARNO and b-arrestin (Arr) and plays a role in the regulation of receptor internalization. (b) The Ral GEF, RalGDS, is localized to the cytosol and maintained in an inactive complex with b-arrestins. GPCR activation results in the membrane translocation of the b-arrestin/RalGDS complex. b-Arrestin (Arr) receptor binding is proposed to promote the b-arrestin/RalGDS complex, freeing RalGDS to activate membrane bound Ral

receptors 1 and 5 is also PLD2-dependent (Bhattacharya et al. 2004b; Du et al. 2004; Koch et al. 2006). However, unlike what is reported for the m-opioid receptor, the PLD2-dependent internalization of metabotropic glutamate receptors 1 and 5 is independent of Arf activity and is rather dependent upon the recruitment of Ral and RalGDS to the receptor (Bhattacharya et al. 2004b). Nevertheless, PLD2 represents a potentially important and novel endocytic adaptor that may work in concert with Arf and b-arrestin proteins to facilitate GPCR endocytosis. Consistent with the role of Arf1 in the regulation of vesicular secretion there is evidence that Arf1 contributes to the resensitization of protease receptors (PARs) (Luo et al. 2007). The activation of PARs by proteases results in their irreversible activation, requiring that the receptors be rapidly targeted to lysosomes for degradation to terminate signaling (Trejo et al. 1998). Thus, resensitization of PAR signaling involves the recruitment of receptors stored in the Golgi apparatus (Hein et al. 1994). Recently, Luo and colleagues (2007) demonstrated that PAR2 is associated with the Golgi resident protein p24A. Activation of cell surface PAR2 receptors results in the Arf1-dependent dissociation of the PAR2/p24A complex, thereby initiating the trafficking of the receptor to the cell surface. Although the molecular mechanism by which Arf1 regulates

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the dissociation of the PAR2/p24A complex is not known, this may represent a novel mechanism by which many intracellularly localized receptors are recruited to the cell surface.

3.2.3.2

Regulation of GPCRs by Class III Arf proteins

As indicated earlier in this review, Arf6 is the sole member of the Class III Arf proteins. The activity of Arf6 is regulated by two Arf GEFs, ADP ribosylation factor nucleotide-binding site opener (ARNO) and EFA6. The first evidence linking Arf6 to the regulation of GPCR signaling is provided by a study examining the mechanisms underlying the b-arrestin-dependent desensitization of the luteinizing hormone (LH) receptor (Mukherjee et al. 2000). In this study, ARNO is shown to promote the release of membrane-bound b-arrestin protein in response to LH receptor activation, allowing b-arrestin to bind to and desensitize the receptor. Moreover, amino-terminal fragments of Arf6 antagonize the release of b-arrestin from the membrane indicating that Arf6 activation by the LH receptor is also important for the regulation of LH receptor desensitization. It is now recognized that b-arrestin forms a complex with both ARNO and Arf6 in response to b2-adrenergic receptor (b2AR) activation and that Arf6 activation is required for the endocytosis of this receptor (Fig. 3.1a) (Claing et al. 2001). Similarly, overexpression of centaurin-a1, an Arf6 GTPase activating protein, attenuates b2AR endocytosis (Lawrence et al. 2005). Arf6 is now recognized to be involved in the regulating the endocytosis of the AT1AR, M2 AChR, M4 AChR, vasopressin type II receptor, m-opioid receptor, endothelin B receptor and metabotropic glutamate receptor 7 (Houndolo et al. 2005; Poupart et al. 2007; Lavezzari and Roche 2007; Reiner and Nathanson 2008).

3.2.4

Rab Family GTPases and GPCRs

Rab GTPases form the largest branch of the Ras family of GTPases with at least 60 family members (Zerial and McBride 2001; Seachrist and Ferguson 2003). Rab GTPases are localized to the surface of distinct membrane bound organelles and any given organelle may contain several Rab species (Fig. 3.2). The Rabs play an essential role in the regulation of protein trafficking between intracellular membrane compartments by controlling vesicular endocytosis, trafficking, fusion, and exocytosis. At least two Rab proteins, Rab1 and Rab2, are localized to the ER-Golgi and regulate the transport of proteins from this compartment. Rab4, Rab5, and Rab11 have all been localized to early endosomes and Rab4 and Rab11 are found on perinuclear recycling endosomes. Rab7 is localized to late endosomes and, possibly, lysosomes. In response to agonist activation, GPCRs are internalized to endosomes where they can be dephosphorylated and either recycled back to the cell surface or targeted to lysosomes (Ferguson 2001). Thus, because Rabs regulate

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Fig. 3.2 Rab GTPases regulate the GPCR endocytosis and recycling both directly and via association with the receptor tail. Rab 5 regulates the endocytosis of GPCRs into clathrin-coated vesicles and mediates the AT1AR-dependent fusion with early endosomes. Rab 11 regulates the recycling of many GPCRs to the cell surface and interacts with the C-tail of the b2AR and thromboxane A2 receptor

trafficking of proteins between intracellular compartments, they are likely to play an important role in regulating the GPCR activity.

3.2.4.1

Rab4 and Rab5 Regulation of GPCR Activity

The internalization and intracellular trafficking of GPCRs is a highly regulated and dynamic process that is not only essential for GPCR desensitization, but is also required to allow dephosphorylation and resensitization of many GPCRs (Ferguson 2001). Treatments that prevent the endocytosis of GPCRs, such as the b2AR, also prevent receptor dephosphorylation and resensitization (Pippig et al. 1995; Zhang et al. 1997). However, prior to the use of dominant-negative and constitutively active Rab mutants, the precise identity of the compartment(s) involved in GPCR dephosphorylation, as well as the vesicular pathway(s) governing GPCR recycling, were unknown. Early studies examining the compartments involved

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in the dephosphorylation and resensitization of b2AR reveal that the receptor is dephosphorylated as the receptor traffics between Rab5- and Rab4-positive early endosomes (Seachrist et al. 2000). Overexpression of a dominant-negative Rab5 mutant prevents both the mobilization of the b2AR to early endosomes and b2AR dephosphorylation. In contrast, the overexpression of a dominant-negative Rab4 mutant does not prevent the dephosphorylation of the b2AR, but prevents the resensitization of the receptor by blocking receptor recycling. In addition to being localized to the Rab4-positive recycling endosomes, the b2AR has also been reported to traffic to Rab11-positive recycling endosomes, and Rab11 activity may dictate whether the receptor is recycled or targeted to lysosomes (Moore et al. 2004). Rab GTPases have been shown to regulate the endocytosis and recycling of several GPCRs. In the case of the neurokinin-1 receptor, Rab5 regulates the trafficking of the receptor to early endosomes, whereas both Rab4 and Rab11 regulate neurokinin-1 receptor recycling (Roosterman et al. 2004). The recycling of M4 AChR, CXCR2 and the thromoxane A2 receptor appears to be preferentially regulated by Rab11-positive recycling endosomes (Fan et al. 2003; Volpicelli et al. 2002; Theriault et al. 2004). In contrast, internalized corticotrophin-releasing factor receptor 1a is predominantly localized to Rab5- and Rab4-positive endosomes in both HEK 293 cells and cortical neurons (Holmes et al. 2006). Agonist activation of the m-opioid receptor results in the internalization of the receptor to Rab5-positive early endosomes (Wang et al. 2008). However, the recycling of phosphorylated receptor is regulated by Rab4-positive recycling endosomes, whereas the recycling of non-phosphorylated receptor appears to be Rab11dependent. Thus, while the majority of GPCRs that have been examined to date are initially internalized to Rab5-positive endosomes, they can be recycled back to the cell surface via Rab4-positive recycling endosomes, Rab11-positive recycling endosomes or both.

3.2.4.2

RabGTPase Association with GPCRs

Rab GTPases may also associate directly with GPCRs. Yeast two-hybrid screening with the AT1AR carboxyl-terminal tail identified Rab5 as an AT1AR-binding partner (Seachrist et al. 2002). Rab5 not only binds to the carboxyl-terminal tail of the AT1AR, but agonist activation of the AT1AR also stimulates Rab5 GDP/GTP exchange, suggesting the possibility that GPCRs might function as GEFs for small GTPases. AT1AR activity leads to the homotypic fusion of early endosomes into large hollow cored vesicular structures, the formation of which can be prevented by the expression of a dominant-negative Rab5 mutant. The association of Rab5 with the AT1AR prevents both AT1AR recycling and targeting to lysosomes. Rab11 has also been shown to interact directly with the carboxyl-terminal tails of both the thromboxane A2 receptor and b2AR (Hamelin et al. 2005; Parent et al. 2008). The association of Rab11 with the carboxyl-terminal tails of both receptors appears to be necessary to mediate receptor recycling. Taken together, these observations

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suggest that, similar to what has been described for Arf proteins, Rab GTPases also form a complex with GPCRs to regulate their trafficking through the intracellular endosomal compartment. 3.2.4.3

Consequence of Altered Rab Protein Expression and Function

Recent studies provide evidence that alterations in Rab protein expression and/or activity may lead to alterations in the intracellular trafficking of GPCRs that may be associated with pathophysiological changes in GPCR signaling in vivo. The treatment of cells with the proton pump inhibitor, bafilomycin A1, prevents the accumulation of the b2AR in lysosomes in response to prolonged agonist treatment (Moore et al. 1999). Moreover, bafilomycin A1 treatment results both in the accumulation of the b2AR in Rab11-positive recycling endosomes and slows the overall rate of b2AR recycling. In addition, the treatment of cells with phosphoinositol 3-kinase inhibitors increases the apparent rate of b2AR endocytosis by promoting the accumulation of the receptor in a non-recycling Rab7-positive late endosomal compartment (Awwad et al. 2007). The overexpression of either Rab7 or a constitutively active Rab11 mutant promotes the exit of the AT1AR from Rab5-positive early endosomes to late endosomes and to Rab11-regulated recycling endosomes (Dale et al. 2004). The effect of Rab7 overexpression is to promote AT1AR degradation, whereas the effect of constitutively active Rab11 mutant overexpression is to allow AT1AR recycling. Consistent with a potential role for either altered Rab expression or function in disease, cardiac Rabs 1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing b2ARs (Wu et al. 2001). Moreover, cardiac b2AR responsiveness and resensitization to catacholamines is reduced in transgenic mice overexpressing a dominant-negative Rab4-S27N mutant and is associated with diminished cardiac inotropy (Odley et al. 2004). Thus, alterations in Rab GTPase expression and/or activity may be associated with disease. Although Rab GTPases appear to be ubiquitously expressed, the relative level of Rab protein expression between cell and tissue types is not clearly defined. Therefore differences in Rab protein expression may underlie observed differences in GPCR signal transduction profiles in different cell types.

3.3

Summary

Over the past decade considerable progress has been made regarding the understanding of the mechanisms by which GPCRs signal in cells. In this regard, it is now appreciated that in addition to heterotrimeric G protein signaling, GPCRs signal via a variety of small G proteins. Moreover, small G proteins play important roles in regulating a variety of fundamental cellular processes regulated by GPCRs. GPCRs activate small GTPases both through classical intracellular signaling cascades, as well as through the direct association with small G proteins. One outstanding issue to be

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determined is whether GPCRs function as GEFs for small GTPases or whether they recruit protein complexes that also contain small GTPase GEFs and effector proteins. Nevertheless, the activation of small G proteins by GPCRs is affected by a diverse variety of mechanisms and suggests that small G proteins may provide a novel target for the manipulation of GPCR signaling transduction in disease. Acknowledgements F.M.R. is the recipient of a Heart and Stroke Foundation of Canada Fellowship. S.S.G.F. is the recipient of a Canada Research Chair in Molecular Neurobiology and is a Heart and Stroke Foundation of Ontario Career Investigator. This work is supported by HSFO grant T5933 to S.S. G.F.

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Chapter 4

Regulation of G Protein Receptor Coupling, Mood Disorders and Mechanism of Action of Antidepressants Moran Golan, Gabriel Schreiber, and Sofia Avissar

4.1

Mood Disorders

Mood disorders encompass a group of psychiatric disorders in which pathological moods dominate the clinical picture. Major depressive disorder (unipolar depression) is reported to be the most common mood disorder, and may manifest as a single episode or as recurrent episodes. Bipolar disorders consist of at least one hypomanic or manic episode. The World Health Organization (WHO) has calculated that of the ten leading causes of disability worldwide, four are mental disorders, while the number one and number six causes are, respectively, major depression and bipolar disorder (Murray and Lopez 1996). The lifetime prevalence of major depression is 10–25% for women and 5–12% for men, and for bipolar disorder is 1–2% (American Psychiatric Association 1994).

4.2

The “Pharmacological Bridge” Approach to the Construction of Pathogenic Hypotheses for Mood Disorders

Most of the present modalities of pharmacological treatments for mental disorders were discovered in the 1950s. The Australian psychiatrist John Cade reported the anti-manic therapeutic potential of lithium in 1949 (Cade 1949). The French psyG. Schreiber (*) Barzilai Medical Center, Ashkelon and Division of Psychiatry, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva, 84105, Israel e-mail: [email protected] M. Golan and S. Avissar Division of Psychiatry, Ben Gurion University of the Negev, P.O. Box 653, Beer Sheva, 84105, Israel A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_4, © Springer-Verlag Berlin Heidelberg 2010

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chiatrists Jean Delay and Pierre Deniker discovered the therapeutic effects of the first antipsychotic medication, chlorpromazine, in 1952 (Delay and Deniker 1952). Similarly, the antidepressant therapeutic effects of imipramine, the first tricyclic antidepressant (Kuhn 1957), and iproniazide, the first monoamine oxidase (MAO) inhibitor antidepressant (Loomer et al. 1957), were discovered respectively by Roland Kuhn and by Nathan Kline in 1957. The introduction of relatively effective and selective drugs for the management of major mental disorders encouraged formulations of biological concepts for the pathophysiology and pathogenesis of psychiatric disorders. The “pharmacological bridge” approach to the construction of biological hypotheses for the pathogenesis of mental disorders is an attempt, from knowledge and hypotheses concerning biochemical mechanisms of action of neuropsychiatric medications, to construct pathogenic and pathophysiological hypotheses, e.g., (1) the dopamine hypotheses of schizophrenia (Creese et al. 1976; Seeman et al. 1976), and (2) the monoamine theories of mood disorders (Schildkraut 1965; Lapin and Oxenkrug 1969). The catecholamine (Schildkraut 1965) and indoleamine (Lapin and Oxenkrug 1969) hypotheses were essentially based on two sets of pharmacological observations. First, reserpine, a medication that decreases blood pressure by depleting biogenic amine stores, was known to precipitate clinical depression in some patients. Second, antidepressant medications which alleviate clinical depression were found to raise the functional capacity of the biogenic amines in the brain. Different variations of the biogenic amine model give somewhat different importance to the relative weight of norepinephrine, serotonin and other neurotransmitters in the development of pathological mood states. The permissive biogenic amine hypothesis suggests that serotonin deficits permit the expression of catecholaminemediated affective states (Prange et al. 1974); the cholinergic–noradrenergic imbalance hypothesis of mania and depression (Janowsky et al. 1972) represents yet another attempt to elucidate the roles of biogenic amines in mood disorders; and the noradrenergic dysregulation hypothesis (Siever and Davis 1985) envisions oscillation from one output mode to the other at different phases of depressive illness. There is an ongoing active search for candidate genes for mood disorders involving monoamine metabolism. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin metabolism, catechol-O-methyltransferase (COMT), which metabolizes norepinephrine and dopamine, monoamine oxidase A (MAO-A), a mitochondrial enzyme that plays an important role in degradative deamination of several different amines, including serotonin, norepinephrine and dopamine, the serotonin transporter, and serotonin receptors, are all considered major candidate genes for association studies in mood disorders (for review see Avissar and Schreiber 2002). Biochemical research in mood disorders has focused, alongside the cascade of events involved in signal transduction, on studies at the level of the primary messenger (the monoamine neurotransmitter) to the level of neurotransmitter receptors, and recently on information transduction mechanisms beyond receptors, involving the coupling of receptors with signal transducers and the regulation of this coupling. The family of heterotrimeric G proteins is a crucial point of convergence

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in the transmission of signals from a variety of primary messengers including monoamines and their membrane receptors to a series of downstream cellular events: intracellular second messenger effector enzymes and ionic channels.

4.3

Regulation of G Protein-Coupled Receptor Signal Transduction

G protein-coupled receptor (GPCR) signaling plays a pivotal role in regulating various physiological functions including neurotransmission and transduction. The magnitudes of these physiological responses are linked intimately to the delicate balance between GPCR signal generation and signal termination. Almost all GPCRs are tightly regulated by a common desensitizing mechanism. The process of agonist-specific, homologous desensitization of receptors is characterized by an increase in the refractoriness of a receptor to signal in response to repeated or sustained exposure to its agonist, limiting both the magnitude and the temporal extent of the receptor signal, thus protecting cells from over-stimulation. Our knowledge concerning the basic mechanisms underlying the phenomenon of desensitization, internalization, down-regulation, and resensitization of GPCRs has advanced far during the last decade. These mechanisms involve the activities of two families of proteins: GPCR kinases (GRKs) (Palczewski 1997; Pitcher et al. 1998; Metaye et al. 2005), and arrestins (Stephen and Lefkowitz 2002; Gainetdinov et al. 2004). G protein signal transduction is regulated at various points; a proximal regulatory point is receptor coupling with G protein. A family of proteins characterized as cytosolic regulators of G protein function, phosducin-like proteins (PhdLP) (Schulz 2001), has been described. The most important characteristic of these proteins appears to be their high affinity sequestration of bg-subunits of G proteins leading to neutralization of Gbg and thus impeding G protein-mediated signal transmission (Muller et al. 1996; Thibault et al. 1997; Bauer and Lohse 1998). A distal regulatory point is GTPase activity. Regulators of G protein signaling (RGS) (Berman et al. 1996) play a crucial role in modulating signaling through G protein pathways by attaching to GTP-bound Ga proteins and shortening the duration of G protein signaling by acting as GTPase activating proteins.

4.3.1

GPCR Kinases (GRKs)

Besides their ability to activate G proteins, GPCRs in their active state serve also as the substrate for protein phosphorylation by a family of protein kinases called GRKs. GRKs are cytosolic serine-threonine kinases, capable of specifically phosphorylating the agonist-occupied form of GPCRs (Benovic et al. 1986), meaning that they can discriminate between the inactive and the active state of the receptor, in part because they are catalytically activated by stimulated receptors (Gainetdinov

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et al. 2004). Receptor phosphorylation by GRKs has been ultimately identified as the initial and critical step in the uncoupling of receptors from this G protein, leading to the attenuation or desensitization of GPCR signaling (Lefkowitz et al. 1990). Seven mammalian genes encoding GRKs have been cloned to date (Palczewski 1997; Pitcher et al. 1998; Wise et al. 2004). GRK1 and GRK7 are specific to the visual system, GRK4 is selectively present in sperm cells, while GRKs 2, 3, 5 and 6 are ubiquitously distributed. Based on sequence and functional similarities, the GRK family has been divided into three subfamilies: (a) the rhodopsin kinase subfamily (GRKs 1 and 7); (b) the b-adrenergic receptor kinase subfamily (GRK2/3); and (c) the GRK4 subfamily (GRKs 4, 5, and 6) (Metaye et al. 2005). All GRKs are translocated to the plasma membrane for their appropriate interaction with receptor domains. It is known that free G protein bg-subunits bind to the 125 amino acid C-terminal domain of GRK2/3 and facilitate their translocation process to the plasma membrane (Koch et al. 1993). It was shown that phosphorylation of the C-terminal domain of GRK2 by mitogen-activated protein kinase (MAPK) affects its ability to phosphorylate GPCRs (Pitcher et al. 1999). In contrast, GRK2 activity and plasma membrane translocation are potentiated in response to serine phosphorylation by protein kinase C (PKC) (Chuang et al. 1995; Winstel et al. 1996) or tyrosine phosphorylation by c-Src (Sarnago et al. 1999). Therefore, it seems that GRK2 activity is regulated by a complex series of protein phosphorylation events. GRKs phosphorylate GPCRs at serine and threonine residues located within either the third intracellular loop or the carboxyl-terminal domain (Ferguson 2001). Mutation of all of the serine and threonine residues within the carboxylterminal tail of the b2-adrenergic receptor (b2-AR) abolished GRK2-mediated phosphorylation of these receptors and thus inhibited the desensitization process (Bouvier et al. 1988). It should be mentioned that different studies showed that the phosphorylation patterns of the serine/threonine residues at the carboxyl terminal of GPCRs by GRKs dictates the stability of the interaction between receptors and arrestins – the family of proteins that facilitates the next steps of the desensitization and internalization processes (Oakley et al. 1999).

4.3.2

Arrestins

The arrestins constitute a family of proteins that are capable of interacting specifically with GPCRs following their activation by agonists and subsequent phosphorylation by GRKs (Stephen and Lefkowitz 2002). Arrestins recognize both GRK phosphorylation sites on the receptor and the active conformation of the receptor following agonist binding, that together drive robust arrestin association with the receptors (Luttrell and Lefkowitz 2002). Arrestin binding leads to uncoupling of the receptor from its cognate G protein in such a way that, despite the continued activation of receptor by agonist, it cannot exchange the GTP group on the G protein

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a-subunit for GDP (Gainetdinov et al. 2004), eventually causing desensitization of GPCR signaling via downstream second-messenger molecules (Lohse et al. 1990; Stephen and Lefkowitz 2002; Gainetdinov et al. 2004; Lefkowitz 2004). Although most of the research regarding the desensitization process has been carried out using the b2-AR as a model, it is now clear that this process regulates the function of many GPCRs, including a- and b-adrenergic, muscarinic cholinergic, serotonergic and dopaminergic receptors (Kristen and Lefkowitz 2001; Luttrell and Lefkowitz 2002). In addition to their role as regulators of GPCR desensitization, b-arrestins also interact with proteins of the endocytic machinery such as clathrin and the adaptor protein AP2, and thus promote internalization of receptors via clathrin-coated vesicles (Goodman et al. 1996; Laporte et al. 1999). b-Arrestins are also involved in both receptor down-regulation (Gagnon et al. 1998) and resensitization (Zhang et al. 1997; Oakley et al. 1999). To date, four members of the arrestin gene family have been cloned (Freedman and Lefkowitz 1996). Arrestin1 and arrestin4, also known as visual arrestin and cone arrestin respectively, are expressed almost exclusively in the retina (Murakami et al. 1993) where they regulate photoreceptors function: rhodopsin and color opsins. By contrast, b-arrestin1 and b-arrestin2, also known as arrestin2 and arrestin3 respectively, are ubiquitously expressed proteins, regulating GPCRs. High b-arrestin1 protein and mRNA levels were found in peripheral blood leukocytes. The abundant expression of b-arrestin1 in peripheral blood leukocytes supports the suggestion of a major role for the b-GRK/b-arrestin system in regulating receptor mediated immune functions (Parruti et al. 1993). In the last few years it was found that b-arrestins also play a pivotal role in intracellular signal transduction. They serve as adaptors to a wide variety of signal proteins such as c-Src, MAPKs, Mdm2, ARNO, NSF and more (Luttrell and Lefkowitz 2002; Gurevich and Gurevich 2003; Shenoy and Lefkowitz 2003). b-Arrestins in their inactive form are phosphorylated cytosolic proteins. b-Arrestin1 undergoes phosphorylation in serine-412 in the carboxyl-terminal (Lin et al. 1997), by extracellular signal regulated kinase (ERK1/2) (Lin et al. 1999). Following agonist stimulation, b-arrestin1 is translocated to the plasma membrane where it undergoes dephosphorylation, a process that is not required for receptor binding and desensitization but is obligatory for receptor internalization. Subsequently, b-arrestin1 is rephosphorylated (Lin et al. 1997) and returns to the cytosol as a phosphoprotein. Similarly, b-arrestin2 also has phosphorylation sites at serine-361 and threonine-383 (Lin et al. 2002). It was found that casein kinase II is responsible for threonine-383 phosphorylation (Lin et al. 2002). Like b-arrestin1, b-arrestin2 is also dephosphorylated at the plasma membrane: again, a process required for receptor internalization but not for its binding or desensitization (Lin et al. 2002). The exact time at which b-arrestins are dephosphorylated on the plasma membrane (before or after receptor binding) and the time they fall off the receptor and the cellular locus of their rephosphorylation are still not known. Figure 4.1 summarizes the pivotal role of b-arrestins as regulators of multiple intracellular signaling pathways.

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R P

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Fig. 4.1 The role of b-arrestin in the regulation of diverse intracellular signaling pathways. In addition to its classical role in GPCR desensitization and internalization, b-arrestin mediates diverse alternative new pathways of intracellular signaling, e.g., ERK, JNK, Akt, Mdm2 ubiquitination, and others

4.4

GRKs and Mood Disorders

Using a convergent functional genomic approach, a series of candidate genes involved in the pathogenesis of mood disorders was identified including G proteincoupled receptor kinase 3 (GRK3), which was also found to be decreased in lymphoblastoid cell lines from a subset of bipolar patients (Niculescu et al. 2000). As already mentioned, a single nucleotide polymorphism (SNP) in the promoter region of GRK3 was found to be associated with bipolar disorder (Barrett et al. 2003). The findings concerning the GRK3 gene are in accordance with evidence from a genome-wide linkage survey suggesting that the chromosome 22q12 region contains a susceptibility locus for bipolar disorder. Membrane-associated GRK2/3 was found to be increased in specimens of post-mortem prefrontal cortices collected from depressed patients (Garcia-Sevilla et al. 1999; Grange-Midroit et al. 2003). Comparison between drug-free and antidepressant-treated depressed subjects showed that GRK2 was reduced in both membrane and cytosolic preparations after antidepressant drug treatment (Grange-Midroit et al. 2003). Acute treatment (1–6 h) with the tricyclic antidepressant desipramine, but not the SSRI fluoxetine, increased membrane-associated GRK2/3 in rat brain. This effect vanished with a prolonged desipramine exposure (24 h–14 days) (Miralles et al. 2002). Major depression was found to be associated with reduced platelet GRK2, while treatment

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with mirtazapine reversed this abnormality (Garcia-Sevilla et al. 2004). Recently, GRK3 was also implicated in the mechanism of action of lithium and carbamazepine in bipolar disorder. Chronically administered lithium and carbamazepine, but not valproic acid, increased the translocation of GRK3 from cytosol to membrane in rat frontal cortex (Ertley et al. 2007).

4.5

Arrestins and Mood Disorders

A substantial body of evidence has accumulated indicating that b-arrestins play a major role in the pathophysiology of mood disorders as well as in the mechanism of action of antidepressants. b-Arrestin1 levels were significantly elevated by serotonin selective (SSRI), norepinephrine selective (NRI) and non-selective reuptake inhibitor antidepressants in rat cortex and hippocampus. This process became significant within 10 days and took 2–3 weeks to reach maximal increase (Avissar et al. 2004). b-Arrestin1 protein and mRNA levels in mononuclear leukocytes of untreated patients with major depression were significantly lower than those of healthy subjects. The low b-arrestin1 protein and mRNA levels were alleviated by antidepressant treatment. The reduction in b-arrestin1 protein and mRNA levels was significantly correlated with the severity of depressive symptomatology (Avissar et al. 2004; Matuzany-Ruban et al. 2005). Normalization of b-arrestin1 measures preceded, and thus predicted, clinical improvement (Matuzany-Ruban et al. 2005). These findings support the implication of b-arrestin1 in the pathophysiology of major depression and in the mechanism underlying antidepressant-induced receptor down-regulation and therapeutic effects. b-Arrestin1 measurements in patients with depression may potentially serve for biochemical diagnostic purposes and for monitoring and predicting response to antidepressants. Lately, lithium action on behavior was suggested to be mediated through a b-arrestin2 signaling complex (Beaulieu et al. 2008). Lithium was found to regulate Akt/GSK3 signaling and related behaviors in mice by disrupting a signaling complex composed of Akt, b-arrestin2, and protein phosphatase 2A. In the absence of b-arrestin2 (knockout mice), lithium failed to affect Akt/GSK3 signaling and induce behavioral changes associated with GSK3 inhibition as it does in normal animals (Beaulieu et al. 2008). Those results further supported a role for b-arrestin2 and GSK in the molecular mechanism of action of lithium.

4.5.1

b-Arrestins’ Alternative Signaling Events and Antidepressants

b-Arrestins can induce a switch in receptor signaling from classical second messengergenerating G protein-mediated pathways by conferring tyrosine kinase activity upon the receptor, leading to activation of the mitogen-associated protein (MAP) kinase cascade (Luttrell et al. 1999). b-Arrestins function as GPCR-regulated scaffolds for

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mitogen-activated protein kinase modules such as apoptosis signal-regulating kinase (ASK), mitogen-activated protein kinase kinase (MKK), C-Jun N-terminal kinases (JNK) and RAF-MEK (MAPK/ERK kinase)-extracellular signal-regulated protein kinases (ERKs) (McDonald et al. 2000; Luttrell et al. 2001). It was found that not only does b-arrestin2 form complexes with individual members of a particular MAPK cassette, but it also retains the activated MAPK in the cytoplasm, thereby directing phosphorylation of specific cytoplasmic substrates (DeFea et al. 2000a; DeFea et al. 2000b; Luttrell et al. 2001). Consequently, it inhibits phosphorylation of nuclear transcription factors, thus inhibiting ERK-dependent transcription (Tohgo et al. 2002; Tohgo et al. 2003). This ability of b-arrestin2 to retain activated scaffolded MAPKs in the cytoplasm is apparently related to the presence of a leucine-rich nuclear export signal in the C-terminus of b-arrestin2, but not of b-arrestin1 (Scott et al. 2002; Wang et al. 2003). The findings described here on the involvement of b-arrestin in the pathophysiology and treatment of depression may have extended implications concerning a possible switch in post-receptor signaling related to tyrosine kinases, Src, and MAPK that may characterize major depressive disorder or be induced by antidepressant treatment. Recent findings on reduced activation and expression of ERK1/2 MAPK in post-mortem brains of depressed suicide subjects (Dwivedi et al. 2001) and on MAPK activation by fluoxetine in cultured rat astrocytes (Mercier et al. 2004) support this suggestion. Studies have indicated that MAPKs are expressed abundantly in the central nervous system and that ERK is involved in long-lasting neuronal plasticity, including long-term potentiation and memory consolidation (Kyosseva 2004). While the role of ERK in neuronal plasticity and behavioral adaptation is beginning to emerge, the role of MAPK signal transduction cascades in major psychiatric disorders is not well understood. Evidence from human post-mortem studies (Kyosseva et al. 1999), as well as from the phencyclidine model of schizophrenia (Kyosseva et al. 2001), indicate that diverse MAPK cascades may be involved in the pathogenesis of schizophrenia. Einat et al. (2003) showed that lithium and valproic acid stimulate the ERK pathway in the rat hippocampus and frontal cortex. Also, inhibition of the ERK pathway by a blood-brain barrier-penetrating MAPK kinase inhibitor caused behavioral changes that were prevented by chronic lithium pretreatment (Einat et al. 2003), thus suggesting that the ERK pathway may mediate the anti-manic effect of mood stabilizers. As was mentioned earlier, MAPK was found to be involved in major depression as well. Dwivedi et al. (2001) found reduced activation and expression of ERK1/2 in post-mortem brains of depressed suicide subjects which was correlated with an increase in the expression of MAPK phosphatase 2 (MKP2). The authors concluded that ERK1/2 is less activated in the post-mortem brains of depressed suicide subjects and this may be because of reduced expression of ERK1/2 and increased expression of MKP2. Given the role of MAPKs in various physiological functions and gene expression, alterations in ERK1/2 activation and expression may contribute significantly to the pathophysiology of depressive disorders. Since their initial characterization, arrestins have been thought of as cytoplasmic proteins that could be recruited to plasma membrane and endocytic compartments following receptor activation. Lately, Kang et al. (2005) have revealed a novel, unexpected function of b-arrestin1 as a cytoplasm-nucleus messenger in GPRC

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signaling, showing that stimulation of a member of the GPCR family, d-opioid receptor, induced nuclear translocation of b-arrestin1. b-Arrestin1 was selectively enriched at specific promoters such as p27 and c-fos, facilitating the recruitment of histone acetyltransferase p300, and resulting in enhanced local histone H4 acetylation and transcription of these genes. The authors suggest that b-arrestin1 acts as a nuclear scaffold that recruits p300 to the transcription factor Cyclic adenosine monophosphate Response Element Binding protein (CREB). This leads to increased acetylation of histone H4 and the reorganization of chromatin, thereby increasing gene expression. The findings described above were specific to b-arrestin1 rather than b-arrestin2. As already mentioned, both b-arrestin1 and 2 are able to shuttle between cytoplasm and nucleus. But in contrast to b-arrestin1, b-arrestin2 possesses a strong nuclear export signal in its C terminus, which hinders its retention in the nucleus (Scott et al. 2002; Wang et al. 2003). Moreover, agonist stimulation failed to induce nuclear accumulation of a b-arrestin1 mutant with b-arrestin2 nuclear export signal on the C terminus. It can thus be concluded that b-arrestin1 may play a more important role in GCPR-mediated nuclear signaling. b-Arrestindependent GPRC signaling lasts longer than conventional G protein-dependent signaling (Ahn et al. 2004; Beaulieu et al. 2005). Thus, the b-arrestin-dependent mechanism for transcriptional control as proposed by Kang et al. (2005) may be used under certain physiological situations when sustained signaling is needed (Beaulieu and Caron 2005). Regulation of c-fos immunoreactivity and the expression of c-fos mRNA by various types of antidepressants in specific rat brain areas is a well described phenomenon (Dahmen et al. 1997; Morinobu et al. 1997; Morelli et al. 1999; Miyata et al. 2005; Slattery et al. 2005; Kuipers et al. 2006). In addition, the antidepressant rolipram, a type IV phosphodiesterase inhibitor, was found to induce expression of p27 in malignant glioma cells (Chen et al. 2002). It should be noted that PDE4-selective inhibition by rolipram was recently found to facilitate the isoprenaline-induced membrane translocation of GRK2, phosphorylation of b2-adrenergic receptors (b2ARs) by GRK2, membrane translocation of b-arrestin and internalization of b2ARs. In the absence of isoproterenol, rolipram-induced inhibition of PDE4 activity acted to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the b2AR (Li et al. 2006). The above findings concerning the regulation of c-fos by antidepressants and of p27 by rolipram could possibly be mechanistically explained by the data on antidepressant-induced elevation of b-arrestin1 protein and mRNA levels (Avissar et al. 2004; Matuzany-Ruban et al. 2005) together with the data on b-arrestin1 selective enrichment at specific promoters such as p27 and c-fos (Kang et al. 2005).

4.6

Phosducin-like Protein

The discovery of phosducin (Phd) in photoreceptor cells of the retina and the ubiquitous distribution of phosducin-like proteins (PhdLP) uncovered the existence of a family of proteins characterized as cytosolic regulators of G protein function

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(Schulz 2001). The most important characteristic of these proteins appears to be their high affinity sequestration of bg-subunits of G proteins (Muller et al. 1996; Thibault et al. 1997), leading to neutralization of Gbg and thus impeding G proteinmediated signal transmission, since Ga cannot reassemble with Gbg to provide a functional G protein trimer (Gabg) (Muller et al. 1996; Bauer and Lohse 1998). A further consequence expected from the rapid binding of PhdLP to Gbg dimers is the inhibition of Gbg-mediated effects, such as activation of GRK2 (Schulz et al. 1996) which will prevent GRK2/3-phosphorylation of GPCR (Pitcher et al. 1992; Schulz et al. 1996; Garzon et al. 2002). Thus, it has been proposed that, following agonistinduced activation of GPCRs, the binding of PhdLP to Gbg dimers offers a temporary protection to agonist-bound receptors not coupled to G-proteins from being acted upon by GRKs. This presumably prevents the uncoupling and internalization (Garzon et al. 2002). In a very recent study (Matuzany-Ruban et al. 2006), PhdLP levels were measured in brain cortices of rats chronically treated with one of five classes of antidepressants: imipramine, venlafaxine, maprotiline, citalopram and moclobemide. None of the antidepressant treatments had any significant effect on PhdLP levels. PhdLP levels were evaluated in mononuclear leukocytes from a group of patients diagnosed with major depressive episode, before the initiation of antidepressant treatment and after four weeks of antidepressant medication. No protein changes were found in leukocytes of either untreated patients with major depressive disorder or after four weeks of the treatment in comparison with healthy volunteers. The fact that phosducin, an important regulatory molecule of signal transduction at the GPCR-G protein level, was found to be involved neither in the mechanism of action of antidepressant treatments nor in the pathophysiology of major depressive disorder, renders further support for the specificity of the involvement of other regulators of GPCR-G protein coupling, i.e., b-arrestins and GRKs, in the pathophysiology of mood disorders and in the mechanism of action of antidepressants.

4.7

Regulators of G Protein Signaling (RGS)

RGS (Berman et al. 1996) play a crucial role in modulating signaling through G protein pathways by attaching to GTP-bound Ga proteins and shortening the duration of G protein signaling by acting as GTPase activating proteins. At least 20 RGS family members have been identified. Presently there is no published information concerning possible involvement of RGS family members either in the pathophysiology of mood disorder or in the mechanism of action of antidepressants. In contrast to this lack of findings in mood disorders, the transcript-encoding RGS4 was the most consistently and significantly decreased in the post-mortem prefrontal cortex of patients with schizophrenia compared with cortices obtained from subjects with no history of mental disorders and subjects with major depressive disorder (Mirnics et al. 2001). RGS4 maps to locus 1q21-22, a chromosome region strongly

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linked to schizophrenia (Brzustowicz et al. 2000), suggesting it as a candidate for a schizophrenia susceptibility gene on this locus. Findings from association and linkage analyses of RGS4 polymorphisms in schizophrenia are in agreement with this suggestion (Chowdari et al. 2002). Linkage and association studies in five independently ascertained samples have suggested that polymorphisms of RGS4 may confer risk for schizophrenia (Chen et al. 2004; Morris et al. 2004; Williams et al. 2004).

4.8

Biological Diagnostic Tests for Mood Disorders – Comparative Evaluation

A critical measure of the extent of advancement of our understanding of pathophysiological mechanisms underlying mental disorders is the usefulness and applicability of biochemical theories of pathogenesis to the creation and establishment of (a) new modalities of pharmacological treatments and/or advanced pharmacological agents applied to the treatment of mental disorders; (b) biological methods for monitoring treatments in mental disorders and for determining the state of a disorder; and (c) biological methods for diagnosis of mental disorders. Indeed, based on pathophysiological hypotheses, new treatment modalities (i.e., anti-obsessive-compulsive medications) and new drugs (e.g., serotonin-selective reuptake inhibitors as new antidepressants or dopamine-serotonin antagonists as new antipsychotics) have been developed. The most notable improvements concern the development of medications with fewer side effects. However, there is a significant gap between advances in medication for mental disorders and the present static situation in diagnosis and monitoring of treatment in these disorders. There are presently no reliable, sensitive and specific objective pathophysiologically based biological diagnostic markers that can serve as “gold standards” (Avissar and Schreiber 2002). The dexamethasone suppression test (DST) has been extensively used to study the hypothalamic–pituitary–adrenal axis in patients with mood disorders. In the late 1960s, abnormal DST was detected in patients with depression. This led to widespread interest in its use in psychiatric research and clinical practice in the 1980s (Carroll 1985). More recent studies have shown that the sensitivity and specificity of various versions of this feedback inhibition test are in fact much lower. The present consensus of opinion is that DST has little value as a diagnostic test for depression, but might be helpful in special clinical situations (i.e., differentiating between psychotic depression and schizophrenia). A differential pattern of GPCR function and of their immunoreactive levels was detected in mononuclear leukocytes of patients and was therefore suggested as a differential diagnostic test with high sensitivity and specificity values for the major mental disorders of mania, depression, schizophrenia and panic disorder (Avissar and Schreiber 2002). Normalization of altered G protein measures in mood disordered patients occurred under lithium, antidepressants and electroconvulsive

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treatment (Avissar et al. 1997), as well as light treatments (Avissar et al. 1999), and in patients with schizophrenia under antipsychotic treatment (Avissar and Schreiber 2002). As state-dependent markers, G protein measures can potentially be used as aid in the biochemical diagnosis of mental disorders and as an aid in the biochemical monitoring of the response to a specific treatment. b-Arrestin1 measurements in mononuclear leukocytes (MNL) of patients with depression might be a better diagnostic assay for detecting depression as the sensitivity and specificity of the b-arrestin1 test were found to be 92.5% and 93.9%, respectively (Muller et al. 1996). These values are far greater than the values previously described for the immunoreactive G protein assay. The dynamics of normalization by antidepressant treatment of the biochemical measures of b-arrestin1 protein and mRNA levels in MNL of patients with depression did not follow, and thus reflect, the clinical improvement of the patients, but rather preceded clinical improvement. The biochemical normalization, which was significant after one week, preceded clinical improvement by 1–2 weeks. It is very difficult to monitor the extent of specific clinical improvement in the early period of the first and second week after initiation of antidepressant treatment. Since clinical response to antidepressant treatments is due both to the specific biochemical antidepressant effects of the medication agent, as well as to placebo effects, and since the placebo effect is usually more pronounced during the early period of treatment initiation, it is very difficult to assess in these early days the specific antidepressant effects of antidepressant treatments. b-Arrestin1 measurements in peripheral blood cells of patients with mood disorder, as a statedependent characteristic, may afford biochemical monitoring of antidepressant effects and prediction of clinical response to antidepressant by 1–2 weeks in advance. The expected significance of the described findings and future studies concerning the involvement of regulatory elements of GPCR-G protein coupling lies in three aspects: 1. Possible identification of new beyond-receptor biochemical sites underlying the mechanism of action of antidepressant pharmacological treatments. Such findings may enable (a) an optimization in antidepressant therapeutic clinical use, and help in establishing biochemical correlates and predictors for drug reactivity and non-reactivity as well as for titrating therapeutic effects and endpoints of treatment; and (b) ability to design new antidepressants to the new target for their mechanism of action. 2. Better understanding of the involvement of beyond-receptor signal transduction elements and regulators in the pathogenesis of mood disorders and establishment of a new integrated pathophysiological model for major depression. 3. Possible identification of biochemical markers with high sensitivity and high selectivity as an aid in the diagnosis of major depression. Such identification could also help to distinguish several biologically different subtypes of mood disorders that cannot be distinguished by the present diagnostic criteria.

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Limitations of the “Pharmacological Bridge” Approach

The pharmacocentric approach governing studies to unravel the etiology of mental disorders is based on using findings concerning underlying mechanisms of drugs with proven efficacy as a guide. Although of indisputably proven success, such an approach has also considerable limitations and disadvantages: (a) Although medications for the treatment of mental disorders have been used clinically for more than 50 years, no consensus has been reached concerning their precise molecular mechanism of action. There is a several-week latency period before onset of the clinical effect of antipsychotics, antidepressants, and mood stabilizers. This latency cannot be explained by the “classical” sites suggested for their biochemical mechanism of action, and calls upon hypotheses of adaptation mechanisms at various cellular levels e.g., receptor desensitization, post-receptor effects at the level of signal transduction, regulation of signal transduction, gene transcription, etc. The detailed mechanisms underlying drug-induced adaptive neural changes are as yet unknown. (b) A considerable percentage of patients do not respond to a specific medication. Individual differences may exist in the responsiveness to certain medications belonging to the same pharmacological group. Targeting etiological hypotheses of a drug’s mechanism of action ignores unresponsive or partially responsive patients and individual variation with regards to drug responsiveness. To solve such problems, the whole research field of pharmacogenetics has been developed. (c) More established knowledge of a drug’s mechanism of action may still be unrelated directly to pathogenesis. Drugs may affect a disordered biochemical system at an indirect site which may be only obliquely related to a pathogenetic site. Concentrating pathophysiological research on a probable or definite underlying mechanism of drug action may prove unfruitful or even misleading with regard to unraveling etiological mechanisms. (d) Hypothesis-driven research based upon classical targets for drugs’ mechanisms of action is not expected to lead to new drug modalities and/or new etiological understandings. Hypothesis-driven research should be complemented by a new strategy that relies on a “bottom-up” search for new drug targets and new etiological mechanisms through the new avenues opened up by biotechnology: genomics, proteomics, compound libraries, etc. It is to be hoped that an admixture of new techniques and strategies of genomics and proteomics, which enable multi-marker, large-scale automated measures, together with the “old” biochemical approaches, will enable the achievement of an objective biological differential diagnostic system for major mental disorders that will also enable objective biological treatment monitoring. The identification of biological diagnostic markers could also help to distinguish several biologically different subtypes of mental disorders undistinguishable by our current diagnostic criteria. Such an achievement is expected to be revolutionary for psychiatry, with a

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magnitude similar to the impact of the discovery of pharmacological modalities of treatment for mental disorders more than 50 years ago. Acknowledgment S. Avissar is supported in part by a 2005 NARSAD Independent Investigator Award and holds of the Eugene Hecht Chair in Clinical Pharmacology.

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Chapter 5

Dysregulation of G Protein-Coupled Receptor Signaling in Cancer JoAnn Trejo

5.1

Introduction

The progression of cancer involves specific genetic changes that alter cellular growth, promote angiogenesis, and stimulate tissue invasion and metastasis (Hanahan and Weinberg 2000). In the majority of cells, signaling by transmembrane receptors is critical for control of tumor growth, angiogenesis, invasion, and metastasis. Moreover, growth factor receptor signaling is often dysregulated in malignant cancer. Indeed, overexpression of the ErbB family of receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and ErbB2 is correlated with poor prognosis of several types of malignant cancer. More recent studies indicate that signaling by G protein-coupled receptors (GPCRs) is also crucial for tumor progression. However, unlike receptor tyrosine kinases, the precise mechanisms by which GPCRs contribute to cancer progression have yet to be fully elucidated. GPCRs are members of the seven transmembrane domain family of receptors, the largest family of signaling receptors in the mammalian genome, which comprises more than 800 members. Most, if not all, seven transmembrane receptors couple to heterotrimeric G proteins, and hence are termed GPCRs. GPCRs elicit signaling responses to diverse extracellular stimuli including chemokines, proteases, neuropeptides, bioactive lipids and prostaglandins as well as other stimuli and control a vast number of biological responses. Moreover, GPCRs are the target of nearly half the drugs currently in use, making members of this receptor family ideal candidates for the development of new therapeutics that can be used in the treatment of a wide range of human diseases. A role for dysregulated GPCR signaling in tumor progression is supported by substantial literature (Dorsam and Gutkind 2007). However, the use of specific GPCR antagonists has been limited as tumor

J. Trejo Department of Pharmacology, University of California, La Jolla, CA, USA e-mail: [email protected] A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_5, © Springer-Verlag Berlin Heidelberg 2010

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growth is driven by signaling pathways that can be activated by multiple tumor cell-associated GPCRs. In fact, the tumor microenvironment generates many different types of GPCR ligands, which can act in an autocrine or paracrine fashion to stimulate GPCR signaling. Thus, understanding the specific mechanisms that lead to uncontrolled GPCR signaling will provide unique opportunities to identify new drug targets and to develop novel strategies that can be used in the prevention and treatment of cancer progression. The mechanism that controls GPCR signaling is different in distinct cell types (Marchese et al. 2008). Thus, defects that cause aberrations in the magnitude, duration and spatial aspects of GPCR signaling are likely to be distinct in various cell and cancer types. This chapter discusses the current understanding of the processes responsible for dysregulation of GPCR signaling in tumor progression.

5.2 5.2.1

G Protein Signaling Regulation of G Protein Signaling

The activation of GPCRs by ligand binding promotes conformational changes within the receptor that facilitates interaction with heterotrimeric G proteins (Fig. 5.1), which are localized predominantly at the inner leaflet of the plasma membrane. There are four main classes of heterotrimeric G proteins (Gs, Gi/o, Gq and G12) that share sequence similarity and regulate specific signaling effectors, and all bind to the Gbg-subunit dimer in the inactive state. Activated GPCRs function as guanine–nucleotide exchange factors (GEFs), which promote GDP release and accelerate GTP binding and cause dissociation of Ga- from Gbg-subunits. Heterotrimeric G proteins are activated upon the binding of GTP to the a-subunit and deactivation occurs when bound GTP is hydrolyzed to GDP by intrinsic GTPase activity (Oldham and Hamm 2007). The dissociation of GDP from the Ga-subunit is the rate-limiting step in G protein activation. The Ga-GTP and free Gbg dimer couple to multiple diverse effector molecules to promote downstream signaling. Regulators of G protein signaling (RGS) proteins enhance the rate of GTP hydrolysis and thereby function as GTPase-accelerating proteins and diminish signaling. The inactive GDP-bound Ga-subunit then reassociates with Gbg, which inhibits GDP dissociation and promotes signal termination. Several studies indicate that aberrant activation of heterotrimeric G protein signaling leads to oncogenic transformation of distinct cell types.

5.2.2

Activating Gs and Gi Protein Mutations in Cancer

A common cause of aberrant G protein signaling involves mutation of the G protein itself. Indeed, specific mutations in G proteins that result in sustained GTP binding

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Fig. 5.1 GPCRs signal to multiple and diverse pathways to promote tumor progression. Many different GPCRs are overexpressed in cancer and respond to diverse activating ligands that are generated in the tumor microenvironment. GPCRs couple to multiple subtypes of heterotrimeric G proteins including G12, Gq, Gs, Gi/o and Gbg-subunits to activate a variety of signaling cascades and cellular responses that control tumor progression. G12 couples to RhoGEF-mediated Rho signaling as well as other effectors to promote tumor progression. Gq and Gi/o induce intracellular Ca2+ mobilization, protein kinase C (PKC) activation, ERK1,2 signaling and other signaling responses. Gs and Gi/o mediate activation or inhibition of adenylyl cyclase activity and thereby differentially modulate protein kinase A (PKA) activation that is linked to tumor cell proliferation in certain types of endocrine tissues. Several recent studies indicate that GPCRs transactivate ErbB family members including EGFR and ErbB2. GPCR signaling stimulates the activity of cell surface metalloproteases such as ADAM, which release ErbB receptor ligands that consequently activate EGFR

causes persistent signaling to downstream effectors even in the absence of a stimulatory hormone. The first oncogenic G protein mutations were identified in the Gas-subunit isolated from human pituitary tumors and dubbed gsp (G stimulatory protein) (Farfel et al. 1999; Radhika and Dhanasekaran 2001). Similar mutations have been found in thyroid adenomas and carcinomas. Many gsp mutations decrease intrinsic GTPase activity and, thus, constitutively activate adenylyl cyclase, resulting in enhanced cyclic AMP (cAMP) production, protein kinase A activation, and induction of cell proliferation. This occurs specifically in certain types of cells in endocrine tissues in which cellular growth is positively regulated by cAMP signaling. In addition to Gas, mutations in Gai2 that cause constitutive activation have been identified in a few adrenal cortex and ovarian adenomas as well as pituitary tumors.

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These activated mutations are denoted gip2 (Gi protein-2). The expression of gip2 in fibroblasts causes oncogenic transformation and induces tumor formation in nude mice. Constitutively active Gi2 promotes oncogenic transformation and cell proliferation through the activation of the mitogen-activated protein (MAP) kinase extracellular-signal regulated kinase (ERK1,2) signaling by either directly or indirectly activating Ras, a small GTPase commonly mutated in malignant cancers of the pancreas, colon, lung, and thyroid and myeloid leukemia (Goldsmith and Dhanasekaran 2007; Radhika and Dhanasekaran 2001). Analogous mutations in Gai1, but not Gai3, also induce fibroblast transformation. The other Gai family members, Gaz and Gao, couple to mitogenic signaling pathways but their contribution to oncogenic transformation has not been firmly established. Similarly, the transforming activity of Gaq has been investigated using an engineered constitutively activated mutant Gaq (Q209L), which persistently activates phospholipase C. Phospholipase C catalyzes the hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) leading to the generation of inositol phosphates and diacylglycerol, important second messengers that mediate persistent protein kinase C activation, Ca2+ mobilization and consequent ERK1,2, Raf and Ras activation.

5.2.3

Wildtype G12 Expression Promotes Oncogenesis

In contrast to other heterotrimeric G proteins, overexpression of wildtype G12 proteins is sufficient to drive oncogenic transformation. The G12 family of heterotrimeric G proteins is comprised of two members Ga12 and Ga13, which are ubiquitously expressed in most cell types. Ga12 was first identified as a transforming oncogene in an expression-cloning screen using a cDNA library generated from a Ewing’s sarcoma-derived cell line (Chan et al. 1993). Strikingly, expression of wildtype Ga12 was shown to be sufficient to induce oncogenic transformation of fibroblasts and is the only family member of heterotrimeric G proteins that promotes transformation without harboring any activating mutations. However, engineered, constitutively active mutants of either Ga12 (Q229L) or Ga13 (Q226L) display considerably more potent transforming activity compared to their wildtype variants (Xu et al. 1993). The G12 protein family controls various biological processes that make important contributions to tumor progression including cell proliferation, migration, invasion and metastasis (Kelly et al. 2007). Moreover, Ga12 and Ga13 proteins differentially regulate these processes in distinct cell types. In recent work, Kelly and colleagues demonstrated that signaling though G12 proteins promotes breast cancer invasion and is up-regulated early in breast cancer progression (Kelly et al. 2006). The G12 proteins have also been implicated in progression of prostate and ovarian cancers and certain types of glioblastomas. The transforming ability of G12 proteins involves multiple signaling effectors including the activation of the small GTPase RhoA that occurs through G12 coupling to specific Rho GEFs (Jaffe and Hall 2002). Interestingly, RhoA is a key regulator of the

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actin cytoskeleton and has been implicated in tumor progression. However, G12 proteins have more potent transforming potential than RhoA indicating that other effectors contribute to the transformation process. G12 protein signaling has also been shown to induce down-regulation of E-cadherin expression (Kelly et al. 2007), a hallmark of cancer progression. Constitutively activated Ga12 protein binds to E-cadherin, which triggers the release of bound b-catenin that then redistributes to the nucleus to regulate gene transcription. However, the exact mechanism by which wildtype G12 protein signaling promotes tumor progression is not known. Moreover, the contribution of other regulators and/or effectors of G12 signaling, such as RGS proteins, RhoGEFs and b-catenin, to G12-promoted cancer progression has yet to be clearly defined.

5.3 5.3.1

GPCRs and Cancer Regulation of GPCR Signaling

In addition to aberrant G protein activation, defects in regulatory processes that control GPCR signaling are also involved in cancer progression. Dysregulation of GPCR signaling in cancer can result from GPCR activating mutations as well as overexpression, mainly to defective receptor trafficking. Three temporally distinct processes – desensitization, internalization and downregulation – directly regulate GPCR signaling. Most activated GPCRs are rapidly desensitized at the cell surface by phosphorylation and arrestin binding (Krupnick and Benovic 1998). GPCR phosphorylation occurs predominantly on serine and threonine residues within the cytoplasmic tail and the third intracellular loop, but rarely on tyrosine residues. Agonist-activated GPCRs are rapidly phosphorylated mainly by GPCR kinases (GRKs). However, other protein kinases can phosphorylate activated GPCRs, which may function to promote rather than terminate signaling in certain cases (Tobin et al. 2008). Most activated and phosphorylated GPCRs then bind the cytosolic protein arrestin, which prevents GPCR–G protein interaction, and thereby uncouples the receptor from signaling. Thus, one function of arrestins is to terminate G protein signaling. A second function of arrestins is to promote GPCR internalization. Arrestins are comprised of distinct N- and C-domains linked by a 12-residue polar core, which engages GPCR-associated phosphates (Moore et al. 2007). A conformational change in arrestin is induced upon binding to GPCRs, exposing the C-terminal domain, which interacts with clathrin and the b2-subunit of the clathrin adaptor protein complex-2 (AP-2) to facilitate GPCR internalization, and thereby remove activated receptor from the plasma membrane. Once internalized, GPCRs are then sorted within an early endosomal tubulovesicular compartment to either a recycling or a lysosomal degradative pathway

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(Marchese et al. 2008). Sorting of GPCRs to a recycling pathway can occur through a default pathway similar to bulk membrane flow or through a regulated process. Within endosomes, receptors dissociate from their ligands, becomes dephosphorylated, and then return to the cell surface in a state capable of responding to ligand. Recent studies indicate that the internalized GPCR–arrestin complex is also capable of signaling to downstream effectors independent of heterotrimeric G proteins (Lefkowitz and Shenoy 2005), as discussed in greater detail below. Thus, arrestins provide novel pathways for intracellular signaling. In contrast to receptor recycling, some GPCRs are sorted from endosomes to lysosomes and degraded, a process important for signal termination. One lysosomal sorting pathway for GPCRs involves the ubiquitin-dependent endosomalsorting complex required for transport (ESCRT) machinery, which is comprised of a complex network of proteins that function coordinately to sort ubiquitinated cargo to a degradative pathway (Piper and Katzmann 2007). In addition, several GPCRs appear to target to lysosomes for degradation independent of ubiquitination and some components of the ESCRT machinery, suggesting that additional lysosomal degradation pathways exist (Marchese et al. 2008). Thus, GPCR trafficking has critical functions in signal termination and propagation as well as receptor resensitization.

5.3.2

Constitutively Activated GPCRs in Cancer Progression

GPCRs isomerize between an active and inactive state, although most GPCRs reside in the inactive state in the absence of an agonist. However, in the presence of an agonist the active conformation of the receptor is stabilized, which then couples to and activates heterotrimeric G proteins. GPCR mutations that increase the rate of isomerization and stabilize the active state conformation independent of an activating ligand lead to constitutive activation of heterotrimeric G protein and effector signaling (Parnot et al. 2002). These types of mutations are commonly referred to as gain-of-function phenotypes. GPCR-activating mutations have been shown to occur in a few specific tissue and cell types since such mutations in more broadly expressed receptors are likely to be embryonic lethal. Indeed, activating mutations in the thyroid-stimulating hormone receptor (TSHR) have been identified in thyroid carcinoma and adenomas and mutations in the luteinizing hormone receptor (LHR) are associated with male precocious puberty (Parnot et al. 2002). These particular GPCR-activating mutations couple to Gs-stimulated increases in cAMP formation and Gbg signaling to phosphoinositide-3 (PI3) kinase and ERK1,2 activation, and promote cellular proliferation in discrete endocrine tissues. In some cases, the constitutively activated GPCRs are also continuously internalized, recycled, and appear to signal persistently at the cell surface, whereas other constitutively activated GPCRs are internalized and are then targeted to lysosomes for degradation. Thus, the fate of constitutively activated GPCRs is different for distinct receptors.

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Virally Encoded Constitutively Active Wildtype GPCRs

In addition to GPCR-activating mutations, several human viruses harbor genes that encode constitutively active wildtype GPCRs that have the capacity to induce oncogenesis. The Kaposi sarcoma-associated herpes virus (KSHV) encodes a GPCR homologous to the human chemokine receptors CXCR1 and CXCR2. The KSHV GPCR is oncogenic, angiogenic and promotes tumor growth via activation of multiple protein kinase signaling cascades (Sodhi et al. 2004). A second viral-associated GPCR is the Epstein–Barr virus (EBV) encoding a GPCR gene dubbed BILF1, and is associated with nasopharyngeal carcinoma and the lymphoproliferative diseases mononucleosis and Burkitt’s lymphoma (Paulsen et al. 2005). The human cytomegalovirus herpes virus encodes a GPCR termed US28, which is constitutively active and displays potent transforming activity when ectopically expressed in fibroblasts (Maussang et al. 2006), but how it contributes to particular human pathological conditions has yet to be fully elucidated.

5.3.4

Constitutively Active Wildtype GPCRs and Oncogenic Transformation

Several human GPCRs have been identified whose overexpression is sufficient to drive oncogenic transformation without any apparent activating mutations. The mas oncogene was discovered in an NIH 3T3 transformation screen using a cDNA library derived from a human epidermoid carcinoma (Young et al. 1986). mas expression is high in the central nervous system; however, endogenous activating ligands have not been identified. A cDNA library generated from the T28 murine T hybridoma cell line led to the discovery of a second transforming GPCR dubbed G2A (Whitehead et al. 2001). G2A is expressed predominantly in hematopoietic tissues and is upregulated by the oncoprotein Bcr-Abl and, as in the case of the mas oncogene, its endogenous activating ligands are not known. Protease-activated receptor-1 (PAR1), a GPCR activated by thrombin and other extracellular proteases, was also identified in a NIH 3T3 transformation screen using a retroviral cDNA expression library generated from 32D and B6StuA1 murine myeloid cell lines (Whitehead et al. 2001). Interestingly, however, the transforming activity of PAR1 appears to involve its overexpression and also activating proteases, since the introduction of a mutation in PAR1 that renders the receptor uncleavable and unactivatable failed to transform cells. Moreover, inhibitors of thrombin, the major effector protease for this receptor, also failed to block PAR1 transforming activity, suggesting that other proteases generated by tumor cells activate PAR1. Indeed, recent work indicates that the matrix metalloprotease-1 (MMP1), also known as interstitial collagenase, generated predominantly by stromal cells, signals through PAR1 to promote breast tumor growth and invasion (Boire et al. 2005), but precisely how MMP1 acts on PAR1 to promote tumorigenesis is not known. Thus, in

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many cases, GPCR overexpression coupled with the presence of activating ligands are critical for promotion of human tumor progression.

5.4

Dysregulated GPCR Trafficking

Defective GPCR trafficking is one mechanism responsible for aberrant GPCR overexpression and signaling, and contributes to malignant transformation. Substantial work on the ErbB family of receptors has established a clear link between defective trafficking and proliferative signaling in cancer progression (Moseson et al. 2008). Aberrant ErbB signaling and overexpression is due to gene amplification as well as defects in receptor endocytosis and lysosomal degradation. The next section discusses the specific defects associated with GPCR trafficking and signaling that promote cancer progression.

5.4.1

GPCR Endocytosis

GPCR internalization is crucial for the control of signal termination and promotes disassembly of signaling complexes by removing activated receptors from G proteins and signaling effectors at the plasma membrane. Thus, defects in GPCR endocytosis can lead to persistent signaling in certain types of invasive cancer. Clathrin-mediated endocytosis is responsible for internalization of most GPCRs in mammalian cells. Clathrin-coated pits form at plasma membrane sites enriched in phosphatidylinositol (4,5)-bisphosphate (PIP2). Clathrin, adaptor proteins, and dozens of regulatory proteins coordinate the assembly and invagination of clathrincoated pits through a highly regulated and dynamic process (Edeling et al. 2006). The GTPase dynamin is essential for release of clathrin-coated pits from the plasma membrane (Schmid 1997). Clathrin-coated pits are high capacity carriers that efficiently internalize many different types of cargo and the function of clathrin adaptors is to enrich select cargo within a forming coated pit. Defects in certain components of the endocytic machinery are likely to contribute to aberrant GPCR trafficking (Moseson et al. 2008).

5.4.1.1

Arrestins and Dysregulation of GPCR Signaling in Cancer

The ubiquitously expressed nonvisual arrestins, arrestin-2 and arrestin-3, (also known as b-arrestin 1 and b-arrestin 2) were the first clathrin adaptor proteins shown to function in GPCR endocytosis (Goodman et al. 1996). The mechanism by which arrestins control GPCR internalization is best characterized for the b2adrenergic receptor (b2AR). Arrestins recognize activated and phosphorylated GPCRs and bind to clathrin and AP-2 to facilitate endocytosis. The b2AR–arrestin

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complex is then internalized through clathrin-coated pits and arrestin rapidly dissociates from the receptor. In contrast to the b2AR, other GPCRs are phosphorylated on multiple serine and threonine residues within their cytoplasmic tail domain, which stabilizes arrestin binding. The angiotensin II type 1A receptor, vasopressin 2 and neurokinin-1 receptor have been shown to co-internalize together with arrestins (Marchese et al. 2008). In addition, arrestin-3 interacts with the E3 ubiquitin ligase murine minute double minute-2 (MDM2), which mediates arrestin-3 ubiquitination, a post-translational modification critical for the endocytic function of arrestin. Interestingly, MDM2 is often overexpressed with Ras and promotes fibroblast transformation and tumor formation (Freedman et al. 1999). Moreover, MDM2 is also a negative regulator of p53, a tumor suppressor, suggesting that arrestin could affect oncogenesis by modulating p53 activity. In addition to arrestins’ endocytic function, these multi-faceted proteins function as scaffolds and bind to multiple signaling effectors including Src, ERK1,2 and JNKs as well as many other effectors, and promote signaling from the plasma membrane and on endocytic vesicles (Lefkowitz and Shenoy 2005). Thus, arrestins provide new signaling pathways for GPCRs that can occur independent of heterotrimeric G protein signaling. In colorectal carcinoma cells, the G protein coupled prostaglandin E2 (PGE2) receptor induces EGFR transactivation via a Src-dependent mechanism and stimulates cellular proliferation and migration. A recent study showed that b-arrestin 1 interaction with Src is critical for PGE2-induced EGFR transactivation, colorectal carcinoma cell migration in vitro and tumor cell metastasis in vivo (Buchanan et al. 2006). Arrestins are also critical for protease-activated receptor-2 (PAR2)-stimulated ERK1,2 activation and migration of invasive breast cancer cells (Ge et al. 2004). However, the specific defect that leads to aberrant arrestin expression and function in various cancer types has not been thoroughly investigated.

5.4.2

Ubiquitination and GPCR Trafficking

Dysregulation of processes that control ubiquitination probably contributes to aberrant receptor signaling and trafficking in cancer. Post-translational modification with ubiquitin, a 76-amino acid polypeptide, is best known for targeting proteins for degradation by the proteosome, but it also serves as a signal for GPCR internalization and endocytic sorting (Marchese et al. 2008). The attachment of ubiquitin to substrate proteins is carried out by an ATP-dependent mechanism that requires the sequential activity of three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3) (Kerscher et al. 2006). Proteins can be modified on a single or multiple lysine residues by one ubiquitin moiety or by poly-ubiquitin chains. The fate of ubiquitinated proteins is determined, in part, by the length of the ubiquitin chain and on the configuration of ubiquitin– ubiquitin linkages. The attachment of a single ubiquitin (mono-ubiquitination) or attachment of ubiquitin to lysine-63 of an adjacent ubiquitin controls endocytic

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sorting, but not proteasomal degradation. Ubiquitin conjugation of proteins is a highly dynamic and reversible process. De-ubiquitinating enzymes (DUBs) are proteases that specifically cleave the isopeptide bond that links ubiquitin chains and ubiquitin protein conjugates. A role for ubiquitination in proper trafficking of the chemokine CXCR4 receptor and PAR1 has been recently demonstrated (Marchese et al. 2003; Wolfe et al. 2007). Moreover, defective trafficking is responsible, at least in part, for increased CXCR4 and PAR1 expression that occurs in multiple types of malignant cancers including breast cancer (Booden et al. 2004; Li et al. 2004).

5.4.2.1

Dysregulation of CXCR4 Trafficking in Malignant Cancer

The chemokine CXCR4 receptor promotes tumor cell proliferation, survival and migration and is overexpressed in several different types of cancer (Dorsam and Gutkind 2007). Interestingly, the selective ligand for CXCR4, stromal cell-derived factor-1 (SDF1) is expressed predominantly in tissues that harbor metastatic foci including the lymph nodes, lungs, bone marrow and liver. Thus, increased expression of CXCR4 is linked to metastasis and poor prognosis. The mechanism by which ubiquitination regulates lysosomal sorting and degradation of CXCR4 has been extensively studied (Marchese and Benovic 2001; Marchese et al. 2003). Activated CXCR4 is rapidly ubiquitinated, sorted to lysosomes and degraded within hours. Sorting of activated CXCR4 to lysosomes is mediated by ubiquitination of lysine residues within the cytoplasmic tail. The ubiquitin ligase atrophin interacting protein 4 (AIP4), also known as Itch, mediates CXCR4 ubiquitination upon agonist stimulation. Although CXCR4 ubiquitination occurs at the plasma membrane, it functions as an endosomal sorting signal to facilitate CXCR4 sorting from endosomes to multivesicular bodies (MVBs) and lysosomes for degradation. Interestingly, AIP4 not only ubiquitinates CXCR4 but also mediates ubiquitination of hepatocyte-growth factor-regulated tyrosine kinase substrate (HRS), an endosomal adaptor protein. Moreover, the endocytic function of AIP4 is negatively regulated by phosphorylation, which is mediated by the cytokine-independent survival kinase (CISK), a serine and threonine kinase activated downstream of PI3 kinase (Slagsvold et al. 2006). A recent study showed that ErbB2 (HER2), an oncogenic tyrosine kinase receptor, which is overexpressed in ~ 30% of breast cancers, impairs CXCR4 degradation and thereby increases CXCR4 expression at the cell surface (Li et al. 2004). ErbB2 enhances CXCR4 expression by increasing protein synthesis and by impairing CXCR4 ubiquitination and lysosomal degradation through a mechanism that may involve PI3 kinase activity. CISK, which is activated by PI3 kinase signaling, blocks AIP4-mediated lysosomal sorting and degradation of CXCR4 (Slagsvold et al. 2006), raising the possibility that CISK may contribute to elevated CXCR4 expression in breast cancer. However, the precise mechanism by which ErbB2 signaling disrupts CXCR4 ubiquitination and lysosomal degradation in particular cancer settings remains to be determined.

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Aberrant PAR1 Trafficking Promotes Cancer Progression

Protease-activated receptors (PARs) are GPCRs that signal in response to extracellular proteases and have been implicated in cancer progression. PAR1 is overexpressed in aggressive melanoma, colon cancer, prostate cancer and invasive breast cancer (Arora et al. 2007). The expression of PAR1 and PAR2 is also increased in stromal fibroblasts of malignant tissues. Overexpression of PAR1 induces hyperplasia of mammary gland epithelial cells (Yin et al. 2003a,b), an oncogenic phenotype. In addition, the targeted expression of PAR1 in mammary glands activates the Wnt and b-catenin pathway (Yin et al. 2003a,b), a hallmark of malignant cancer. PAR2 has also been recently shown to promote development of mammary adenocarcinoma in polyoma middle-T mice (Versteeg et al. 2008). PARs have the capacity to transduce signals in response to multiple tumor-generated proteases (Arora et al. 2007). Tumors are replete with proteases including urokinase-plasminogen activator (uPA), which generates plasmin. Plasmin cleaves and activates PAR1 and is also critical for cleavage and activation of MMPs. MMP1 appears to also signal through PAR1 to promote breast tumorigenesis (Boire et al. 2005). PAR1 contributes to tumor progression through a variety of mechanisms including cancer cell growth, invasion and metastasis. Moreover, Arora and colleagues recently showed that proteolytic activation of PAR1 causes persistent transactivation of EGFR and ErbB2 in invasive breast carcinoma, but not in normal mammary epithelial cells (Arora et al. 2008). PAR1-stimulated EGFR and ErbB2 activation led to prolonged ERK1,2 signaling and cellular invasion and ablation of PAR1 expression in human breast adenocarcinoma-impaired tumor growth as assessed by mammary fat pad xenografts. In addition to PAR1 overexpression, tumor cells display aberrant PAR1 trafficking, which causes persistent signaling and cellular invasion (Booden et al. 2004). One phenomena contributing to persistent signaling is slowed receptor internalization and/or recycling and a lack of lysosomal degradation. These defects appear to be specific to breast cancer cells, because PAR1 ectopically expressed in normal mammary epithelial cells displays proper trafficking and signal termination. The mechanisms responsible for dysregulated PAR1 trafficking are not known. Wolfe and colleagues recently reported a direct role for ubiquitination in the regulation of PAR1 internalization in HeLa cells and fibroblasts (Wolfe et al. 2007). PAR1 appears to be ubiquitinated under basal conditions, and deubiquitinated following activation. Ubiquitination negatively regulates PAR1 constitutive internalization and thereby functions to retain the receptor at the cell surface in the absence of agonist. Interestingly, ubiquitination of PAR1 also appears to function in agonist-induced internalization by specifying a distinct clathrin adaptor requirement that occurs independent of arrestins and AP-2. The identity of the clathrin adaptor that mediates internalization of ubiquitinated PAR1 is not known. PAR1 is de-ubiquitinated after activation and sorts from endosomes to lysosomes independent of ubiquitination, HRS and tumor-susceptibility gene 101 (TSG101), components of the ESCRT machinery (Gullapalli et al. 2006; Wolfe et al. 2007). Thus, de-ubiquitinated rather than ubiquitinated PAR1 efficiently transits the

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endosomal–lysosomal system. The sorting of PAR1 to lysosomes is dependent, however, on sorting nexin-1 (SNX1) (Wang et al. 2002), a protein that localizes to early endosomes and is known to function in membrane trafficking (Cullen 2008). SNX1 is likely to regulate PAR1 sorting early in the endocytic pathway; however, the identities of other endocytic adaptor proteins that mediate ubiquitin-independent lysosomal sorting of PAR1 remain to be determined.

5.4.3

Other GPCRs Linked to Cancer Progression

In addition to CXCR4 and PAR1, other GPCRs are overexpressed in various types of tumor cells and contribute to cancer progression. However, unlike CXCR4 and PAR1, the mechanisms that lead to aberrant overexpression and signaling remain poorly understood. The bioactive lipid lysophosphatidic acid (LPA) acts on GPCRs and promotes ovarian cancer (Mills and Moolenaar 2003). In addition, a GPCR called GPR30 was recently identified and shown to mediate responses to estrogen in human breast cancer (Thomas et al. 2005), but precisely how GPR30 contributes to cancer progression remains to be determined. In contrast to most GPCRs, the activating ligand KiSS-1 binds to GPR54 and functions as a tumor suppressor by inhibiting metastasis (Lee et al. 1996). The molecular basis for the potent antimetastatic activity of the Kiss-1/GPR54 signaling cascade is not known. Smoothen and Frizzleds are seven transmembrane receptors expressed in tumor cells and mediate Hedgehog and b-catenin signaling, respectively. Smoothen and Frizzleds also promote progression of certain types of cancers (Dorsam and Gutkind 2007), but precisely how the canonical G protein signaling and regulatory machinery is involved in eliciting responses in cells to Smoothen and Frizzled has not been definitively determined.

5.5

Defective Trafficking Machinery and Cancer Progression

GPCR trafficking is critical for the temporal and spatial control of signaling and is often dysregulated in cancer resulting in GPCR overexpression. There have been no mutations identified in GPCRs that appear to promote dysregulated receptor trafficking; rather, aberrant receptor signaling and/or endocytic sorting machinery are likely to be responsible for misregulation of receptor trafficking in malignant cancer. This may be particularly important for GPCRs that require ubiquitination for efficient internalization and lysosomal degradation. Indeed, the stability and function of c-Cbl, an E3 ubiquitin ligase that ubiquitinates EGFR, is regulated by Src as well as the Nedd4 family of E3 ubiquitin ligases. The dysregulation of these pathways in cancer cells leads to Cbl down-regulation and increased expression of EGFR (Moseson et al. 2008). Cbl has also been shown to ubiquitinate PAR2 (Jacob et al. 2005). However, whether similar dysregulation of the machinery that controls

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the ubiquitination status of CXCR4 and PAR1 occurs in various types of malignant cancers remains to be determined. The Huntington-interacting protein 1 (HIP1) and related HIP1R are clathrin adaptor proteins that bind to clathrin and the actin cytoskeleton to facilitate clathrinmediated endocytosis (Engqvist-Goldstein et al. 2004). HIP1 is overexpressed in various cancers including prostate and glial carcinomas and is correlated with increased expression of EGFR and other receptor tyrosine kinases (Bradley et al. 2007; Rao et al. 2003). Moreover, ectopic expression of HIP1 in fibroblasts induces transformation and promotes tumor formation in nude mice (Rao et al. 2003). Other aberrantly expressed clathrin adaptor proteins include Eps15, Disabled 1 and Numb (Moseson et al. 2008). Whether these clathrin adaptors directly or indirectly regulate GPCR trafficking in tumor cells has not been determined. In addition, genetic studies in Drosophila support a function for aberrant regulation of the ESCRT machinery in cancer progression, in which loss of TSG101 and VSP25 disrupts polarity and promote a mesenchymal phenotype (Moberg et al. 2005). However, the tumor suppressor function of TSG101 in mammalian systems has not been clearly established. Increased expression of TSG101 has been reported in certain breast cancers and thyroid carcinomas (Oh et al. 2007). In addition to defects in endosome–lysosomal sorting machinery, certain aberrations in recycling components including Rab15 may be also involved in defective receptor trafficking and dysregulated signaling (Moseson et al. 2008), but how these contribute to defective GPCR trafficking is not known.

5.6

Conclusions

GPCRs have critical functions in the control of tumor cell growth and angiogenesis associated with tumor progression as well as in mediating cellular responses to multiple inflammatory molecules providing a link between tumor progression and inflammation. One paradigm by which GPCR signaling promotes tumor progression involves transactivation of ErbB family members (Fig. 5.1). Hyperactivation of ErbB signaling is associated with multiple malignant cancers and correlates with poor prognosis. Humanized antibodies generated against ErbB2/HER2 termed trastuzumab (Herceptin) reduce ErbB2 activity and expression at the cell surface and increase survival of patients with metastatic breast cancer (Slamon et al. 2001). However, many patients who achieve initial response to trastuzumab-based therapies acquire resistance and many ErbB2/HER2 positive breast cancers do not respond to trastuzumab therapy (Nahta and Esteva 2007), Thus, identifying other pathways that lead to hyperactivation of ErbB signaling is critical for improving treatment options for patients with metastatic breast cancer. Recent work indicates that aberrant GPCR signaling mediates hyperactivation of ErbB signaling in breast and colorectal carcinoma to promote tumor progression (Arora et al. 2008; Buchanan et al. 2006). Thus, new strategies that combine drugs that target ErbB2 signaling as well as specific GPCRs may prove to be more effective regimens for treatment of certain types of malignant cancers.

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In many cases, aberrant GPCR signaling derives from receptor overexpression coupled with the abundance of activating ligands generated in the tumor microenvironment. Thus, identifying the molecular machinery responsible for defective GPCR trafficking in cancer cells will provide novel targets for the development of new drugs that can be used in the treatment of cancer progression. In addition, GPCRs are allosteric proteins that adopt many distinct conformations, some of which promote receptor internalization independent of heterotrimeric G protein activation (Kenakin 2007). The identification of new drugs (termed allosteric modulators) that promote GPCR internalization and down-regulation independent of cellular signaling could be potentially useful therapeutics for aberrantly overexpressed GPCRs. In some cases, inverse agonists can stabilize the inactive GPCR state and thereby decrease signaling activity of certain constitutively activated GPCRs (Parnot et al. 2002). In summary, GPCRs have proven to be entities amenable to drug modulation, and future research aimed at the development of drugs that specifically target cancer-associated GPCRs will likely be beneficial to patient treatment strategies and outcomes.

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Part II

Growth Factors

Chapter 6

Insulin Signaling in Normal and Diabetic Conditions Patrice E. Fort, Hisanori Imai, Raju Rajala, and Thomas W. Gardner

6.1

Introduction

Insulin is a pleiotropic anabolic hormone that stimulates the uptake and storage of carbohydrates, fatty acids, and amino acids into glycogen, fat, and protein, respectively. The signal transduction pathway by which insulin action stimulates various cells to accomplish their biosynthetic roles has been extensively investigated and summarized in several excellent reviews (Flati et al. 2008; Muscogiuri et al. 2008; Saltiel and Pessin 2002). The pancreatic “organelle,” the islets of Langerhans, is composed of clusters of cells organized into ovoid structures dispersed throughout the pancreas which account approximately for 2% of the pancreatic mass. An islet of Langerhans is composed of a, b, d, and PP cells, secreting the hormones glucagon, insulin, somatostatin, and pancreatic polypeptide, respectively. Thus, the islets are organized to produce hormones that have opposing actions on blood glucose levels. Insulin action begins with the synthesis and maturation of the 110 amino acid preproinsulin peptide, the product of a single insulin gene on the human chromosome 11. Maturation of the preproinsulin peptide into proinsulin requires cleavage of the 24 amino acid signal sequence that directs synthesis of the peptide into the endoplasmic reticulum (ER). In the ER, proinsulin is folded and three disulfide bonds stabilize the insulin A and B chains of 21 and 30 amino acids, respectively. The b cells then store proinsulin in secretory granules, from which it is released into the circulation when the cells are stimulated. The final step of insulin maturation takes place in the secretory granule, and consists of the removal of the C (connectT.W. Gardner (*), P.E. Fort, and H. Imai Departments of Ophthalmology and Cellular and Molecular Physiology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA e-mail: [email protected] R. Rajala Departments of Ophthalmology, University of Oklahoma School of Medicine, Oklahoma City, OK, USA A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_6, © Springer-Verlag Berlin Heidelberg 2010

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ing) chain (35 amino acids) that links the A and B chains. Insulin, C peptide, and uncleaved proinsulin are all secreted into circulation via the portal vein. Insulin binding and activation of its receptor triggers the recruitment and activation of proteins or other molecules which in turn recruit other adapter and enzymatic proteins to transmit the signal and affect the biological processes. The first part of this chapter concentrates on the “classical” insulin signaling using skeletal muscle, liver, and adipose tissue as examples, while the second part focuses on the special features of insulin signaling in the central nervous system (CNS), especially the retina. Each part depicts the ligand/receptor specificities, and the downstream effectors of the pathway, followed by functional information. Finally, insulin signaling disruption and insulin therapy will be discussed in the context of diabetes.

6.2 6.2.1

Conventional Insulin Signaling Receptor/Ligand Interactions

Like other hormones, insulin exerts its biological actions by first binding to its specific receptor on the cell membrane. Insulin, insulin-like growth factor-1 (IGF-1, also called somatomedin C) and insulin-like growth factor-2 (IGF-2, somatomedin A) activate cell signaling pathways through tyrosine kinase holoreceptors (tetrameric a2b2-receptors) composed of two hemi-receptors (half-receptors, a,bheterodimers). Insulin, IGF-1, and IGF-2 bind to these holoreceptors with different specificities and affinities depending on the combination of specific half-receptors. Insulin receptors exist as two isoforms called IR-A and IR-B, IR-A being an exon 11 splice variant of IR-B. IR-A is expressed predominantly in the CNS, hematopoietic cells, fetal tissues and various malignant cells, whereas IR-B is predominantly expressed in muscle, adipose tissue, and liver. In muscle and other tissues subject to acute effects of insulin, signals are transduced primarily through IR-B homodimers. Both extracellular a-subunits of the tetramer bind insulin and the two transmembrane b-subunits transmit the signal through their tyrosine kinase activity (Taha and Klip 1999). Insulin binding to the a-subunit induces the transphosphorylation of one b-subunit by the other; specific tyrosine residues (Y1158, Y1162 and Y1163) are phosphorylated, leading to an activation loop, resulting in the increased catalytic activity of the receptor tyrosine kinase. The receptor also undergoes autophosphorylation at other tyrosine residues (Y972, Y1328, 1334) in the juxtamembrane regions and the intracellular tail. Tyrosine phosphorylation of the b-subunits induces specific recruitment of SH2 (Src Homology domain 2) and PTB (phosphotyrosine binding domain) domain-containing proteins. These domains recognize phosphorylated tyrosine residues (Taniguchi et al. 2006). The a-subunit (molecular weight (MW) about 135 kDa) is entirely in the extracellular compartment and binds insulin; the b-subunit (MW about 95 kDa) contains a large hydrophobic fragment (inserted into the membrane), traverses the cell

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membrane and extends into the cytoplasm. Mammalian muscle IR kinase activity and b-subunit tyrosine phosphorylation decreases in the fasting state and increases in the refed animals, correlated with alterations in plasma insulin levels (Burant et al. 1986). Various postreceptor steps regulate cell sensitivity to insulin, and alterations of insulin sensitivity are not necessarily associated with changes in insulin receptor number, phosphorylation or kinase activity. Immediate protein substrates of IR kinase include the insulin receptor substrates (IRS 1–4), Grb-2 associated binder (Gab-1), Shc, and Cbl, which in turn recruit other adapter and enzymatic proteins. The complexity and diversity of the system is demonstrated by the report in Gene Ontology of 146 genes related to the insulin receptor pathway (http://amigo.geneontology.org).

6.2.2

Post-receptor Pathways

Following insulin binding, the insulin receptor initiates intracellular insulin signaling by phosphorylating tyrosines on various cellular substrates. In mammals, at least eleven intracellular substrates of the IR have been identified (Taniguchi et al. 2006). These include the family of IRS (Insulin Receptor Substrate) proteins (IRS1-4) and Shc (Src Homology Collagen protein). IRS proteins are characterized by similar domains including a PH (Pleckstrin Homology) domain and a phosphotyrosine-binding domain; they contain numerous tyrosine residues that can be phosphorylated by the IR. Four distinct IRS proteins are expressed in various mammals, and two additional IRS proteins have been detected in humans (IRS5/DOK4 and IRS6/DOK5). These additional IRS proteins are involved in insulin signaling but are not involved in phosphatidylinositol 3¢-kinase (PI3-K) activation (Cai et al. 2003). With MWs between 60 and 180 kDa, IRS proteins show differential tissue distribution (Sun et al. 1997). IRS-1 and IRS-2 are widely distributed, IRS-3 is restricted to brain, liver, adipocytes and fibroblasts (Sciacchitano and Taylor 1997), and IRS-4 is expressed mainly in embryonic tissues and adult muscle (Schreyer et al. 2003). The IRS protein isoforms also differ in their cellular compartmentalization and activation kinetics (Inoue et al. 1998). IRS action downstream from the IR is tightly regulated by both stimulatory and inhibitory mechanisms. IRS proteins are activated by tyrosine phosphorylation and negatively regulated by protein tyrosine phosphatases (Gonzalez-Rodriguez et al. 2007). Other inhibitory mechanisms include blockade of their binding to the IR (Ueki et al. 2002), and serine phosphorylation mediated by a PI3-K-mediated negative feedback that involves IRS phosphorylation through the MAPK and/or p70S6K pathways (Jiang et al. 2004). In mammals, Shc is also an insulin receptor substrate. There are 46-, 52- and 66-kDa isoforms of Shc that arise from alternative splicing of the primary Shc transcript (Pelicci et al. 1992). Upon insulin stimulation, the activated insulin receptor interacts with Shc. The NPXY (Asparagine Proline X Tyrosine, X is any amino acid) motif around the tyrosine-960 residue of the insulin receptor binds to the N-terminal PTB domain of Shc. Subsequently, the 52-kDa, and to a lesser extent,

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the 46-kDa Shc isoforms are tyrosine phosphorylated. Insulin predominantly phosphorylates the Shc tyrosine-317 residue (Sasaoka and Kobayashi 2000). Phosphorylation of Shc by the IR has been reported in various transformed cell models. Most reports suggest that in mammals IRS-1 is the major substrate leading to stimulation of glucose transport in muscle and adipose tissues, whereas in liver IRS-1 and IRS-2 have complementary roles in insulin signaling and metabolism. In contrast, Shc does not appear to be directly involved in metabolic signaling of insulin but plays a critical role in insulin-induced mitogenesis. Interestingly, tyrosine phosphorylation of Shc (52 kDa) depends on the nutritional state; it increases after starvation, refeeding, and insulin injection. In mammals, tyrosine phosphorylation of IRS and Shc proteins makes these proteins docking sites for various signaling proteins containing a SH2 domain (mostly the PI3-K regulatory subunit and Grb2). These intracellular interactions lead to the activation of the two main IR signaling pathways: PI3-K/Akt and MAPK.

6.2.2.1

PI3-K/Akt

Mammalian PI3-K is a heterodimer composed of one catalytic subunit of 110 kDa (p110) and an SH2-containing regulatory subunit of about 85 kDa (p85) (Hirsch et al. 2007); the two are associated noncovalently through specific binding motifs. In mammals, eight regulatory subunits of PI3-K (of different sizes) and three p110 isoforms have been described. IRS proteins interact with all PI-3K regulatory subunits, and thereby activate the catalytic PI3-K subunits (Taniguchi et al. 2006). Once docked to IRS, PI3-K phosphorylates the membrane phospholipid, phosphatidylinositol 4,5-biphosphate (PIP2), thereby producing phosphatidylinositol 3,4,5-triphosphate (PIP3), which in turn, acts as a second messenger to activate PDK1/2 (3-phosphoinositide-dependent kinase-1/2) and three known isoforms of Akt/PKB. PDK1 mediates the phosphorylation of the activation loop site in Akt/PKB. Activated Akt directly phosphorylates and inactivates glycogen synthase kinase-3 (GSK-3b), Bad, and the forkhead transcription factor, Foxo1. Inhibition of the PI3-K/ Akt pathway blocks almost all of insulin’s metabolic actions, including stimulation of glucose transport, glycogen synthesis and lipid synthesis (Cheatham et al. 1994). Thus, this pathway plays a pivotal role in the metabolic actions of insulin. The biological effects of the PI3-K pathway can be negatively regulated at the level of PIP3 by phospholipid phosphatases, including the phosphatase and tensin homologue (PTEN) and SH2-containing 5¢-phosphatase-2 (SHIP2), which dephosphorylate and inactivate PIP3 (Sleeman et al. 2005; Wijesekara et al. 2005). The degree of PI3-K activation in mammalian liver and muscle depends upon the nutritional state (prolonged fasting versus refeeding), and can be increased in response to insulin injection or inhibited following insulin immuno-neutralization. This response is in parallel with changes observed at early steps of the insulin signaling cascade (tyrosine phosphorylation of IR b-subunit, IRS-1 and Shc). The p85 regulatory subunit of PI3-K itself can, in mammals, exert strong inhibitory control of IRS signaling (Taniguchi et al. 2006). Interestingly, a recent work has shown that

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the conventional insulin signaling pathway in chicken liver, but not leg muscles, is sensitive to feeding/fasting conditions (Dupont et al. 2008).

6.2.2.2

MAPK

In mammals, the MAPK extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway is activated by insulin through IRS or Shc tyrosine phosphorylation (Taniguchi et al. 2006). This pathway is composed of a set of three sequentially acting kinases. The activation of a MAPKKK kinase (Raf) leads to the phosphorylation and activation of a MAPKK kinase called MEK1/2, which then stimulates ERK1/2 MAPK activity through dual phosphorylation on threonine and tyrosine residues.

6.2.3

Insulin Signaling Function

The following paragraphs depict the diverse array of biological responses elicited by insulin signaling in different tissues, including glucose homeostasis control, carbohydrate, lipid, and protein metabolism regulation (Saltiel and Kahn 2001).

6.2.3.1

Glucose Uptake

Insulin promotes glucose uptake by muscle and adipose tissue via stimulation of GLUT4 vesicles from intracellular sites to the plasma membrane. The PI3-K/Akt pathway is pivotal to glucose transport in several tissues, especially through GLUT4 translocation. GLUT4 is a member of the facilitative glucose transporter (GLUT) family, characterized by preferential expression in muscle and fat tissues, where it is responsible for insulin-stimulated glucose uptake (Shisheva 2008). The insulin-derived signaling pathways regulating the net gain in surface GLUT4 include activation of PI3-K, PDK1/2, atypical protein kinase C (PKC) (Farese et al. 2005, 2007), Akt/PKB and AS160, a Akt/PKB substrate, and other effectors described earlier. In addition, GLUT4 translocation is also downstream of a PI3K-independent pathway involving c-Cbl (Baumann et al. 2000). Insulin recruits c-Cbl via the adaptor protein CAP (c-Cbl-associated protein) and stimulates its tyrosine phosphorylation in metabolically responsive cells (Ribon et al. 1998). Upon Cbl phosphorylation, the Cbl/CAP complex is translocated to the plasma membrane domain enriched in lipid rafts or caveolae and forms a complex with a number of proteins including flotillin, TC10, CRKII and other accessory proteins involved in vesicular trafficking and membrane fusion (Chiang et al. 2001). The PI3-K/Akt pathway and the CPA/Cbl complex represent two compartmentalized parallel pathways leading to GLUT4 translocation and glucose uptake.

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Glycogen Synthesis and Gluconeogenesis

Insulin suppresses hepatic glucose output by stimulating glycogen synthesis and inhibiting glycogenolysis and gluconeogenesis. Insulin directly controls some of the important enzymatic checkpoints that regulate these metabolic processes via phosphorylation and dephosphorylation. In the insulin signaling pathway, GSK-3b is active in the absence of insulin and phosphorylates (and thereby inhibits) glycogen synthase and several other substrates. Insulin binding to the receptor activates a phosphorylation cascade, leading to inhibitory phosphorylation of GSK-3b by Akt. Thus, insulin activates glycogen synthase by promoting its dephosphorylation through inhibition of GSK-3b. The concerted action of insulin, glucagon, and glucocorticoids regulates expression of genes important for glycolysis, glycogenolysis, and gluconeogenesis (Pilkis and Granner 1992). The gene expression regulation is related to the forkhead transcription factor family (FKHR or Foxo1) which is phosphorylated in an insulindependent manner by Akt kinase (Accili and Arden 2004; Quinn and Yeagley 2005). Under basal conditions, Foxo1 resides in the nucleus, and upon insulin stimulation and phosphorylation by Akt, Foxo1 is excluded from the nucleus to the cytoplasm, thereby providing a powerful mechanism by which insulin downregulates genes including IGF-binding protein-1, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase. Peroxisome proliferator activated receptor g coactivator 1 (PGC-1) represents another transcriptional coactivator that plays an important role in the regulation of genes involved in hepatic gluconeogenesis (Herzig et al. 2001).

6.2.3.3

Lipid Metabolism

Insulin’s anabolic effects include promoting lipid synthesis and suppressing lipolysis. Moreover, in the liver, insulin works reciprocally with glucagon to control the balance of glucose and lipid metabolism. Recent studies indicate that the transcription factor steroid regulatory element-binding protein (SREBP)-1c is a major mediator of insulin action on the expression of glucokinase and lipogenesis-related genes in the liver (Foretz et al. 1999). In addition to promoting lipogenesis in the liver, insulin also stimulates lipid synthesis enzymes (fatty acid synthase, acetyl-CoA carboxylase) and inhibits lipolysis in adipose tissue. The anti-lipolytic effect of insulin is primarily mediated by inhibition of hormone sensitive lipase through a mechanism that involves activation of a cAMP-specific phosphodiesterase (Sztalryd et al. 1995).

6.2.3.4

Protein Synthesis

Insulin stimulates protein synthesis via two major kinds of effects: the rapid activation of existing components of the translational apparatus, and the longer term

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increase in the capacity of the cell or tissue for protein synthesis, which includes an increase in ribosome number (Proud 2006). The rapid activation of protein synthesis by insulin is mediated primarily through the GSK-3b inactivation mediated by activated Akt. Insulin, by inhibiting GSK-3b, elicits the dephosphorylation and activation of eIF2B, a protein required for recycling of eIF2, itself necessary for all cytoplasmic translation initiation events. Insulin-activated Akt also phosphorylates the tuberous sclerosis complex TSC1–TSC2 complex to relieve its inhibitory action on mTOR (mammalian target of rapamycin), a key controller of translation initiation and elongation. The cap-binding factor eIF4E can be sequestered in inactive complexes by 4E-BP1 (eIF4E-binding protein 1). By activating mTOR, insulin induces phosphorylation of 4E-BP1 and its release from eIF4E, allowing eIF4E to form initiation factor complexes. It also induces dephosphorylation and activation of eEF2 to accelerate elongation. Insulin inactivates eEF2 kinase by increasing its phosphorylation at several mTOR-regulated sites. Insulin also stimulates synthesis of ribosomal proteins by promoting recruitment of their mRNAs into polyribosomes.

6.2.3.5

Cell Fate

Besides its action on the anabolic processes described above, insulin also affects cell fate by controlling proliferation and differentiation. Foxo1 is a transcriptional enhancer that regulates genes involved in glucose production as well as cell cycle regulation and apoptosis. In response to insulin, Foxo1 is phosphorylated by Akt which induces its exclusion from the nucleus, and promotes cell survival. Insulin activation of the PI3-K/Akt pathway also has a key role in Wnt signaling via GSK3b that is critical for determination of cell fates during embryonic development (Harwood 2001). On the other hand, insulin activation of the Ras-MAPK pathway plays a central role in cell fate determination. Ras is particularly critical to insulin’s ability to promote the differentiation of 3T3 L1 cells to adipocytes (Benito et al. 1991). ERK1/2 MAPK also regulates the expression of genes including some in the PI3-K pathway, controlling cell growth and differentiation (Avruch 1998).

6.2.4

Insulin Signaling, Diabetes and Therapies

Diabetes results from impaired action of the insulin receptor due to deficient ligand availability consequent to pancreatic b-cell failure (Type 1 or insulin-deficient diabetes) or to insulin resistance (Type 2 diabetes). Insulin causes its effects by stimulating the uptake of glucose, amino acids and fatty acids into skeletal muscle, adipose and liver, or by facilitating the oxidation of these substrates for ATP production, depending on the overall energy state of the organism. Systemic insulin resistance implies reduced effectiveness of insulin action (Kahn 1978), measured as

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glucose uptake into cells in response to insulin, or as reflected by the plasma insulin/glucose ratio. Excessive nutrient ingestion causes a physiologic downregulation of further nutrient uptake into storage tissues, presumably because liver, adipose and/or the brain sense “satiety” and less need for additional nutrients to synthesize ATP or macromolecules. The mechanisms by which insulin-sensitive cells downregulate insulinstimulated glucose uptake have been studied extensively and include: (1) reduction of GLUT4 glucose transporter expression and translocation; (2) serine phosphorylation of IRS-1 by protein kinase C (PKC); (3) activation of suppressors of cytokine signaling (SOCS) proteins; (4) release of inflammatory cytokines (IL-1, 6) and leptin from adipose tissue; (5) activation of ER stress response pathways (Hotamisligil 2006); (6) phosphatases such as protein tyrosine phosphatase 2B; and (7) glycosylation of insulin signaling pathway intermediates (Buse 2006). These mechanisms are not mutually exclusive and are likely redundant. Moreover, the distinction between insulin deficiency and insulin resistance is not clear-cut. For example, insulin-deficient patients or animals can be insulin-resistant if they receive insufficient insulin replacement or if they have excessive nutrient intake and develop abdominal obesity (Greenbaum 2002). Conversely, prolonged b-cell stimulation to compensate for nutrient excess can lead to b-cell failure and insulin deficiency in patients with Type 2 diabetes. At present the mechanisms by which insulin deficiency and insulin resistance contribute to organ damage are uncertain. Therapies to counteract these fundamental defects of metabolism include physiologic steps to reduce dietary nutrient intake and increase physical activity, or pharmacologic means such as activation of proliferating peroxisome activating (PPAR) receptors alpha and gamma by thiazolidinediones, blockage of inflammatory pathways with salicylates, or experimental inhibition of PKC activity. However, current pharmacologic approaches for Type 2 diabetes have modest effectiveness and substantial side-effects, such as the increased risk of cardiac and hepatic dysfunction in patients receiving thiazolidinediones (Rubenstrunk et al. 2007). In Type 1 diabetes, subcutaneous insulin administration by injection or pump is not physiologically sound because insulin is delivered to the systemic circulation rather than the portal circulation. Moreover, intensive insulin therapy is limited by the risk of hypoglycemia (Cryer 1999). Therefore, improved physiologic means of controling insulin action are needed.

6.3 6.3.1

Insulin Signaling in the Retina and Brain History of the Insulin Receptor

The IR and IGF-1R proteins and insulin-like peptides are evolutionarily ancient, found in sponges prior to the Cambrian era (Skorokhod et al. 1999), thus developing prior to the origin of the pancreas or islets as found in fish or mammals.

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Moreover, the expression of insulin and IR/IGF-1R in primitive organisms is primarily in the CNS, and mutation of the IR in Caenorhabditis elegans and Drosophila leads to brain and eye defects (Song et al. 2003b). Thus, it appears that the IR/IGF-1R system and its ligands developed to promote cell growth and differentiation in animals with little need for control of blood nutrient levels, and evolved later to regulate blood nutrient levels in response to pancreas-derived hormones. Indeed, both neurons and b cells express Thy-1 and islet-1 proteins and are surrounded by oligodendrocytes. The next section details IR signaling pathways in the retina and their importance for diabetic retinopathy.

6.3.2

Receptor/Ligand Interactions

Budd et al. reported preproinsulin mRNA expression in rat retina (Budd et al. 1993), but whether insulin protein is secreted by retinal cells and contributes to the basal tyrosine phosphorylation of retinal IRs in an autocrine or paracrine fashion is unknown. IGF-1 and IGF-2 mRNA are expressed in the retina which suggests that locally produced IGF-1 or IGF-2 may contribute to basal IR phosphorylation in retina. Moreover, crosstalk between insulin, the IGFs, and their cognate receptors has been shown previously (Louvi et al. 1997). We have shown that retinal IR could be activated by IGF-2 stimulation (Reiter et al. 2003). Insulin receptors (IR) and insulin signaling proteins are widely distributed throughout the CNS (Havrankova et al. 1978a). Insulin and IGF-1 receptors are expressed at high levels in many brain areas, and in both glial and neuronal cells. Insulin binding characteristics have been described in the mammalian brain including the retina (Havrankova et al. 1978b). However, our understanding of insulin action in the retina has lagged considerably compared to insulin-responsive tissues. Waldbillig and Rodrigues (Waldbillig et al. 1987a, b) first characterized the mammalian retinal IR. Our labs have more recently demonstrated that the level of retinal IR kinase activity in vivo is similar to that in brain (Reiter et al. 2003). In contrast, the tyrosine phosphorylation and kinetic activity of the IR from liver and skeletal muscle fluctuate with circulating insulin levels during periods of fasting and feeding, which in turn affects the physiology of downstream mediators of insulin signaling. Tyrosine phosphorylation of brain and retinal IR is unaltered between fasted and fed rats (Simon et al. 1986a, b). Likewise, the kinetic activity of the IR in retina mirrors brain IR activity and is resistant to changes associated with fasting, which diminishes liver IR activity. Taken together with the studies by Waldbillig and Rodrigues, in vivo retinal and brain IR activity are similar and are maintained in a tonic state. The differences in IR activity regulation between peripheral and neural tissues may result from how circulating insulin is delivered to tissues of the body, and suggests that the blood-retinal barrier may stabilize insulin uptake into the retina. One major structural difference between IRs expressed in retina and liver, skeletal muscle, or adipose tissue is the extent of glycosylation. The extent of glycosylation of the a and b subunits of IRs expressed on neuronal cells is less than that in liver

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and skeletal muscle (Heidenreich and Brandenburg 1986) (Reiter et al. 2003). Enzymatically catalyzed IR glycosylation is important for normal IR function and mutations of the four N-glycosylation sites on the IRb lead to loss of kinase activity (Leconte et al. 1992). Although glycosylation is required for IR function, an explanation for variability of the glycosylated IR among tissues currently remains elusive. A second difference involves exon 11 expression on the a-subunit, which is the only sequence variance in the mature peptide (Ebina et al. 1985; Ullrich et al. 1985). This splice variant expression varies between tissues and influences ligand binding affinities. Brain and retina exclusively express the exon 11 lacking form (type A) of the receptor, whereas the majority of liver IRs contains the exon 11 (type B) (Seino and Bell 1989). Consistent with the “housekeeping” nature of the GC-rich IR promoter, and the lack of a TATA box (Araki et al. 1987), the IR is expressed constitutively and broadly throughout the retina on neuronal, endothelial, and retinal pigmented epithelial (RPE) cells. (Rajala et al. 2002, 2008) demonstrated the expression of IR in the plasma membrane of the rod outer segment. Ghalayini et al (Ghalayini et al. 1998) previously reported that light stimulates tyrosine phosphorylation of several proteins in the ROS in vivo. To determine whether light has an effect on the insulin receptor pathway, Rajala et al. used different animal models defective in elements of the phototransduction cascade. Their studies suggest that in the ROS, the phototransduction cascade is coupled to the activation of IR phosphorylation. Photobleaching of rhodopsin is necessary for light-dependent IR phosphorylation, while the visual transduction cascade downstream of rhodopsin is not. Their results indicate the existence of a light-mediated IR pathway in the retina that differs from the known insulin-mediated pathway in non-neuronal tissues. A third level of regulation of hormone action is by IR and IGF-1 receptor combinations. Holo-receptors can be composed of combinations of two insulin halfreceptors (IR), two IGF-1 half-receptors, or as hybrids composed of IR and IGF-1 half-receptors. IGF-I hemi-receptors can hetero-dimerize with either the A or B isoform IR hemi-receptor leading to the formation of A-type and B-type insulin/ IGF-1 hybrid receptors. IR-A:IGF-1R and IR-B:IGF-1R hybrids have different binding affinities for insulin, IGF-1 and IGF-2. The IGF-1R hemi-receptor typically dominates the downstream response to ligand binding to insulin hybrid receptors. IGF-1 binds to IGF-1 homo- or hybrid holo-receptors and induces auto-phosphorylation within the cytoplasmic domain of the receptor followed by the binding of various adapter molecules that contribute to the specificity of downstream signaling. Further investigation is required to determine the functions of IR/ IGF-1R hybrids in retina and the CNS.

6.3.3

Post-receptor IR Pathways

Similar to conventional insulin signaling pathways, phosphorylation of the IR in the retina correlates with high phosphorylation and kinase activity levels of downstream

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proteins such as the PI3-K/Akt pathway. Ex vivo studies showed that insulin increases IR b-subunit, IRS2, PI3-K, and Akt phosphorylation and/or activity (Reiter et al. 2003). Interestingly, insulin stimulation of ex vivo retina does not induce IRS1 phosphorylation, so most of the signaling goes through IRS2 in the retina, pointing out another difference with insulin signaling in peripheral tissues. All three Akt isoforms have been demonstrated in the retina, although the Akt1 isoform appears to be preferentially activated, and the ERK1/2 pathway exhibited basal phosphorylation that was not further responsive to insulin in ex vivo retina (Reiter et al. 2003). One of the major differences between the IR signaling in the retina and conventional IR signaling in peripheral tissues is the light sensitivity of the retinal IR described above. Rajala’s group demonstrated that the light-induced tyrosine phosphorylation of IR was followed by light-induced binding of p85 to the insulin receptor and activation of PI3-K (Rajala et al. 2002, 2007). Photoreceptor-specific, ligand-independent IR activation via G proteins mediated by photon capture by photoreceptors is probably unique to the retina and provides important new understanding of IR regulation.

6.3.4

IR Regulation/Function

The IR is highly conserved and the high degree of IR signaling homology between C. elegans, Drosophila, Mus musculus, and humans suggests functional conservation in the mammalian retina. The IR regulates neuronal survival in C. elegans (Wolkow et al. 2000). In Drosophila, the IR serves the important function of guiding retinal photoreceptor axons from the retina to the brain during development, and the IR influences the size and number of photoreceptors (Brogiolo et al. 2001; Song et al. 2003a). Mutations in either IR autophosphorylation sites, or its binding partner, Dock, result in a severe photoreceptor axonal misguidance phenotype in Drosophila. In humans, defects in IR signaling in the CNS are associated with Alzheimer’s disease and Parkinson’s disease (de la Monte and Wands 2004; Takahashi et al. 1996). The lack of IR activation leads to neurodegeneration in brain/neuron-specific IR knock-out mice (Schubert et al. 2004), and IR deletion impairs synapse formation in Xenopus (Chiu et al. 2008). Deletion of IRS-2 leads to accelerated cell death of inner and outer retinal neurons (Yi et al. 2005) and delayed cerebral myelination downstream of the IGF-1R but not the IR. These studies clearly suggest that the IR and IGF-1R pathways are important for neuronal survival and maintenance, although further study is needed to determine a causal relationship with human brain disease. Previous studies have suggested a role for retinal and brain insulin signaling in the regulation of neuronal growth and differentiation (Heidenreich 1993; Robinson et al. 1994). Neurons metabolize glucose in an insulin-independent manner, and ablation of IR in brain results in increased food intake, moderate diet-dependent obesity, and hypergonadotropic hypogonadism, associated with impaired maturation

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of ovarian follicles in females and reduced spermatogenesis in males (Bruning et al. 2000). These studies indicate that IRs play a role in the control of appetite and reproduction, and have important implications for preventing obesity. Retinal endothelial cells and pericytes also display specific binding for insulin, and treatment of these cells with insulin in vitro rapidly stimulates proliferation (King et al. 1985). The IR in retinal pigment epithelial cells is structurally similar to the peripheral form (Waldbillig et al. 1991), and localizes exclusively to the basolateral surface, suggesting a possible role in unidirectional insulin transport from the choroidal circulation to the photoreceptors (Sugasawa et al. 1994). Deletion of IR and IGF-1R on vascular endothelium reduces retinal neovascularization in neonatal mice exposed to a hyperoxic environment, but does not affect unstressed animals (Kondo et al. 2003).

6.3.5

Nervous System Insulin Signaling, Diabetes and Localized Therapies

The brain and retina have been thought to be insensitive to fluctuations in plasma insulin levels and insulin was not thought to have a role in diabetes complications. However, there is increasing recognition that the brain plays an important part in metabolism via hepatic glucose regulation, energy expenditure and lifespan, with central roles for insulin and leptin (Porte et al. 2005). Moreover, recent evidence suggests that persons with diabetes have an increased risk of cognitive impairment (Brands et al. 2005; Klein 2003), and rats with experimental diabetes exhibit features similar to those of humans with Alzheimer’s disease (Li et al. 2007). Mice with insulin-deficient diabetes have reduced brain IR signaling and cognitive function (Jolivalt et al. 2008). We have reported similar reduction of basal IR pathway activity, but no change in IGF-1R activity, in the retinas of diabetic rats and mice (Barber et al. 2005; Reiter et al. 2006). Rajala et al. (2009) showed that reduction of retinal IR phosphorylation is associated with activation of the phosphatase, PTP1B, so the loss of IR ligands may not necessarily contribute to the reduction of receptor activity. However, systemic and local administration of insulin restores retinal IR activity (Reiter et al. 2006), and local administration of insulin likewise restores peripheral nerve function (Brussee et al. 2004), indicating that insulin probably has direct pathophysiologic actions on neural tissues. Understanding the complex alterations in retinal and brain IR signaling pathways may lead to improved means of restoring the homeostatic balance of the normal retina (Antonetti et al. 2006). That is, augmenting deficient insulin actions may serve to maintain CNS cell viability in the face of diabetes and suppress the inflammatory response that is integral to diabetes and its complications. One approach could involve localized delivery of insulin or other neuroprotective factors to the eye and/or brain to augment the effects of systemic treatment to protect the tissues from neurodegeneration. Despite major improvements in lifespan and

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living conditions related to systemic insulin administration, diabetes still leads to high morbidity and mortality related to complications including retinopathy. Several groups have been developing new delivery systems as well as drugs in order to prevent the development of diabetic retinopathy (Chantelau et al. 2008; Kane et al. 2008; Schwartz and Flynn 2007). Numerous targets have been tested, including, but not restricted to, steroids and anti-VEGF agents to prevent or restore vascular permeability; PKC or growth hormone inhibitor to prevent angiogenesis; and ER stress or TNFa inhibitors as well as a Bax inhibitor to prevent neuronal cell death; all of which might be promising but show limitations suggesting that multiple therapies are probably necessary (Oshitari et al. 2008).

6.4

Conclusions

Detailed knowledge of the classical insulin signaling pathway in muscle, liver, and adipose tissues has provided an important understanding of diabetes and contributed to the development of clinically useful therapies, such as thiazolidinediones and insulin analogues. Insulin signaling, however, extends beyond glucose metabolism in “insulin-sensitive” tissues, and has significant roles in brain, retina and the peripheral nervous system. The projected dramatic rise in risk of vision-threatening diabetic retinopathy over the next four decades demonstrates the need for new ways to prevent diabetes-associated neurodegenerative complications (Saaddine et al. 2008). Details that remain to be elucidated include the regulation of critical functional nodes such as Akt isoforms and the mTOR components, TORC1 and TORC2, in CNS neuronal cell survival mechanisms. In addition, it will be essential to understand the impact of systemic metabolic dysregulation of lipids and amino acids on these pathways in order to devise safe and effective treatments for diabetes.

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Chapter 7

Epidermal Growth Factor (EGF) Receptor Signaling and Cancer Elizabeth S. Henson and Spencer B. Gibson

7.1

Introduction

Tyrosine kinase receptors are a type of cell surface receptor that binds to growth factors. Upon growth factor binding to the receptor, the kinase domain within the receptor becomes activated leading to tyrosine phosphorylation and intracellular signaling (Bublil and Yarden 2007). One of the best-understood families of receptor tyrosine kinases is the EGF receptor tyrosine kinase family (also known as the ErbB receptor family). These receptors bind to many growth factors including EGF and have been extensively studied for their functions in development, physiology, and cancer. ErbB receptors activate many signal transduction pathways such as the RAS/Erk pathway, PI3K/AKT pathway and JAK/STAT pathway (Scaltriti and Baselga 2006). Furthermore, ErbB receptors can also translocate to the nucleus and activate transcription (Wieduwilt and Moasser 2008). Other receptor signaling pathways can crosstalk with the ErbB receptor signaling pathway amplifying their ability to signal to cells. This occurs by changing ErbB ligand expression levels or activation of non-receptor tyrosine kinases leading to phosphorylation and activation of ErbB receptors. In cancer, ErbB receptors are deregulated through either overexpression of the receptors, or mutations in the receptors, leading to increased signaling; this leads to increased proliferation and cell survival that are hallmarks of cancer. This correlates with increased resistance to chemotherapy, higher tumor grade, and poor prognosis in cancer patients (Wieduwilt and Moasser 2008). Treatments that target the ErbB receptors are emerging and some have been established as first line treatments in a number of cancers. In this chapter, we review the ErbB receptor family’s structure, function, and signaling and their role in cancer progression and treatment. S.B.Gibson (*) and E.S. Henson Department of Biochemistry and Medical Genetics, Manitoba Institute of Cell Biology, University of Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, R3E 0V9, Canada e-mail: [email protected]

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_7, © Springer-Verlag Berlin Heidelberg 2010

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ErbB Receptor Family

The EGF receptor family is made up of four receptors, ErbB1 (EGFR, HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). The receptors are evolutionarily conserved and contain functional redundancy to ensure signaling is maintained and effective (Rowinsky 2004). The receptors contain a ligand-binding domain, with the exception of ErbB2, and an intracellular tyrosine kinase domain. ErbB3 has a non-functional tyrosine kinase domain with no catalytic activity indicating, it acts as a co-receptor with other ErbB receptors (Bazley and Gullick 2005).

7.2.1

Ligands of the ErbB Receptor Family

7.2.1.1

ErbB Receptors and Their Ligand Binding Partners

There are families of ligands that specifically bind ErbB receptors, with EGF , tumor growth factor (TGF)-a, amphiregulain, heparin binding (HB)-EGF, betacelluline, epigen, and epireculin binding to ErbB1; neuregulin 1, 2 and C binding to ErbB3; and betacelluline, epigen, neuregulin 1–4 and tomoregulin binding ErbB4. No ligand has been identified for ErbB2 (Rowinsky 2004).

7.2.1.2

Production of ErbB Receptor Ligands

ErbB receptor ligands are produced as transmembrane precursors. Their ectodomains are processed by proteolysis leading to shedding of soluble growth factors. Ligand production can be either paracrine, via stromal cells, or autocrine. Autocrine production of ErbB receptor ligands is through a variety of mechanisms including activation of G protein coupled receptors (GPCR), Frizzled (FZD), or the estrogen receptor. Activation of these receptors leads to the activation of the ADAM family of metalloproteinases causing the cleavage of the pro-form of ErbB receptor ligands into their active form; this is called ectodermal shedding. The mechanisms behind the regulation of ectodermal shedding are unknown (Hynes and Lane 2005).

7.2.2

Receptor Structure

7.2.2.1

ErbB Receptors and Their Dimerization Partners

Ligands bind to their respective receptor and induce receptor dimerization leading to a signaling cascade. ErbB1 forms either homodimers or heterodimers with ErbB2–4, but its preferred partner is ErbB2. In contrast, ErbB2 is the preferred binding partner for all other ErbB family members. ErbB3 and ErbB4 also bind to

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all other ErbB family members but the ErbB2/ErbB3 dimer is the most active signaling dimer in the ErbB receptor family (Hsieh and Moasser 2007). 7.2.2.2

ErbB Receptor Structure and Phosphorylation Sites

The structure of the ErbB receptors has been carefully studied using crystallography. ErbB receptors are single-chain transmembrane glycoproteins spanning approximately 620 amino acids that include an extracellular ligand-binding domain, a transmembrane domain of 23 residues, a 40 amino acid juxtamembrane domain, a tyrosine kinase domain approximately 260 amino acids long, and a tyrosinecontaining C-terminal tail that is 232 amino acids (Fig. 7.1). 7.2.2.3

Ligand Binding and Receptor Dimerization

The extracellular ligand-binding domain is made up of four subdomains, the L1 (leucine-rich repeats 1), CR1 (cysteine-rich repeats 1), L2 and CR2. The CR1 domain also contains a hairpin loop that has been shown to be essential to receptor function (Wieduwilt and Moasser 2008). When ErbB3 or ErbB4 are in their ligandfree, inactive tethered form, the hairpin loop in CR1 interacts with CR2 sequester-

Extracellular domain L1 (I) CR1 (II) L2 (III) CR2 (IV)

Tyrosine Kinase domain N-lobe

Transmembrane Juxtamembrane

C-lobe C-terminal Tail

Main tyrosine phosphorylation sites in C-terminal region of ErbB receptor family members ErbB1

Y992 Y1042 Y1068 Y1086 Y1148 Y1173

ErbB2

ErbB3

ErbB4

Y1139 Y1196 Y1226/7 Y1248

Y1035 Y1178 Y1180 Y1203 Y1241 Y1257 Y1270 Y1309

Y875 Y1035 Y1056 Y1081 Y1128 Y1162 Y1188 Y1242 Y1258

Fig. 7.1 EGF receptor structure and phosphorylation sites. ErbB receptor family members share a high degree of homology. They are single transmembrane glycoproteins which include an extracellular domain, a transmembrane domain, a juxtamembrane domain and a two-lobed tyrosine kinase domain, with a C-terminal tail. The activation of these receptors results in the phosphorylation of specific tyrosine (Y) residues in this C-terminal tail, which lead to the activation of downstream signaling pathways (Hynes and Lane 2005)

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CR1

L1

CR1

CR 1

Receptor dimerization

Ligand binding L1

L2 L2 L2

Extended inactive

ob e C-l

N-l ob e

C-lobe

be

N-lobe C-lobe

N-lo

be

N-lobe

Tethered inactive

L2 C-lo

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Fig. 7.2 Ligand binding and receptor dimerization. In the ligand free state, ErbB receptors are held in a tethered, inactive conformation by a hairpin loop in the CR1 (cysteine-rich repeats 1) domain that interacts with the CR2 domain (cysteine-rich repeats 2). When ligand binds to the L1 (leucine-rich repeats 1) or L2 (leucine-rich repeats 2), a conformational change into the extended form reveals the hairpin loop in CR1, allowing dimerization with other receptors. Upon dimerization, conformational changes in the intracellular tyrosine kinase domain lead to receptor autophosphorylation and activation of downstream signaling pathways (Rogers et al. 2005)

ing the dimerization loop. Crystal structure studies have revealed that when EGF binds to ErbB1 in the L1 or L2 domain, a conformational change into an extended form exposes the dimerization loop and allows interaction with receptor domains (Hubbard 2005). Furthermore, it has been shown that ErbB2 is locked in the extended conformation, ready to interact with other receptors, but the ligandbinding domain is blocked by the close proximity of the L1 and L2 domains. When the ligand binds to the receptor, it also induces conformational changes in the intracellular tyrosine kinase domain leading to receptor autophosphorylation. The tyrosine kinase domain has a bi-lobed structure, termed N and C, and ATP is bound between the lobes. The receptor transactivation takes place when the N-lobe of one receptor interacts with the C-lobe of the other receptor’s tyrosine kinase domain. This leads to phosphorylation of specific tyrosine resides within the receptor’s C-terminal tail initiating signal transduction (Bublil and Yarden 2007) (Fig. 7.2).

7.3 7.3.1

ErbB Receptor Signaling Pathways Generalized ErbB Receptor Signal Transduction Pathways

ErbB receptors are normally activated by autocrine or paracrine production of ligands. Activation of ErbB receptors leads to dimerization and activation of the

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ligand receptor

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Cellular Function (proliferation , cell survival, differentiation) Fig. 7.3 Generalized ErbB receptor signal transduction pathway. ErbB receptors bind a ligand, which leads to receptor dimerization and tyrosine autophosphorylation of the C-terminal tail of the receptor. Adaptor proteins bind to the receptor, and then activate a kinase that, in turn, phosphorylates and activates other kinases. This leads to cellular functions such as proliferation, cell survival and differentiation. These pathways are negatively regulated by phosphatases that remove phosphorylation from proteins

tyrosine kinase domain through trans-tyrosine phosphorylation of their kinase domains and C-terminus tails. For example, six tyrosine residues within the ErbB3 C-terminus tail are phosphorylated by its co-receptors allowing ErbB3 to signal without a functioning kinase domain. These tyrosine residues are docking sites for signaling molecules that contain an SH2 domain. Adaptor proteins that contain an SH2 domain and bind to tyrosine phosphorylated ErbB receptors are Grb2, Nck, and Shc. These adaptor proteins also bind to other proteins through tyrosine phosphorylation of residues on the adaptor protein mediated by the ErbB receptors, or constitutively bind to other proteins through domains such as the Src homology 3 (SH3) domain that binds to proline rich regions in other proteins. This leads to activation of a kinase cascade and activation of transcription factors causing specific cellular functions such as proliferation and cell survival. There are three major kinase cascades activated by ErbB receptors, these include the RAS/Erk pathway, the PI3K/AKT pathway and the JAK/STAT pathway (Henson and Gibson 2006) (Fig. 7.3).

7.3.2

MAPK Pathway (RAS/Erk)

The mitogen-activated protein kinase (MAPK) superfamily is activated by a diverse set of stimuli, which include ErbB receptors. ErbB receptors activate the

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MAPK kinase pathway by phosphorylation of mitogen-activated protein kinase kinase kinases (MKKKs), which phosphorylate mitogen-activated protein kinase kinases (MKKs), which ultimately phosphorylate MAPKs. MAPK includes extracellular signal related kinases (ERKs), c-jun terminal kinases (JNKs) and p38 MAPKs. These MAPKs can translocate into the nucleus and activate transcription factors through phosphorylation (Henson and Gibson 2006).

7.3.2.1

ErbB Receptor Activation of the RAS/Erk Pathway

ErbB receptors activate the RAS/Erk pathway when the adaptor proteins Grb2 or Shc bind to the tyrosine phosphorylated ErbB receptors through their SH2 domains. This recruits the SH3 domain-containing guanylyl nucleotide-release protein son of sevenless (SOS) to the plasma membrane. When SOS localizes to the plasma membrane, SOS associates with the small G protein RAS that is bound to the plasma membrane by a prenylation site in its C-terminal end. This association causes activation of Ras through the exchange of nucleotide GDP for GTP within RAS. Activation of RAS leads to activation of the MKKK, Raf-1. Raf-1 then phosphorylates and activates the MKK, MEK1/2, which in turn phosphorylates and activates Erk1/2 on tyrosine and threonine residues within its kinase domain. This activation leads to phosphorylation of numerous substrates, including Rsk, MSK1, cytosolic phospholipase A2 and transcription factors c-Myc, NF-IL6, Tal-1, Ets-2 and Elk. Increased gene transcription leads to increased proliferation, and higher levels of anti-apoptotic Bcl-2 family members and inhibitors of apoptosis proteins. Rsk, which is activated by Erk1/2, also causes the phosphorylation and inactivation of BAD, a pro-apoptotic Bcl-2 family member. The RAS/Erk signaling pathway can also be activated through the activation of RAS by the PI3K/AKT pathway (Brown and Sacks 2008). This is called crosstalk, and is important in signaling pathways and will be discussed in more detail later (Fig. 7.4). 7.3.2.2

Negative Regulation of the RAS/Erk Pathway

Tyrosine phosphatases, such as PTP1B, lead to the dephosphorylation of the ErbB receptors and adaptor proteins such as Shc, which lead to a reduction in the RAS/Erk pathway. Dual specificity phosphatase (DSP) controls the MAPK signaling pathway through specifically dephosphorylating the tyrosine and threonine residues in MAPK proteins. The phosphatases are regulated by subcellular location, induction of expression and tissue distribution. The non-catalytic amino terminus domains of DSPs interact specifically with MAPK. In the RAS/Erk pathway, the MSK-3 phosphatase binds to Erk and dephosphorylates and inactivates it; however, MSK-3 fails to interact with and dephosphorylate JNK or p38. Also ErbB receptor activation of the RAS/Erk pathway increases the expression of MSK-3 which reduces activation of this pathway further (Wieduwilt and Moasser 2008).

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Fig. 7.4 RAS/Erk signaling pathway. Upon ligand binding, the RAS/Erk pathway is activated by the adaptor protein Grb2. Grb2 is recruited to the tyrosine phosphorylated ErbB1 through its Src homology 2 (SH2) domain. Grb2 then brings in the guanyl nucleotide-release protein SOS to the plasma membrane by Grb2 binding to SOS through its Src homology 3 (SH3) domain. SOS exchanges GDP for GTP in the small G protein RAS, activating it. The mitogen-activated protein kinase kinase kinase (MKKK) Raf then binds to the activated RAS. This activates Raf-1 which leads to the phosphorylation of the mitogen-activated protein kinase kinase (MKK) Mek1/2. Mek1/2 then activates the mitogen activated protein kinase (MAPK) Erk1/2 through phosphorylation. This leads to the activation of a variety of downstream signaling pathways including transcription factors, and ultimately to increased proliferation and cell survival

7.3.3

PI3K/AKT Pathway

Phosphoinositide 3-kinases (PI3K) belong to a conserved family of lipid kinases that phosphorylate the 3¢-hydroxyl group of phosphatidylinositides. The best known member of this family is phosphatidylinositol-3,4,5-triphosphate; this secondary messenger recruits the serine/threonine kinase AKT leading to its activation. This causes cellular proliferation and survival (Yuan and Cantley 2008).

7.3.3.1

ErbB Receptor Activation of the PI3K/AKT Pathway

ErbB receptors activate the PI3K pathway though the recruitment of PI3K to the plasma membrane. This occurs through binding of the SH2 domain of the p85 subunit of PI3K to tyrosine-phosphorylated ErbB receptors and primarily occurs on ErbB3 upon tyrosine phosphorylation. Alternatively, adaptor proteins such as Shc

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Fig. 7.5 PI3K/AKT signaling pathway. Upon ligand binding to ErbB1, the adaptor protein Shc binds to the tyrosine phosphorylated receptor through its SH2 domain. This recruits lipid kinase PI3K through tyrosine phosphorylation of Shc by ErbB1. Upon recruitment to the receptor, PI3K phosphorylates the phosphatidylinosides PI4,5P and PI4P. This generates a 3¢-phosphatidylinositol phosphate PI3,4,5P or PI3,4P (shaded). These are referred to as PIPs. PIPs bind to the pleckstrin homology (PH) domain in the serine threonine kinase AKT. This causes AKT to become phosphorylated and subsequently activated. Activated AKT phosphorylates multiple substrates including caspase 9, Bad and FOXO leading to their inactivation and subsequent increase in cell survival. AKT-mediated phosphorylation of mTOR, Creb, and NFkB leads to activation and subsequent increase in cell survival

bind tyrosine phosphorylated ErbB receptors and become tyrosine phosphorylated themselves. This allows recruitment of the SH2 domain in p85 subunit of PI3K to the receptor. The p110 catalytic subunit of PI3K then phosphorylates membranebound phosphatidylinositides (PI-4-P and PI-4,5-P2) generating 3¢-phosphatidylinositol phosphates (PI-3,4P2 and PI-3,4,5-P3 also called PIPs). PIPs act as high-affinity binding sites for proteins with pleckstrin homology domains (PH). Phosphoinositide-dependent kinase 1 (PDK1) and AKT both contain a PH domain that binds to phosphatidylinositides generated by PI3K. This binding allows both PDK and AKT to become localized to the plasma membrane where PDK1 phosphorylates a threonine residue in the kinase domain of AKT, thereby activating its kinase activity (Yuan and Cantley 2008) (Fig. 7.5). AKT, also called protein kinase B, is a serine threonine kinase that controls cell survival through phosphorylation of a variety of downstream targets. It has been shown that AKT signaling is central to ErbB receptor signaling. If the PI3K/AKT pathway is blocked, ErbB2 overexpressing cells become sensitive to undergo cell death (apoptosis). If ErbB1 activation is inhibited, AKT activation decreases and apoptosis increases. AKT substrates can be divided into three types: apoptotic proteins, transcription factors and protein kinases. By phosphorylating both BAD and caspase 9, which are pro-apoptotic proteins, AKT inhibits their apoptotic activity and promotes cell survival. The transcription factors that are targeted by AKT

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include NFkB, HIF-1a and CREB. These transcription factors are activated by AKT phosphorylation, and lead to an increase in the transcription of pro-survival genes (Franke 2008). NFkB is a transcription factor that plays an important role in immune, inflammatory and cell survival responses. ErbB receptor activation of AKT regulates NFkB activation by phosphorylating the inhibitor of NFkB kinase (IKK), which in turn phosphorylates IkB, targeting it for proteosomal degradation. NFkB, in its inactive state, is constitutively associated with IkB. Upon IkB degradation, NFkB translocates to the nucleus and induces gene expression of anti-apoptotic proteins. When NFkB activity is inhibited, the cells are sensitized to a wide variety of apoptotic stimuli (Gibson et al. 2002).

7.3.3.2

Negative Regulation of the PI3K/AKT Pathway

The lipid phosphatase PTEN is specific for the phosphate on the 3¢ position of the sugar ring of phosphatidylinositol 3,4,5 triphosphate and phosphatidylinositol 3,4 biphosphate. These lipid signaling molecules are downstream activators of AKT, and their down-regulation is critical in the normal functioning of the cellular signaling machinery. The important role PTEN plays in regulating the PI3K/AKT pathway is demonstrated in cancer where it is often mutated, leading to increased activation of the PI3K/AKT pathway and cell survival (Keniry and Parsons 2008).

7.3.4

JAK/STAT Pathway

The role of Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway has been studied extensively in cytokine receptor signaling. Engagement of the cytokine receptor activates the association of JAK, which then phosphorylates the receptor’s cytoplasmic domain allowing the recruitment of a STAT through its SH2 domain. This leads to the phosphorylation of STAT and subsequent homodimer formation. The dimer translocates to the nucleus and activates gene expression. It has been shown that STAT3 is constitutively up-regulated in many cancers and regulates transcription of many genes that are involved in proliferation, cell survival and angiogenesis. These genes include cyclin D1, c-Fos, c-Myc, Bcl-xL and VEGF (Henson and Gibson 2006).

7.3.4.1

ErbB Receptor Activation of the JAK/STAT Pathway

ErbB receptors activate the JAK/STAT pathway through binding of JAK and STAT to the tyrosine phosphorylated receptor. JAK2 and STAT3 are associated with activated ErbB1. Upon association, JAK2 tyrosine phosphorylates STAT3 causing the formation of a STAT3 homodimer that translocates to the nucleus

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Fig. 7.6 JAK/STAT pathway. JAK phosphorylates the ErbB receptor allowing recruitment and activation of a STAT. This phosphorylation leads to the formation of a STAT homodimer that translocates to the nucleus, activating gene transcription. STAT3 and ErbB1 have also been shown to form a complex that can translocate to the nucleus directly. This pathway leads to increased cell survival and proliferation

where it activates gene transcription. In addition to tyrosine phosphorylation of STAT3, serine phosphorylation of STAT3 also occurs, maximizing its transcriptional activity. Genes up-regulated by STAT3 include Bcl-2 family members Bcl-2, Bcl-xL and Mcl-1 as well as p21 and cyclin D1. STAT3 is involved in epithelial cell polarity and adhesion. Besides STAT3, STAT5 is activated in a similar manner and binds to activated ErbB1 or ErbB4 (Lo et al. 2008) (Fig. 7.6).

7.3.4.2

Negative Regulation of the JAK/STAT Pathway

STAT3 activates the transcription of a wide variety of genes that play a role in cell survival, proliferation, and cell cycle regulation. Negative regulation of this pathway is necessary to terminate these functions. The upstream suppressors of cytokine signaling (SCOS) inhibit JAK activity thereby blocking STAT activation. STATs also have proteins that specifically inhibit them – protein inhibitors of activated STATs (PIAS). These proteins are highly conserved and function by interfering with DNA binding and recruiting transcriptional co-repressors such as histone deacetylases (HDACs) to the promoters to inhibit gene transcription. Protein tyrosine phosphatases also play a role in regulating STAT3 activity, and STAT3 signaling is downregulated by the members of SH2-domain containing the tyrosine phosphatase family (SHP-1 and SHP-2) which directly dephosphorylate STAT3 (Wormald and Hilton 2004).

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Crosstalk with Other Signaling Pathways

The ErbB signaling network interacts with other receptor signaling networks including GPCR, integrin, Wnt, and other growth factor receptor pathways (Wieduwilt and Moasser 2008). In addition, signals induced by hormones, neurotransmitters, lymphokines, and stress inducers also crosstalk with ErbB receptors. An example of crosstalk between two different receptor signaling networks occurs when ligands such as lysophosphatidic acid (LPA), bombesin, angiotensin-II, thrombin or endothelin bind to their GPCR, leading to tyrosine phosphorylation of ErbB1 and ErbB2 at their C-terminus tails by non-receptor tyrosine kinases. Specifically, the non-receptor tyrosine kinase c-Src has been implicated in the transactivation of ErbB2 by LPA receptors. Besides G-coupled receptors, integrins, hormones and cytokine receptors have also been shown to activate non-receptor tyrosine kinases such as c-Src and JAK, leading to tyrosine phosphorylation of ErbB receptors. For example, growth hormone stimulation of JAK2 leads to the phosphorylation and activation of ErbB1 and prolactin activates JAK2 leading to phosphorylation and activation of ErbB2. ErbB receptors are also activated by nonErbB ligands that activate the ADAM family of matrix metalloproteinases. The matrix metalloproteinases cleave membrane-bound ErbB receptor ligands, freeing them to bind to their ErbB receptors, activating them further. The Frizzled ligands Wnt1 and Wnt5a can transactivate ErbB1 by stimulating the matrix metalloproteinase-dependent cleavage of membrane-bound ErbB receptor ligands. Furthermore, a6b4 integrin leads to up-regulation of ErbB2 and ErbB3 expression by activation of transcription factors (Ohtau et al. 2006). Increased levels of ErbB3 promote the formation of the preferred ErbB2/ErbB3 heterodimer and increased downstream signaling. Thus, ligand activation of ErbB receptors must be taken in context with other receptors and signaling pathways.

7.3.6

Nuclear Signaling by ErbB Receptor Family Members

ErbB receptor family members can translocate to the nucleus and effect gene transcription. Targeting to the nucleus requires three clusters of basic amino acids in the juxtamembrane domain of ErbB receptors. This targeting domain shares homology with known nuclear localization sequences (Wieduwilt and Moasser 2008). There may also be interactions between the receptors and proteins involved in nuclear importation, or the receptors could be internalized, and their endosomes targeted directly to the nuclear envelope. Full-length ErbB1, ErbB2 and ErbB3 have been found in the nucleus of cells. Furthermore, truncated and constitutively active ErbB2 and the truncated C-terminal form of ErbB4 have also been found in the nucleus. It has been shown that ErbB1 will translocate to the nucleus of breast cancer cells upon EGF treatment. Upon entering the nucleus, ErbB1 binds to DNA and acts as a transcriptional activator. It has been shown that increased levels of

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ErbB1 in the nucleus, correlates with poor prognosis in breast cancer patients (Scaltriti and Baselga 2006). Nuclear ErbB receptors lead to increases in the expression of genes that are important in cell survival and proliferation. These genes include cyclin D1, B-myb, cyclooxygenase-2 and the iNOS/NO pathway. In particular, nuclear ErbB1 binds to STAT3 and induces expression of the nitric oxide synthesis pathway, which contributes to cell survival. It also cooperates with E2F1 at the B-myb promoter, inducing its expression. B-myb activates cdc2, a G2-phase regulator. Its expression goes down at G0/early G1, and activated in mid to late G1 through S phase thereby regulating cell cycle and proliferation (Wieduwilt and Moasser 2008). This provides an alternative signaling pathway to cell surface ErbB receptor signaling.

7.3.7

Negative Regulation of ErbB Receptor Activation

Negative regulation of the ErbB receptors is critical since prolonged activation of these receptors leads to diseases such as cancer. ErbB receptor signaling can be immediately attenuated though dephosphorylation of a tyrosine residue in its C-terminal tail, inhibition of its tyrosine kinase activity and internalization of activated receptors followed by degradation (Wieduwilt and Moasser 2008). Tyrosine phosphatase PTP-1B dephosphorylates the C-terminal tails of ErbB receptors preventing binding of adaptor proteins. Proteins such as mitogeninduced gene six (MIG-6), also known as RALT, is up-regulated through the RAS/Erk pathway upon ErbB receptor stimulation, and acts as a negative feedback regulator through binding to the tyrosine kinase domain of the ErbB1 and ErbB2 receptors and inhibiting their kinase activity (Bublil and Yarden 2007; Gotoh 2008). There are also negative feedback loops in which receptors undergo ligand-induced internalization and ubiquitination. This targets the receptor to a pre-lysosomal compartment within the cell for degradation. ErbB1 is regulated by the E3 ubiquitin ligase, c-Cbl, which binds to a specific phosphotyrosine of ErbB1, enhancing ubiquitination, endocytosis and lysosomal degradation. In addition, heat shock proteins Hsp90 and Hsp70 act as negative regulators of ErbB2 through the E3-ubiquitin ligase CHIP, promoting ErbB2 degradation. Another form of negative regulation is sequestration of ErbB receptors and their ligands away from each other leading to attenuation of their signaling. For example, the ErbB2–ErbB3 dimer is inhibited by p85-s, a secreted protein consisting of the extracellular portion of ErbB3. p85-s inhibits neuregulin activation of ErbB2, ErbB3 and ErbB4 by sequestering the ErbB ligand away from its receptors (Citri et al. 2003). There is also an alternatively spliced version of ErbB2, herstatin, which contains a segment of the extracellular domain fused to a novel carboxylterminus. Herstatin binds to ErbB2 and inhibits heterodimerization and activation with ErbB3 (Azios et al. 2001). These mechanisms for negative regulation of ErbB receptor activation ensure an effective cellular response to receptor activation (Fig. 7.7).

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Fig. 7.7 Down-regulation of ErbB receptor signaling pathways. All pathways can be down-regulated by receptor endocytosis and subsequent degradation by proteosome or lysosome pathway. This is partially mediated by adaptor protein Cbl that binds to ErbB1 through its SH2 domain. MIG6 directly inhibits the ErbB1 tyrosine kinase activity. The RAS/Erk pathway is negatively regulated by tyrosine phosphatases such as PTP-1B that dephosphorylates the ErbB receptors and Msk-3, a DSP that acts directly on Erk1/2. The PI3K pathway is negatively regulated by the lipid phosphatase PTEN. Members of the SH2-domaincontaining tyrosine phosphatase family, SHP-1 and SHP-2, negatively regulate the JAK/STAT pathway through de-phosphorylation of STAT3

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ErbB Receptors and Hallmarks of Cancer

Cancer is a disease of deregulation, and involves dynamic changes in the genome. Tumorigenesis in humans is a multi-step process, with genetic alterations that drive the transformation of cells from normal to malignant derivatives. Tumor cells show multiple genetic aberrations at a variety of sites. The aberrations vary from simple point mutations to changes in the number of chromosomes. Each change on the path to a malignant phenotype provides the cells with a growth advantage until a normal cell has been converted into a cancer cell. The hallmarks of cancer include growth factor autonomy, evasion of apoptosis, insensitivity to anti-growth signals, unrestricted cell proliferation, tissue invasion, and sustained angiogenesis. There are three primary hallmarks of cancers that are associated with deregulation of the ErbB receptor family members: overexpression or mutation of ErbB receptors that transduce growth factor independent signaling, alteration of downstream cell signaling machinery leading to evasion of apoptosis (cell survival), and unrestricted proliferation (Hanahan and Weinberg 2000).

7.4.1

Deregulation of ErbB Signaling Pathways in Cancer

Deregulation of ErbB receptors is associated with a variety of human cancers, including breast, head and neck, lung, and glioma. High ErbB1 expression is found in the majority of epithelial derived cancers, and 30% of all human solid tumors. Deregulation of the ErbB receptors can begin with the amplification of a gene, and overexpression of the protein product leading to aberrant function. Overexpression may enable hyperresponsiveness to concentrations of ligand that would not normally trigger a proliferative response. When ErbB1 is overexpressed, receptor activation leads to downstream signaling cascades, excessive cell division, and the formation of tumors. ErbB2 overexpression plays a role in most epithelial-derived tumors. ErbB2 is ligand independent, and plays a significant role in signaling due to its ability to form dimers with all members of the ErbB family. Additionally, ErbB2 is the preferred binding partner for all other ErbB members. The amplification status of ErbB3 has not been extensively investigated in malignancies, but its expression is often increased in cancers. New evidence suggests that ErbB3 may play a role in resistance to targeted therapies by promoting cell survival. In contrast, it seems that ErbB4 is anti-proliferative, and as such, overexpression of ErbB4 is not seen in malignancies (Hsieh and Moasser 2007). Interestingly when high throughput binding assays were done on ErbB receptors, a large set of putative binding proteins were identified. Furthermore, it has been shown that both ErbB1 and ErbB2 can recruit a wide range of signaling molecules when the affinity threshold is lowered. This provides an intriguing possibility that when the receptors are deregulated and overexpressed as they are in cancer, both the strength and the diversity of the signaling pathways activated are changed (Bublil and Yarden 2007) (Fig. 7.8).

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Fig. 7.8 Deregulation of ErbB signaling pathways in cancer. The hallmarks of cancer include growth factor autonomy and evasion of apoptosis, insensitivity to anti-growth signals, unrestricted cell proliferation, tissue invasion and sustained angiogenesis. In cancers where ErbB family members are playing a role, receptors are overexpressed or mutated leading to growth factor-independent signaling. This increases the downstream kinase cascade, altering downstream cell signaling machinery leading to up-regulation of cell survival proteins and down-regulation of cell death proteins. Furthermore, there is an increase in proliferation and a disruption in the cell cycle checkpoints

7.4.2

Cancers Where ErbB Receptor Family is Overexpressed and Mutated

7.4.2.1

Breast Cancer

Breast cancer affects one in nine women. The five-year survival rate for breast cancer is approximately 85%. At diagnosis, treatment decisions and prognostic information are gleaned from the levels of estrogen receptor (ER). Women who are negative for ER have a poor prognosis since they will not respond to anti-estrogen therapies. Of that group, a subset of approximately 30% has amplification of the ErbB2 gene leading to overexpression of ErbB2. This group of patients has a very poor prognosis, with shorter disease-free survival, resistance to chemotherapy and increased distant metastasis due to increased RAS/Erk and PI3K/AKT signaling (Hicks and Kulkarni 2008). 7.4.2.2

Squamous Cell Carcinoma of the Head and Neck (SCCHN)

Squamous cell carcinoma of the head and neck (SCCHN) tends to be aggressive, with a five-year survival rate of approximately 50%. ErbB1 expression is critical in

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the pathogenesis of head and neck cancers (SCCHN), and is found in over 90% of tumors (Rogers et al. 2005). ErbB2 has not been found to contribute to pathogenesis in SCCHN, but may play a role in progression. It has been shown that the higher the density of ErbB1 receptor expression in SCCHN cells, the lower the sensitivity to radiation. One possible mechanism for this is that inhibitory phosphatases that act on the ErbB1 receptor are repressed by radiotherapy (Reuter et al. 2007).

7.4.2.3

Lung Cancer

Lung cancer has become the leading cause of cancer deaths worldwide. Approximately 85% of people diagnosed with lung cancer will die of their disease in the first five years. It is a heterogeneous disease, with several different tumor types, the most common being non-small cell lung carcinoma (NSCLC). Each tumor type has a different treatment regime and prognostic outcome. Both ErbB1 and ErbB2 are overexpressed in most tumor types and ErbB1 has mutations that increase its kinase activity in a subset of patients (Molina et al. 2008).

7.4.2.4

Glioblastoma Multiforma

The most common primary intracranial malignancy is high-grade gliomas. Glioblastoma (GBM) is a grade IV tumor, and is the most lethal of all the brain tumors. The current standard of care is treatment with temozolomide either sequentially or concurrently with radiation therapy (Mrugala and Chamberlain 2008). Despite ongoing research efforts, the median survival of patients with glioblastoma is only 15 months, and few patients survive beyond three years. ErbB1 is amplified in 40–50% of GBMs and overexpressed in more than 60% of patients. ErbB1 is often mutated in GBMs, and the most common mutation is EGFRvIII, which lacks a portion of the extracellular receptor domain. This mutant protein is ligand independent and constitutively phosphorylated (Rowinsky 2004). EGFRvIII-positive GBM tumors are associated with a poor prognosis and resistance to therapy. Up to 50% of gliomas have either mutated or overexpressed ErbB1. Interestingly, 40% of patients with amplification of ErbB1 have the constitutively active mutation EGFRvIII; however, no mutations in the tyrosine kinase domain have been detected in GBM patients (Brandes et al. 2008).

7.4.2.5

Other Malignancies Where ErbB Receptors May Playing a Role

There are several other malignancies where ErbB receptors are altered. These include esophageal, gastric, colorectal, pancreatic, liver, endometrial, ovarian and prostate cancer (Uberall et al. 2008). The role of ErbB receptors in these cancers remains to be fully understood.

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7.4.3

Targeting ErbB Receptor Family Overexpression for Therapy

7.4.3.1

Targeting ErbB Receptor Overexpression Using Antibody Derived Therapies

Monoclonal antibodies against the ErbB family of receptors have been extensively studied and they have evolved as a targeted therapy against cancer cells. These monoclonal antibodies could have multiple mechanisms of action, including interaction with components of the immune system through antibody-dependent cellular toxicity or complement-dependent cytotoxicity, or the elimination of a critical cell surface antigen. The monoclonal antibodies developed against the ErbB family of receptors are highly effective at attenuating or altering downstream signal transduction pathways. They do so by either blocking the interaction between the receptor and its activating ligand, or by preventing receptor heterodimerization. Virtually every clinically effective unconjugated mAb disrupts the signaling that promotes proliferation and the survival of the targeted cell population (Adams and Weiner 2005). This makes ErbB receptors an ideal target for this type of therapy. The first targeted monoclonal antibody against the ErbB receptor family was the ErbB2 targeting antibody, trastuzumab (Herceptin), FDA approved in 1998. Herceptin is used in the treatment of ErbB2 over-expressing breast cancers, in combination with traditional chemotherapies such as paclitaxel. Herceptin increases disease-free survival, and causes tumor regression. The mechanism of action is still not well understood, but Herceptin binds to ErbB2 in the juxtamembrane region of the extracellular domain and often causes internalization and degradation of the receptor. A second antibody that targets the dimerization portion of the extracellular domain of ErbB2 has been developed, pertuzumab, which is in ongoing clinical trials. Monoclonal antibodies against ErbB1 have also been developed, cetuximab (Erbitux) and panitumumab (Vectibix). Both bind the extracellular domain of ErbB1 and have been approved for clinical use (Wieduwilt and Moasser 2008) (Fig. 7.9).

7.4.3.2

Targeting ErbB Receptor Overexpression Using Tyrosine Kinase Inhibitors

The tyrosine kinase function of the ErbB family of receptors is necessary for their activity, making it a natural target for therapeutics. A challenge in designing these therapies is that the kinase domain of ErbB receptors shares significant homology with other human tyrosine kinases. However, targeting the quinazoline structure has proven to be highly selective for the ErbB receptor family members. Three compounds are used in the clinic, gefitinib (Iressa), erlotinib (Tarceva), and lapatinib (Tykerb). Iressa and Tarceva target ErbB1, whereas Tykerb targets both

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Pertuzumab Cetuximab

CR1

ErbB1 L1

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CR2

L2

L1

CR 1

L2 Matuzumab

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Gefitinib Erlotinib

tyrosine kinase domain

CI-1033

Lapatinib

Labatinib

tyrosine kinase domain

CI-1033 EKB-569

EKB-569

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Targeted antibody

Fig. 7.9 Targeting the ErbB1 and ErbB2 receptors. Targeted therapies to ErbB1 and ErbB2 have been developed. There are two main types, the targeted antibody and the TKI. Cetuximab, matuzumab, panitumumab are antibodies that target the L2 region of ErbB1. Pertuzumab is an antibody that targets the dimerization loop in the CR1 region of ErbB2 while the antibody, trastuzumab targets the juxtamembrane region. Tyrosine kinase inhibitors gefitinib and erlotinib target the tyrosine kinase region of ErbB1. Labatinib, CI-1033, and EKB-569 target the kinase domains of both ErbB1 and ErbB2 (Rogers et al. 2005)

ErbB1 and ErbB2. Tyrosine kinase inhibitors (TKIs) bind competitively within the ATP pocket, blocking downstream signaling. A challenge has been to overcome the high intracellular concentrations of ATP, which necessitate higher concentrations of reversible inhibitors to maintain continuous blockage of phosphorylation. This has been the rationale behind developing TKIs that bind irreversibly in the ATP binding site of the receptor. There are two that are currently being investigated, CI-1033 and EKB-569, which inhibit both ErbB1 and ErbB2 (Rowinsky 2004). The types of cancer ErbB receptor-targeted therapies that have been investigated in clinical trials are listed in Table 7.1.

7.4.4

Role of Mutations in the ErbB Receptor in Targeted Therapies

7.4.4.1

ErbB1 Truncation Mutation, EGRFVIII

Response to the TKIs gefitinib (Iressa) or erlotinib (Tarceva) in recurrent glioma is poor, though a response has been noted in patients that co-express EGFRvIII and PTEN. In addition, ErbB1-targeted drugs, matuzumab and cetuximab, fail

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Table 7.1 Cancers with EGF pathway deregulation and their targeted therapies. Targeted therapies against ErbB receptor-driven cancers are in various stages of clinical trial. Therapies that have been approved for treatment as of 2008 are shown in italics Cancer Deregulation Targeted therapy Head and neck ErbB1 Cetuximab, gefitinib, erlotinib Glioma ErbB1, mutant subtype EGFRVIII Gefitinib, erlotinib Cetuximab, gefitinib, Lung (NSCLC) ErbB1, mutations in ErbB1 erlotinib, panitumumab, correlated to response to pertuzumab, matuzumab, therapy CI-1033, EKB-569 Trastuzumab, pertuzumab, Breast ErbB1 and ErbB2. ErbB2 is lapatinib, CI-1033 strongly correlated to higher grade and poor prognosis Gastric ErbB1 marker of poor prognosis Cetuximab, panitumumab ErbB1 and/or ErbB2 Pertuzumab (ovarian, prostate), Colorectal, cetuximab (pancreatic), pancreatic, matuzumab (gynecological, hepatocellular, pancreatic) prostate, ovarian, endometrial

to bind to EGFRVIII rendering them inappropriate for treating EGFRVIII-positive tumors. Research efforts have been focused on targeting tumors with the EGFRvIII variant, which lacks exons 2–7, since this is only found in malignant tissue, making it an ideal target. Early clinical trials suggest that these vaccines might need to be used in the context of combinational therapies since not all cells in an EGFRvIIIpositive tumor express the mutant variant (Sonabend et al. 2007). GBMs represent a significant clinical challenge, with tumor resistance and treatment failure being the inevitable outcome. The mechanisms of tumor resistance are poorly understood, and are probably a complex interaction between multiple redundant pathways downstream of ErbB receptors including, but not limited to, constitutive PI3K/ATK signaling, PTEN loss, and the activation of downstream AKT target mTOR (Sonabend et al. 2007).

7.4.4.2

Mutations in the Tyrosine Kinase Domain

Targeted therapies showed early promise; however, in several phase three clinical trials gefitinib (Iressa) vs. placebo failed to show a survival benefit. Erlotinib showed a statistically significant clinical benefit, and has been approved by the FDA for use in patients with locally advanced or metastatic NSCLC who have failed first-line therapies. Studies showed that there was a subgroup of patients, specifically ones who were female, had never smoked, with adenocarcinoma, or were Asian, who had a better response to gefitinib or erlotinib (Jimeno and Hidalgo 2005). This prompted further investigations into the molecular

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mechanisms involved. NSCLC shows different mutational frequencies between different populations, with activation mutations found in 10–15% of cases in the North America and Europe, and 30–50% of cases in Asia. Lung cancers with mutations in the kinase domain (KD) of ErbB1 are highly responsive to TKIs. Data from in vitro studies showed that KD mutations enhance coupling of the mutant receptor to pro-survival pathways. Clinically, patients with mutations in their kinase domain show increased ErbB1 copy numbers, which suggests that these mutant ErbB1 alleles are selectively amplified and perhaps are required for NSCLC survival. Tumor cells with kinase domain mutations in ErbB1 preferentially activate the PI3K/AKT pathway, as well as the JAK/STAT pathway. Interestingly, mutant ErbB1 does not bind the p85 subunit of PI3K. Instead, it activates the pathway through adaptor protein GAB1, which then binds another adaptor protein, Grb2. ErbB2 mutations may be clinically relevant: when ErbB2 KD mutants were introduced into normal human breast and lung cells, the mutant ErbB2 was constitutively active and conferred oncogenic properties on the cells. Furthermore, in these cells, both ErbB1 and multiple downstream signaling pathways were activated. In the treatment of SCCHN, it has been shown that resistance to radiation may be reversed by treatment with either ErbB1 receptor-targeted monoclonal antibody cetuximab, or the TKIs gefitinib (Iressa) or erlotinib (Tarceva). It is interesting to note that, unlike lung cancer, in SCCHN there are no activating mutations in ErbB1 that can be used to predict response to therapy. Whether amplification of ErbB1 can be used as a predictor of response to therapy is currently under investigation (Ladanyi and Pao 2008). 7.4.4.3

Targeting ErbB Signaling Networks for Combinational Therapies

The ErbB signaling pathways have developed networks that buffer mutational changes allowing the signaling system to continue to function properly. Activation of ErbB receptors may lead to the accumulation of genetic abnormities by preventing cell death after mitotic failure (Zhang et al. 2007). Mutations in ErbB receptors could also lead to changes in signaling patterns. G-coupled protein agonists have been shown to transactivate ErbB receptors in normal and cancer cells. Furthermore, in some cancers, particularly prostate cancer, the deregulation of G-coupled proteins and their ligands has been linked to tumor development. Of note, ErbB1 activation has been well documented in prostate tumors, suggesting a possible link between these different types of receptors. The ErbB receptor family is part of a complex signaling network. By targeting proteins that interact directly with ErbB receptors, inhibition of their function may be achieved. Hsp90 stabilizes ErbB2 at the cell membrane, and patients with higher levels of Hsp90 have a poor prognosis. If Hsp90 is inhibited with geldanamycin, ErbB2 is degraded, attenuating the signal (Wieduwilt and Moasser 2008). ADAM family metalloproteinases cleave EGF ligands from the cell membrane, activating the pathway. These proteins have been shown to be responsible for TKI resistance

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in lung cancer, by increasing ligand concentrations, and activating the ErbB1– ErbB3 dimer. If ADAM17 is targeted in NSCLC using a selective inhibitor INCG3619, sensitivity to gefitinib is restored (Wieduwilt and Moasser 2008). Thus, understanding changes in signaling networks within cancer cells will provide rationale for combinational treatment within the ErbB signaling network (Fischgrabe and Wulfing 2008).

7.4.5

Mechanisms of Resistance to ErbB Targeted Therapy

Similar to conventional chemotherapy, cancer cells treated with ErbB receptortargeted therapies develop drug resistance. This resistance could be intrinsic to the cancer cell or acquired during treatment. For example, Herceptin response rates in breast cancer patients overexpressing ErbB2 is approximately 35%. This resistance could be due to alteration in ErbB receptor ligand regulation either by autocrine or paracrine mechanisms. Herceptin fails to block ErbB2 dimerization with other members of the family and subsequent signaling events. This allows ErbB receptor ligands to maintain ErbB signaling in the presence of Herceptin. Another antibody against ErbB2, pertuzumab, binds to ErbB2 and prevents dimerization indicating effectiveness in tumors with low ErbB2 expression. Currently these antibody-based therapies are being investigated in combination in clinical trials to prevent drug resistance. Other mechanisms of resistance to Herceptin therapy are increased ErbB2 expression through enhanced protein stability, reduced degradation and increased recycling of ErbB2 to the plasma membrane. In addition, it has been observed that ErbB2 is found in the serum of patients and could sequester Herceptin away from ErbB2-expressing cancer cells. Similar to Herceptin resistance, cancer cells treated with TKIs against ErbB receptors often are drug resistant. Mutations in the ErbB1 receptor (EGFRvIII) are often found in cancers and confer resistance to gefitinib and cetuximab. Resistance also occurs downstream of ErbB receptors. Mutations in PTEN allow increased activation of the PI3K/ AKT pathway, which renders cancer cells resistant to ErbB receptor TKIs. In addition, up-regulation of other receptor tyrosine kinases could give resistance to TKI by activating downstream pathways such as the PI3K/AKT pathway independent of ErbB receptors. These receptors include the insulin growth factor receptors (IGFR) and c-Met receptors (Hynes and Lane 2005). Similar to ErbB1 and ErbB2, ErbB3 is also overexpressed in cancer cells but is not directly targeted by antibody or TKI-based therapies. Indeed, ErbB receptor TKIs only transiently suppress the transphosphorylation of ErbB3 suggesting other receptor tyrosine kinases phosphorylate ErbB3. In fact, amplification of the tyrosine kinase receptor c-Met can restore ErbB3 signaling through tyrosine phosphorylation of its C-terminal tail leading to activation of the PI3K/AKT pathway (Wieduwilt and Moasser 2008). This illustrates the requirement for combinational therapy to treat ErbB receptorexpressing cancers.

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Concluding Remarks

ErbB receptors are a family of receptor tyrosine kinases that provide multiple functions in development, cellular proliferation and cell survival. They activate a network of signal transduction pathways leading to changes in gene transcription. These pathways are tightly regulated by phosphorylation status, subcellular localization of receptors, and formation of protein complexes. This dynamic signaling network ensures the cell response to ErbB receptor ligands leading to the appropriate cellular function. When this network is deregulated, diseases such as cancer can occur. This happens when overexpression of ErbB receptors, and/or mutations in ErbB receptors takes place, leading to changes within cells that represent hallmarks of cancer (evasion of apoptosis, proliferation and ligand-independent signaling). Targeted therapies against ErbB receptors have been shown to be effective but resistance remains a major issue. The use of ErbB receptor inhibitors in combination with inhibitors of the ErbB signaling network gives the promise for effective therapies for ErbB-expressing cancers in the future.

References Adams GP, Weiner LM (2005) Monoclonal antibody therapy of cancer. Nat Biotechnol 23:1147–1157 Azios NG, Romero FJ, Denton MC, Doherty JK, Clinton GM (2001) Expression of herstatin, an autoinhibitor of HER-2/neu, inhibits transactivation of HER-3 by HER-2 and blocks EGF activation of the EGF receptor. Oncogene 20:5199–5209 Bazley LA, Gullick WJ (2005) The epidermal growth factor receptor family. Endocr Relat Cancer 12(Suppl 1):S17–27 Brandes AA, Franceschi E, Tosoni A, Hegi ME, Stupp R (2008) Epidermal growth factor receptor inhibitors in neuro-oncology: hopes and disappointments. Clin Cancer Res 14:957–960 Brown MD, Sacks DB (2008) Compartmentalized MAPK pathways. Handb Exp Pharmacol 186:205–235 Bublil EM, Yarden Y (2007) The EGF receptor family: spearheading a merger of signaling and therapeutics. Curr Opin Cell Biol 19:124–134 Citri A, Skaria KB, Yarden Y (2003) The deaf and the dumb: the biology of ErbB2 and ErbB3. Exp Cell Res 284:54–65 Fischgrabe J, Wulfing P (2008) Targeted therapies in breast cancer: established drugs and recent developments. Curr Clin Pharmacol 3:85–98 Franke TF (2008) Intracellular signaling by AKT: bound to be specific. Sci Signal 1:pe29 Gibson EM, Henson ES, Villanueva J, Gibson SB (2002) Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release. Cancer Res 62:488–496 Gotoh N (2008) Feedback inhibitors of the epidermal growth factor receptor signaling pathways. Int J Biochem Cell Biol 41:511–515 Hanahan D, Weinberg RA (2000) The Hallmarks of Cancer. Cell 100:57–70 Henson ES, Gibson SB (2006) Surviving cell death through epidermal growth factor (EGF) signal transduction pathways: implications for cancer therapy. Cell Signal 18:2089–2097 Hicks DG, Kulkarni S (2008) HER2+ breast cancer: review of biologic relevance and optimal use of diagnostic tools. Am J Clin Pathol 129:263–273

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Hsieh AC, Moasser MM (2007) Targeting HER proteins in cancer therapy and the role of the nontarget HER3. Br J Cancer 97:453–457 Hubbard SR (2005) EGF receptor inhibition: attacks on multiple fronts. Cancer Cell 7:287–288 Hynes NE, Lane HA (2005) ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 5:341–354 Jimeno A, Hidalgo M (2005) Promises and pitfalls in the prediction of antiepidermal growth factor receptor activity. Expert Rev Anticancer Ther 5:727–735 Keniry M, Parsons R (2008) The role of PTEN signaling perturbations in cancer and targeted therapy. Oncogene 27:5477–5485 Ladanyi M, Pao W (2008) Lung adenocarcinoma: guiding EGFR-targeted therapy and beyond. Mod Pathol 21:S16–S22 Lo H, Cao X, Zhu H, Ali-Osman F (2008) Constitutively activated STAT3 frequently coexpresses with epidermal growth factor receptor in high-grade gliomas and targeting STAT3 sensitizes them to Iressa and alkylators. Clin Cancer Res 14:6042–6054 Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA (2008) Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 83:584–594 Mrugala MM, Chamberlain MC (2008) Mechanisms of Disease: temozolomide and glioblastoma - look to the future. Nat Clin Pract Oncol 5:476–486 Ohtau H, Dempsey PJ, Eguchi S (2006) ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors. Am J Physiol Cell physiol 291:C1–C10 Reuter CW, Morgan MA, Eckardt A (2007) Targeting EGF-receptor-signalling in squamous cell carcinomas of the head and neck. Br J Cancer 96:408–416 Rogers SJ, Harrington KJ, Rhys-Evans P, P OC, Eccles SA (2005) Biological significance of c-erbB family oncogenes in head and neck cancer. Cancer Metastasis Rev 24:47–69 Rowinsky EK (2004) The erbB family: targets for therapeutic development against cancer and therapeutic strategies using monoclonal antibodies and tyrosine kinase inhibitors. Annu Rev Med 55:433–457 Scaltriti M, Baselga J (2006) The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res 12:5268–5272 Sonabend AM, Dana K, Lesniak MS (2007) Targeting epidermal growth factor receptor variant III: a novel strategy for the therapy of malignant glioma. Expert Rev Anticancer Ther 7:S45–S50 Uberall I, Kolar Z, Trojanec R, Berkovcova J, Hajduch M (2008) The status and role of ErbB receptors in human cancer. Exp Mol Pathol 84:79–89 Wieduwilt MJ, Moasser MM (2008) The epidermal growth factor receptor family: biology driving targeted therapeutics. Cell Mol Life Sci 65:1566–1584 Wormald S, Hilton DJ (2004) Inhibitors of cytokine signal transduction. J Biol Chem 279:821–824 Yuan TL, Cantley LC (2008) PI3K pathway alterations in cancer: variations on a theme. Oncogene 27:5497–5510 Zhang H, Berezov A, Wang Q, Zhang G, Drebin J, Murali R, Greene MI (2007) ErbB receptors: from oncogenes to targeted cancer therapies. J Clin Invest 117:2051–2058

Chapter 8

Leptin Signaling Pathway Hiroyuki Shimizu

8.1

Introduction

Leptin was discovered in 1994 in genetically obese (ob/ob) mice (Zhang et al. 1994). Since the discovery of leptin, many investigations have been involved in studying the mechanism of leptin action in the brain and periphery. Leptin is one of the most important anorexigenic peptides related to the brain–adipose axis (Shimizu and Mori 2005). It is synthesized and released from adipose tissue, and reaches the brain through the blood–brain barrier (BBB). It inhibits appetite, and enhances sympathetic activities, resulting in the reduction of body weight (Hallas et al. 1995, Campfield et al. 1995). The anorexigenic effects of leptin are mediated by both neuropeptide Y-containing neurons and pro-opiomelanocortin (POMC)containing neurons in the arcuate nucleus of the hypothalamus (Schwartz et al. 1996a; Thornton et al. 1997; Haekansson et al. 1998; Yaswen et al. 1999). Clinical investigations demonstrated that the loss of leptin signaling causes severe obesity in humans (Montague et al. 1997; Clement et al. 1998). Leptin administration reduces appetite, and reduces body weight in leptin-deficient obese animals and humans (Halaas et al. 1995; Licinio et al. 2004). Circulating leptin concentration shows a strong, positive correlation with the body mass index, percentage of body fat, and total body fat weight. The level is significantly higher in obese people, regardless of the distribution of adiposity in the body (McGregor et al. 1996; Shimizu et al. 1997b). However, leptin fails to inhibit appetite in obese people. A clinical trial using leptin demonstrated that serum leptin concentrations 20–30 times higher than physiological should be necessary for a significant reduction of body weight in moderately obese subjects (Heymsfield et al. 1999). Such clinical results clearly demonstrate that appetite-suppressing effects of leptin

H. Shimizu Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma, Japan e-mail: [email protected] A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_8, © Springer-Verlag Berlin Heidelberg 2010

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are diminished in obese subjects, and that obese subjects are resistant to endogenous and exogenous leptin. Since the improvement of leptin sensitivity is an important key point for the treatment of obesity, the mechanism of leptin resistance has been under investigation for a long time. In this chapter current knowledge about the leptin signaling mechanism in the hypothalamus is summarized from the standpoint of central and peripheral leptin resistance, in addition to the leptin signaling pathway in the peripheral tissues.

8.2 8.2.1

Leptin Signaling in the Brain Animal Models and Leptin Resistance

In the brain it has been suggested that there may be two kinds of leptin resistance from the standpoint of mechanism: central and peripheral resistance. The mechanism by which leptin resistance may be caused can be divided into three steps: transport across the BBB, leptin receptor abnormalities, and post-receptor abnormalities. The disturbance of leptin transport across the BBB is the cause of the peripheral leptin resistance, and the others are those of central resistance. The mechanism of leptin resistance in animal models of obesity is summarized in Table 8.1. It is well known that high-fat diets cause obesity in humans and many animal models. Leptin resistance is associated with diet-induced obesity (DIO) (Widdowson et al. 1997). In DIO, both central (impaired leptin signal transduction) and peripheral (an impaired ability to cross the BBB) mechanisms may contribute to the development of leptin resistance (El-Haschimi et al. 2000). However, diet-induced obese AKR mice show a resistance only to peripheral, but not to central administration Table 8.1 Supposed mechanism of leptin resistance in animal model of obesity Peripheral resistance Central resistance 1. Dietary High-fat diet n–3 PUFA 2. Genetic

(+) (+)

(+) (−)

Diabetic (db/db) obese mouse Zukcer fatty (fa/fa) rat New Zealand obese mouse Osborne–Mendel rat 3. Others

(−) (−) (+) (+)

(+) (+) (−) ?

Aging Hyperleptinemia Continous central leptin infusion

(+) (−) ?

(+) (+) (+)

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of leptin (Van Heek et al. 1997). There may be a strain difference in the mechanism of the development of high-fat-induced leptin resistance. Leptin gene expression increases with age, and in spite of high leptin concentrations, older rats become obese (Li et al. 1997). Therefore, it is thought that agerelated leptin resistance exists (Li et al. 1997). Aged F344×BN rats show both peripheral and central leptin resistance (Zhang and Scarpace 2006). Impaired transport of leptin across the BBB of old CD-1 mice develops with obesity, and is reversible with modest weight reduction (Banks and Farrell 2003). Genetic animal models of obesity also show leptin resistance by different mechanisms. Genetically diabetic (db/db) mice and Zucker fatty (fa/fa) rats have genetic abnormalities in leptin receptors, leading to development of central leptin resistance (Chen et al. 1996; Phillips et al. 1996). In the Koletsky rat, the transport of intravenous leptin across the BBB is greatly reduced, indicating the existence of peripheral leptin resistance (Banks et al. 2002). Intracerebroventricular administration of recombinant mouse leptin inhibits food intake, in spite of no anorexigenic response to peripherally administered leptin in New Zealand obese (NZO) mice (Halaas et al. 1997). Therefore, NZO mice also show peripheral leptin resistance, in which leptin transport to the brain is thought to be disturbed.

8.2.2

Peripheral Leptin Resistance

Leptin is transported across the BBB, and it is thought that a leptin receptor may mediate its transport, because there is a marked decrease in the transport rate into the brain in rats lacking all leptin receptor isoforms (Kastin et al. 1999). Genetically diabetic (db/db) mice that lack only long-type leptin receptor (Ob-Rb) but have intact short-type receptor showed normal leptin transport rates into the brain (Maness et al. 2000). However, neither NZO nor DIO mice with peripheral leptin resistance exhibited significant decreases in short-form leptin receptor (Ob-Ra) mRNA expression in isolated cerebral microvessels, indicating no involvement of leptin receptor abnormalities in the leptin insensitivity of those models (Hileman et al. 2002). Circulating soluble leptin receptor (Ob-Re) levels are negatively correlated with the body mass index, and are significantly lower in obese subjects (Shimizu et al. 2002). Overexpression of soluble leptin receptor enhances the weight-reducing effect of leptin in genetically obese (ob/ob) mice (Huang et al. 2001). These data indicate that soluble leptin receptors may be involved in the determination of leptin sensitivity. Soluble receptors of IL-6 and leptin may enhance the anorexigenic effects of their ligands, possibly by enhancing its receptor binding or accelerating the transport of its ligand across the BBB (Schobitz et al. 1995). There is a possibility that the reduction of circulating Ob-Re in obese subjects may partially contribute to the leptin insensitivity. It has been reported that the ratio of cerebrospinal fluid to serum leptin concentrations is relatively low in obese patients (Schwartz et al. 1996a,b; Caro et al.

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1996). This clinical observation indicates that the transport of leptin to the central nervous system is disturbed in obese patients, and is important in the leptin resistance of humans. The ratio of cerebrospinal fluid to serum leptin concentrations was significantly higher in the S5B/Pl rats, which are resistant to DIO and sensitive to exogenous leptin administration, than in Osborne–Mendel rats which are susceptible to DIO and relatively resistant to leptin (Ishihara et al. 2004). Nutritional status appears to contribute to the development of peripheral leptin resistance. Fetal malnutrition shows impaired response to acute peripheral leptin administration with neonatal leptin surge (Yura et al. 2005). In both fetal malnutrition mice and mice with premature leptin surge caused by exogenous leptin administration, the transport of leptin into the brain was disturbed, because these models are sensitive to central leptin administration. The mRNA expression of the shortform leptin receptor (Ob-Ra) is significantly reduced in these models, indicating the possible involvement of Ob-Ra in the disturbed leptin transport across the BBB caused by fetal malnutrition. On the other hand, triglycerides (TG) induce leptin resistance at the BBB (Banks et al. 2004). Both starvation and DIO elevate serum TG concentration, and decrease the transport of leptin across the BBB, whereas short-term fasting decreases serum TG concentration and increases the transport. Treatment with gemfibrozil reverses both hypertriglyceridemia and impaired leptin transport. TG also contribute to the lipopolysaccharide-induced reduction of leptin transport across the BBB without any change of Ob-Ra mRNA expression in isolated brain microvessels (Nonaka et al. 2004). These data indicate an importance of circulating TG concentration in peripheral leptin resistance. In NZO mice with peripheral leptin resistance, hypothalamic leptin receptor mRNA is expressed as abundantly as in lean mice, and changes in leptin receptor expression do not play an important role in the development of peripheral leptin resistance in NZO mice (Igel et al. 1997). However, the ratio of n–3 polyunsaturated fatty acid (PUFA) to n–6 PUFA was significantly higher in NZO mice than New Zealand black (NZB) controls due to the changes associated with lipid metabolism-related enzyme expression in adipose tissue (Takahashi et al. 2001). Circulating n–3 PUFA levels are significantly higher in massively obese subjects (Oh-I et al. 2005). Therefore, we examined the role of n–3 PUFA in the development of peripheral leptin resistance (Oh-I et al. 2005). Only n–3 PUFA blocked the anorexigenic effect of intraperitoneally administered leptin, but other fatty acids failed to show a significant attenuation of the leptin effects. However, intracerebroventricularly administered leptin significantly inhibited feeding behavior in n–3 PUFA-fed animals. Cerebrospinal leptin concentration was significantly lower in n–3 PUFA-fed animals despite circulating leptin concentrations being no lower than in the control animals, indicating that the leptin transport to the brain was disturbed in these animals. This was additionally confirmed by leptin transport assays using human leptin in rats. These results indicated that n–3 PUFA caused peripheral leptin resistance. It is known that proteins are transported across the BBB by two possible mechanisms; transcellular and paracellular pathways (Fig. 8.1) (Broadwell et al. 1988; Quagliarello et al. 1991). However, the exact mechanism of the leptin transport

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Leptin

Vessel

Trans-cellular transport

Para-cellular transport

Endothelial Cells

Ob-R

Tight junction protein

Central Nervous System

Fig. 8.1 Leptin transport system across the BBB between the hypothalamic endothelial cells

system into the brain has not been fully clarified. To investigate the exact mechanism of peripheral leptin resistance by n–3 PUFA, we measured expressions of leptin receptors, intracellular signaling proteins, and hypothalamic tight junction proteins. Expressions of leptin receptors and intracellular signaling proteins was not changed. Only hypothalamic expression of occludin, one of the tight junction proteins, was increased by n–3 PUFA. It was confirmed that occludin expression was increased in vascular fraction isolated from rat hypothalamus fed with n–3 PUFA. Next, we examined whether the reduction of hypothalamic occuludin expression may recover the anorexigenic effects of peripherally administered leptin by using morpholino-oligonucleotide antisense against occludin. Intraperitoneally administered leptin significantly inhibited food consumption in n–3 PUFA-fed rats treated with morpholino-oligonucleotide antisense against occludin. These data strongly indicate that occludin plays an important role in the induction of peripheral leptin resistance by n–3 PUFA. In addition, hypothalamic occludin expression increased in high-fat diet-fed mice, but was not changed in genetically diabetic (db/db) mice, indicating a possible involvement of hypothalamic occludin expression in DIO. In addition, it was demonstrated that epinephrine enhances leptin transport into the brain by working at a1-like adrenergic receptors on the luminal side. However, epinephrine was effective on leptin transport only after intravenous or intraperitoneal injection, but not intracerebroventricular administration (Banks 2001).

8.2.3

Central Leptin Resistance

Leptin, released from adipose tissue, is transported across the BBB, especially in the arcuate nucleus of the hypothalamus where the BBB is poorly developed, and

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it binds to its specific receptor, which belongs to the class I cytokine family (Tartaglia et al. 1995). The long form of the leptin receptor (Ob-Rb) is activated by the formation of a homodimer. Genetic defects in long-form-type leptin receptor (Ob-Rb) cause obesity in genetically diabetic (db/db) mice, Zucker fatty (fa/fa) and Koletsky rats (Chen et al. 1996; Phillips et al. 1996; Wu-Peng et al. 1997). Leptin receptor mRNA and protein expressions were diminished in the hypothalamus of aged Wistar rats (Fernandez-Galaz et al. 2001). Food restriction in old rats lowered adiposity, and recovered responsiveness to centrally administered leptin with an increase of hypothalamic leptin receptor expression, indicating that adiposity plays a key role in the development of leptin resistance associated with aging (Fernandez-Galaz et al. 2002). Central recombinant adeno-associated viralmediated leptin gene delivery causes complete insensitivity to intracerebroventricular infusion of leptin (Scarpace et al. 2003). Central recombinant adeno-associated viral leptin gene therapy diminished maximal leptin signaling capacity in the hypothalamus, and this leptin-invoked leptin resistance perturbs the regulation of energy homeostasis in response to high fat exposure, producing augmented energy consumption (Scarpace et al. 2005). Hormonal status may be associated with leptin resistance. It was demonstrated that human C-reactive protein (CRP) directly inhibits the binding of leptin to its receptor and blocks its ability to signal in cultured cells, and that infusion of human CRP into leptin-deficient (ob/ob) mice blocked the effects of exogenously administered leptin upon satiety and weight reduction (Chen et al. 2006). In addition, the actions of human leptin were completely blunted in mice that express a transgene encoding human CRP. Since circulating CRP concentrations are positively correlated with body adiposity, CRP may contribute to the development of leptin resistance in obese subjects. Obesity accompanies hyperglucocorticoidism, and adrenalectomy prevents the development of obesity in experimental models (Bray 2000). Glucocorticoids play a role in the development of central leptin resistance (Zakrzewska et al. 1997). The anorexigenic effects of intracerebroventricularly administered leptin are enhanced in adrenalectomized rats, and glucocorticoid supplementation dose-dependently inhibits those effects. Withdrawal of glucocorticoid by adrenalectomy increased the expression of leptin receptor mRNA and its signaling molecules expression (Madiehe et al. 2001), indicating the interaction between glucocorticoid and central leptin resistance. In addition, females are more sensitive to the anorexigenic effects of leptin, and there appears to be increased leptin signaling in the arcuate nucleus of females (Clegg et al. 2006). The intracellular mechanism of central leptin resistance is summarized in Fig. 8.2. The intracellular signal transduction of leptin is mediated predominantly through the phosphorylation of the Janus kinase 2 (Jak2) – signal transducer and activator of transcription 3 (STAT3) pathway (Tartaglia et al. 1995; Vaisse et al. 1996). STAT3 activation is reduced within the arcuate nucleus of DIO mice (Munzberg et al. 2004). Centrally administered leptin partially restores STAT3 activation in the animals of DIO, although STAT3 activation by peripherally administered leptin is decreased (El-Haschimi et al. 2000). The absence of leptininduced activation of STAT3 in AgRP/NPY neurons makes mice susceptible to

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IRS-2 PI3K

cf. PTEN

Feeding

SH2-B ERK

c-fos

SHP2 985

SOCS3

1138 STAT3

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PTP1B

Fig. 8.2 Possible mechanism of central leptin resistance in animal models of obesity

further leptin resistance (Gong et al. 2008). The arcuate nucleus in the hypothalamus is selectively resistant in DIO mice, perhaps by elevated suppressor of cytokine signaling (SOCS)-3 expression in this nucleus (Munzberg et al. 2004). Rats selectively bred to develop DIO had less leptin-induced immunoreactive phosphorylated STAT3 expression in the arcuate, ventromedial, and dorsomedial nuclei of the hypothalamus than those bred to be diet-resistant (Levin et al. 2004). The SOCS-3 pathway causes leptin-inducible inhibition of leptin signaling, and has been suggested as a possible mediator of central leptin resistance in obesity (Bjorbaek et al. 1998). Chronic infusion of leptin into the third ventricle increases hypothalamic expression of SOCS-3 mRNA and its protein (Pal and Sahu 2003). The level of SOCS-3 is especially increased in the arcuate nucleus of DIO mice (Munzberg et al. 2004). Neural cell-specific SOCS-3-deficient mice showed enhanced leptin sensitivity and resistance to DIO (Mori et al. 2004). Heterozygous SOCS-3 deficiency enhanced weight loss and hypothalamic leptin receptor signaling in response to exogenous leptin administration, and protected against the development of DIO (Howard et al. 2004). Failure of STAT3 phosphorylation by leptin and increase of SOCS-3 expression in the hypothalamus can partially explain central leptin resistance in the rats of DIO. Another inhibitory molecule, PTP1B, is also involved in the regulation of leptin receptor signaling (Cheng et al. 2002). PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2, and PTP1B-deficient mice have leptin hypersensitivity (Zabolotny et al. 2002). Overexpression of PTP1B in a mouse hypothalamic cell

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line, GTI-7, resulted in a dose-dependent decrease in endogenous Jak2 and STAT3 tyrosine phosphorylation, and led to a decrease in SOCS-3 mRNA accumulation (Kaszubska et al. 2002). Furthermore, leptin-deficient mice lacking PTP1B show an enhanced response toward leptin (Cheng et al. 2002). The selective decrease in hypothalamic PTP1B resulted in decreased food intake, reduced body weight, and improved leptin signaling in the hypothalamus of DIO rats (Picardi et al. 2008). PTP-1B should, therefore, be important in the development of central leptin resistance, in addition to SOCS-3. An activation of insulin receptor substrate (IRS) mediates the activation of phosphatidylinositol 3-kinase (PI3K) (Zhao et al. 2002). Hypothalamus-specific IRS2 knockdown mice, in which IRS2 expression was markedly reduced in the arcuate nucleus, displayed obesity and leptin resistance (Kubota et al. 2004). IRS2 is important in leptin signal transduction in the arcuate nucleus. A PI3Kphosphodiesterase 3B-cyclic AMP pathway may be involved in the development of central leptin resistance (Sahu and Metlakunta 2005). Central infusion of PI3K inhibitor eliminates leptin-induced anorexia (Niswender et al. 2001). In addition, it was demonstrated that pharmacologic inhibition of PI3K prevented a rapid depolarization of POMC neurons by leptin, and that targeted disruption of PI3K blunted the suppression of feeding elicited by central leptin administration (Hill et al. 2008). These authors concluded that PI3K signaling in POMC neurons is essential for the acute suppression of food intake elicited by leptin, but is not a major contributor to the regulation of long-term energy homeostasis. Recent studies demonstrated that leptin acts to increase levels of phosphatidylinositol 3,4,5-triphosphate; however, they have reported that leptin did not increase PI3K, but inhibited the lipid and protein phsophatase activity of PTEN in the presence of active PI3K (Ning et al. 2006). The activation of PI3K pathway by leptin may be mediated by the inhibition of PTEN phosphorylation. From the standpoint of leptin resistance, the PI3K pathway, but not the STAT3 pathway of leptin signaling, is impaired during the development of DIO in FVB/N mice, indicating that a defective PI3K pathway of leptin signaling in the hypothalamus may be one of the mechanisms of central leptin resistance and DIO (Metlakunta et al. 2008). Leptin infused into the mediobasal hypothalamus of rats inhibited white adipose tissue lipogenesis, independently of STAT3 signaling (Buettner et al. 2008). Inhibition of PI3K signaling with PI3K inhibitor LY294002 in the mediobasal hypothalamus prevented the suppression of acetyl-CoA carboxylase and fatty acid synthase expression in white adipose tissue, indicating the involvement of PI3K in the inhibition of lipogenesis by leptin. In addition, SHP2 is a positive regulator of mitogen-activated protein (MAP) kinase (ERK) in leptin signaling (Banks et al. 2000; Bjorbaek et al. 2001). SHP2 down-regulates Jak2/Stat3 activation by leptin in the hypothalamus (Zhang et al. 2004). Inhibition of hypothalamic (AMPK) is necessary for the anorexigenic effects of leptin, because constitutively active AMPK blocks the effects of leptin (Minokoshi et al. 2004). The recent finding that inhibition of a2-AMPK activity by leptin was not observed in the paraventricular, arcuate, and medial hypothalamus of DIO mice may indicate that defective responses of AMPK to leptin may contribute

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to resistance to leptin action on food intake and energy expenditure in obese states (Martin et al. 2006). Recently, SH2-B, a Jak2-interacting protein, was identified as a key regulator of leptin sensitivity (Ren et al. 2005). SH2-B binds simultaneously to both Jak2 and IRS2, promoting leptin-stimulated activation of the PI3K pathway in cultured cells (Zhang et al. 2004). Leptin-stimulated activation of hypothalamic Jak2 and phosphorylation of hypothalamic STAT3 and IRS2 were impaired in SH2-B(–/–) mice, whereas long-form leptin receptor and SOCS-3 expressions were not changed, and deletion of SH2-B may severely impair leptin sensitivity in hypothalamic NPY/ AgRP neurons (Duan et al. 2004). Overexpression of SH2-B counteracted PTP-1Bmediated inhibition of leptin signaling in cultured cells. SH2-B may be indispensable in mediating leptin effects.

8.2.4

Therapeutic Targets For Leptin Resistance

Here we discuss the possibility of clinical treatment of leptin resistance and obesity. In obese subjects, the transport of leptin into the brain is disturbed, but the brain may be as sensitive to leptin as in lean subjects. From the standpoint of peripheral leptin resistance, the development of longer and more permeable analogs, antagonists enhancing the activity of leptin, and intrathecal delivery of leptin are possible candidates for the treatment of leptin resistance (Banks and Lebel 2002). Intranasal leptin administration caused longer inhibition of appetite without a significant increase of circulating leptin concentrations in normal rats (Shimizu et al. 2005). The administration of leptin or analogs into the nasal cavity may be a possible route for leptin administration, by which leptin could effectively reach the brain without the influence of peripheral leptin resistance. Changes of nutritional status should be important in the improvement of leptin resistance. Withdrawal of high-calorie diet for only three days makes leptin-resistant DIO-prone mice sensitive to leptin (Berriel Diaz et al. 2006), confirming that withdrawal of high-calorie diet helps recover from leptin resistance, indicating calorie restriction in leptin-resistant obese subjects. Metformin restores leptin sensitivity in high-fat-fed obese rats with leptin resistance (Kim et al. 2006). Metformin treatment increases cerebrospinal fluid leptin concentrations in both standard and high-fat diet-fed obese rats. The improvement of the transport of leptin into the brain by metformin may correct leptin resistance in the rats of DIO. The combination of metformin treatment with leptin administration could be useful in the treatment of obesity (Kim et al. 2006). Since SHP2 downregulates Jak2/Stat3 activation by leptin in hypothalamus, SHP2 may have potential as a neuronal target for the treatment of central leptin resistance (Zhao et al. 2002). Treatment with melanotan II, a synthetic melanocortin 3/4 receptor agonist, is another therapeutic approach to leptin resistance in DIO (Pierroz et al. 2002). Ciliary neurotrophic factors reduce food intake by increasing STAT3 phosphorylation, and suppressing hypothalamic AMPK signaling in the arcuate nucleus of

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leptin-resistant obese mice, independently of leptin signaling (Steinberg et al. 2006). Anorexigenic, bioactive substances, independent of the leptin pathway, may be useful for the treatment of leptin-resistant obese subjects.

8.3

Leptin Action in the Periphery

Leptin also shows plenty of direct actions on various peripheral tissues, in addition to its indirect actions via central nervous system. We have demonstrated that leptin stimulates lung cancer cell proliferation in vitro by the activation of the MAP kinase cascade (Tsuchiya et al. 1999). Leptin also phosphorylates and activates tyrosine hydroxylase, and subsequently stimulates catecholamine synthesis through MAP kinase in the adrenal medulla (Shibuya et al. 2002). Furthermore, leptin prevents apoptosis of trophoblastic cells by activation of the MAP kinase pathway in placenta (Perez-Perez et al. 2008). Therefore, the MAP kinase pathway should be important in the effects of leptin on peripheral tissues such as cell proliferation and anti-apoptotic action. On the other hand, it is thought that same signaling proteins as insulin is involved peripheral leptin signaling pathway, similarly with the hypothalamic leptin signaling pathway. Leptin has been reported by several investigators to be associated with both in vitro and in vivo insulin secretion. Leptin has been shown to inhibit insulin secretion through a PI3K-dependent mechanism in pancreatic b-cells (Kieffer and Habener 2000). Disruption of leptin receptor expression upregulates the phosphorylation of PTEN, a major negative regulator of the PI3K/Akt signaling, and enhances phsophorylation of p70 S6 kinase and PKB/Akt, which are important for determining b-cell size and survival, respectively (Morioka et al. 2007). However, disruption of leptin receptor expression led to attenuated acute insulin secretory response to glucose, poor compensatory islet growth, and glucose intolerance in animals fed a high-fat diet. We have previously demonstrated that leptin stimulated insulin secretion and preproinsulin mRNA expression in HIT-T 15 cells (Shimizu et al. 1997a,b). Leptin’s effects on insulin secretion may be modified by the circumstantial condition of insulin secretory cells. In addition, preproinsulin gene expression is inhibited by the induction of SOCS-3 by leptin in pancreatic b-cells (Laubner et al. 2005). SOCS-3 may be also involved in the modulation of insulin synthesis by leptin in the periphery. In addition, leptin activates PI3K and PDE3B to inhibit cyclic AMP production increased by glucagons in hepatocytes (Zhao et al. 2000). Leptin has been also shown to increase interleukin-8 production in synovial fibroblasts via the leptin receptor (long form)/Jak2/STAT3 pathway, as well as the activation of the IRS1/ PI3K/Akt/ NF-kB-dependent pathway (Tong et al. 2008). Leptin directly activates resident macrophages to form ADRP-enriched lipid droplets and enhances eicosanoid production via a mechanism that is dependent on activation of the PI3K/ mammalian target of rapamycin (mTOR) (Maya-Monteiro and Bozza 2008). The PI3K pathway, including PTEN, appears to play an important role in the effects of leptin on peripheral tissue.

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153 Leptin

Ob-Rb cell membrane

IRS-1 PI3K

PTEN ERK 1/2

mTOR

Catecholamine synthesis

AKt NF-κB

Cell proliferation

Anti-apoptotic action

IL-8 production

Fig. 8.3 Possible mechanism of action of leptin on peripheral tissues

From the recent observations described above, the putative mechanism of action of leptin on peripheral tissues is summarized in Fig. 8.3.

8.4

The Future of Leptin

Since the discovery of leptin, there is accumulating data on the mechanism of leptin resistance. Both central and/or peripheral mechanisms may be involved in the development of leptin resistance in various kinds of obesity models. It is necessary to override the leptin resistance in order to use leptin clinically for the treatment of massively obese people. Further progress should be necessary in this field to override leptin resistance in human obesity; a new approach such as intranasal leptin administration should be clinically available in the future. The mechanism of leptin action on the peripheral tissues may also contribute to better understanding of leptin signaling mechanism in the hypothalamus.

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Chapter 9

Signaling in Normal and Pathological Angiogenesis Michael R. Mancuso and Calvin J. Kuo

9.1

Introduction

The circulation of blood provides the essential function of delivering oxygen and nutrients and removing carbon dioxide and other metabolic waste products to and from tissues throughout the body and also provides a key route through which the immune system functions. In many multicellular organisms oxygenated blood travels from the heart through a series of arteries into capillary beds where gas, nutrients, and metabolic waste exchange occurs with the underlying tissue. Deoxygenated blood exits the capillary beds into veins where it travels back to the heart and ultimately to the lungs to expel CO2 and become re-oxygenated. All blood vessels, including arteries, capillaries, and veins, are lined by endothelial cells that are supported by mural cells (called vascular smooth muscle cells (vSMC) on arteries and pericytes on veins and capillaries) that are crucial for stabilization of the vasculature. Both the endothelial cells and mural cells are surrounded by a vascular basement membrane on the abluminal surface of the blood vessel that provides a scaffold for cell adhesion and a rich source of growth factors (Fig. 9.1).

9.1.1

Angiogenesis

Angiogenesis is defined as the growth of new blood vessels from pre-existing ones (Fig. 9.2) and is regulated by a precise balance of pro- and anti-angiogenic regulators. Note that angiogenesis differs from vasculogenesis where angioblasts migrate

C.J. Kuo (*) and M.R. Mancuso Divisions of Hematology, Department of Medicine, Stanford University School of Medicine, CCSR 1155, 269 Campus Dr, Stanford, CA, USA e-mail: [email protected]

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_9, © Springer-Verlag Berlin Heidelberg 2010

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towards and adhere to each other to form tubes de novo. During the process of angiogenesis, parenchymal and stromal cells, such as macrophages, in an ischemic (lacking desired amount of blood flow) region of tissue, secrete vascular endothelial growth factor (VEGF) and other mitogens to signal for the recruitment of new blood vessels (Fig. 9.2). These secreted growth factors diffuse to nearby quiescent (resting) blood vessels to signal for endothelial cell migration into the avascular region. The endothelial cells form vascular tubes while simultaneously migrating into the ischemic tissue through undergoing cell-type specific specialization: tip cells form filopodia to sense and migrate towards chemoattractants such as VEGF while stalk cells proliferate and form vascular tubes (Fig. 9.2). The newly formed

Fig. 9.1 Schematic cross-section through a capillary showing spatial orientation of endothelial cells, pericytes, and vascular basement membrane

Fig. 9.2 Angiogenesis, the growth of new blood vessels from pre-existing ones, begins when endothelial cells (light gray) of quiescent vessels sense mitogens such as VEGF (black circles) and migrate by a process known as “vascular sprouting.” The emerging vascular sprout is composed of two types of endothelial cells: (1) Tip cells are migratory cells that sense mitogens through extension of filopodia. (2) Stalk cells are proliferative cells that form vascular tubes behind migrating tip cells. The newly formed vascular tube then undergoes maturation through recruitment of mural cells (dark gray) by secretion of PDGF (dark gray diamonds)

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vascular tube is known as an immature blood vessel or neovessel and is composed solely of endothelial cells. Eventually, the endothelial cells recruit pericytes through secretion of platelet-derived growth factor-B (PDGF-B) to stabilize the vascular tube and form a mature blood vessel capable of supporting blood flow to perfuse the adjacent tissue (Fig. 9.2) with the ultimate goal of providing every cell in any given tissue in the body with access to a capillary network. This chapter describes the molecular mechanisms that regulate both normal physiologic angiogenesis as seen during embryonic development, wound healing, and menses, and pathologic angiogenesis as seen in cancer, macular degeneration, and diabetic retinopathy.

9.2 9.2.1

Endothelial Cell Growth and Migration Vascular Endothelial Growth Factor

VEGF signaling is a key regulator of endothelial cell growth, migration, and survival and is also a potent inducer of vascular permeability (Ferrara et al. 2003). VEGF was originally isolated from bovine pituitary gland for its mitogenic effects on endothelial cells in vitro. There are six VEGF ligands (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F) and three VEGF receptors (VEGFRs) that make up the VEGF family (Fig. 9.3). Additionally, two co-receptors, neuropillin-1 (NRP-1) and neuropillin-2 (NRP-2), are required for signaling through the VEGFRs (Fig. 9.3). Further, the ligand placental growth factor (PlGF) can also signal through VEGFR-1 (Fig. 9.3) as discussed below.

Fig. 9.3 VEGF family interaction with the VEGF receptors. Note that VEGFR-1 is also expressed as a soluble form (sVEGFR1) to sequester ligands during embryogenesis

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VEGF-A

VEGF-A (also called “VEGF”) is the most well-characterized of the VEGF ligands (Ferrara et al. 2003). It was originally isolated for its vascular permeability induction properties and endothelial cell mitogen by two separate groups. The Vegf-a gene contains eight exons that are alternatively spliced to yield four isoforms in humans, VEGF121, VEGF165, VEGF189 and VEGF206, which encode proteins of 121, 165, 189, and 206 amino acids respectively after signal peptide cleavage (Fig. 9.4). VEGF165 is the predominant isoform and exists as a homodimeric glycoprotein 45 kDa in size. All of the VEGF-A isoforms, except for VEGF121, contain a heparinbinding domain which is crucial for VEGF function and also allows for adherence of VEGF to the extracellular matrix. VEGF121 is an acidic protein that is freely soluble while VEGF189 and VEGF206 are highly basic and adhere tightly to the extracellular matrix. VEGF165 has intermediary properties giving it optimal characteristics of bioavailability and potency as one of the major regulators of angiogenesis through its interaction with VEGFR-1 and VEGFR-2. The VEGF165 isoform exists as an extracellular matrix-associated protein amenable to proteolytic release by plasmin and/or matrix metalloproteinases (MMP) to render it freely soluble. VEGF-A is one of the most critical regulators of angiogenesis such that mice lacking even a single allele of VEGF-A die before birth. Vegf-A expression is regulated by hypoxia inducible factor (HIF), a transcription factor whose expression level, and therefore function, is regulated by the presence of oxygen (Pugh and Ratcliffe, 2003). When oxygen is present, HIF is constantly expressed but is then hydroxylated at an asparagine and several proline side-chains which prevent HIF from activating target genes such as VEGF. Further, the hydroxylated side-chains mediate interaction with the ubiquitin ligase Von Hippel–Lindau (VHL) which subsequently ubiquitinates HIF. This ubiquitination event tags HIF for degradation by the proteosome (Fig. 9.5). If a cell suddenly encounters an environment with decreased levels of oxygen (hypoxia), HIF is no longer hydroxylated and accumulates to activate VEGF expression (Fig. 9.5). As discussed above, the activation of VEGF expression ultimately leads to the recruitment of blood vessels to bring oxygen to the cell. Once oxygen levels have returned to normal, hydroxylation of HIF resumes followed by ubiquitination by VHL, to lower levels of the transcription factor and thus lower the level of VEGF expression. Thus, the oxygen-dependent hydroxylation of HIF serves as an indirect oxygen sensor for cells to identify conditions when vascular recruitment is necessary.

9.2.1.2

VEGF-B

VEGF-B exhibits structural similarity to VEGF-A (Olofsson et al. 1999; Olofsson et al. 1996). The Vegf-b gene contains seven exons that are alternatively spliced to yield two VEGF-B isoforms, VEGF-B167 and VEGF-B186, that yield proteins that are 167 and 186 amino acids after signal peptide cleavage. VEGF-B186 is further processed to give rise to a 34 kDa homodimeric glycoprotein. VEGF-B can bind to

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Fig. 9.4 Alignment of cDNA of the VEGF-A Isoforms. The Vegf-a gene contains eight exons (boxes with numbers) that are alternatively spliced to yield the different isoforms. Regions of Vegf-a cDNA encoding important regions of the VEGF-A protein, such as receptor binding sites, are indicated at the top

Fig. 9.5 Regulation of VEGF expression by HIF. The HIF gene is constantly expressed. In the presence of oxygen (normoxia), the HIF protein is hydroxylated, signaling for VHL-mediated ubiquitination of HIF and its subsequent degradation by the proteosome. In the absence of oxygen (hypoxia), HIF is no longer degraded by this pathway and translocates to the nucleus to induce expression of target genes

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both VEGFR-1 and VEGFR-2; however, the biological relevance of VEGF-B is not clear. It is speculated that the heterodimerization of VEGF-B with VEGF-A may add an additional level of specificity to VEGF signaling. However, VEGF-B is not thought to have any overlapping functions with VEGF-A despite the overlapping affinities for VEGFR-1 and VEGFR-2, since loss of a single allele of VEGF-A in mice causes failure of embryonic angiogenesis while mice lacking VEGF-B are phenotypically normal.

9.2.1.3

VEGF-C and VEGF-D

VEGF-C and VEGF-D constitute a subgroup of the VEGF ligand family characterized by unique extensions flanking the N- and C-terminal sides of the VEGF homology domains – which in VEGF-C and VEGF-D are 63% identical. VEGF-C is the only VEGF family member that is not alternatively spliced (Tammela et al. 2005). Instead, the Vegf-c gene encodes a precursor protein that undergoes proteolytic processing. The C-terminus of VEGF-C is cleaved upon secretion but remains bound to the N-terminal portion via disulfide linkages. Proteolytic processing of the amino-terminal propeptide releases the mature active form. VEGF-D is also expressed as a precursor protein that is processed at its N- and C-terminal ends to yield an active form. Both VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3 and play important roles in lymphangiogenesis (the growth of new lymphatic vessels). Mice lacking either VEGF-C or VEGF-D exhibit defects in lymphangiogenesis.

9.2.1.4

VEGF-E and VEGF-F

VEGF-E and VEGF-F are two of the least studied members of the VEGF ligand family. VEGF-E was initially discovered in the viral genome of the orf virus while VEGF-F was initially discovered in snake venom (Otrock et al. 2007). Both VEGF-E and VEGF-F bind selectively to VEGFR-2 and are being investigated for use as research tools and possibly for therapeutic purposes.

9.2.1.5

Placental Growth Factor

PlGF, as the name suggests, was originally identified in the placenta (Otrock et al. 2007). There are four known splice variants of the PlGF gene with only PlGF-2 having the ability to bind to heparan sulfate proteoglycans. PlGF selectively binds VEGFR-1 and is thought to play an important role in macrophage recruitment, a key physiologic function of VEGFR-1 signaling (as described below). Further, PlGF is 42% homologous to VEGF-A and can heterodimerize with VEGF-A to add increased specificity to VEGFR-1 signaling. Mice lacking PlGF have no apparent phenotype; however, preclinical studies indicate that blocking PlGF could be thera-

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peutically beneficial for treating vascular-dependent diseases such as cancer through inhibiting macrophage-driven tumor angiogenesis.

9.2.1.6

Neuropillins

Neuropillins (NRP) are co-receptors required for activation of the VEGF receptors. For example, VEGF-A binds NRP-1 in addition to either VEGFR-1 or VEGFR-2 to signal through these receptors (Fig. 9.3). Similarly, NRP-2 facilitates VEGFR-3 signaling (Fig. 9.3).

9.2.1.7

VEGFR-1

VEGFR-1 (Flt-1) is a receptor tyrosine kinase (RTK) with seven immunoglobulin repeats in the extracellular domain and two RTKinase domains in the intracellular region (Fig. 9.3) (Ferrara et al. 2003; Shibuya 2006). VEGFR-1 is approximately 80% identical to VEGFR2 (below); however, there are several key differences between VEGFR-1 and VEGFR-2 in terms of biological function. VEGFR-1 has a tenfold higher binding affinity for VEGF-A (KD = 10–20 pM) than VEGFR-2 (75– 125 pM). During embryogenesis, VEGFR-1 is produced as a soluble truncated form (sVEGFR-1) (Fig. 9.3) that binds and sequesters VEGF-A, thereby serving as a negative regulator of angiogenesis. However, in adulthood, full-length VEGFR-1 induces the migration of monocytes and other cell types to indirectly stimulate angiogenesis (e.g., macrophages secrete VEGF and other pro-angiogenic factors). VEGFR-1 dimerizes in response to binding to PlGF, VEGF-A, and VEGF-B to undergo autophosphorylation of tyrosine residues 1169, 1213, 1242, 1327, and 1333 in the intracellular domain. Notably, the autophosphorylation pattern of the VEGFR-1 intracellular domain differs according to the specific ligand (i.e., VEGF-A, VEGF-B, PlGF) binding to the extracellular domain. Phosphoryation of Y1169 allows binding of PLCy1 to regulate cell proliferation via Mitogen-Activated Protein Kinase (MAPK)/Extracellular Regulated Kinase (EPK) pathways similar to that seen in VEGFR-2 signaling (below). VEGFR-1 signaling has also been shown to activate phosphoinositol-3-kinase (PI3K); however, the precise mechanism for this activation is currently not known.

9.2.1.8

VEGFR-2

VEGFR-2 (KDR, Flk1) signaling regulates the majority of the angiogenic activities induced in endothelial cells by VEGF (Ferrara et al. 2003; Holmes et al. 2007; Shibuya and Claesson-Welsh 2006; Shibuya et al. 1999). Similar to VEGFR-1, VEGFR-2 contains seven immunoglobulin repeats in the extracellular domain and two tyrosine kinase catalytic motifs in the intracellular domain. VEGFR-2 binds VEGF-A, VEGF-C, VEGF-D, and VEGF-F and signals with the co-receptor

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neuropilin-1 (NRP-1) (Fig. 9.3). Upon binding to VEGF, VEGFR-2 undergoes receptor dimerization and autophosphorylation at tyrosine residues within the intracellular domain leading to multiple signaling cascades regulating proliferation, survival, migration, and permeability of vascular endothelial cells. Y951, Y1054, Y1059, Y1175, and Y1214 are major phosphorylation sites while Y1305, Y1309, and Y1319 are minor phosphorylation sites. Y1054 and Y1059 are important for regulation of tyroinse kinase activity. The physiologic events induced by VEGFR-2 signaling (Fig. 9.6) are as follows: 1. Endothelial cell proliferation. VEGFR-2 primarily induces endothelial cell proliferation through activation of the (ERK-1/2) pathway. Phosphorylation of Y1175 of the intracellular domain creates a binding site for phospholipase C-g1. Activated phospholipase C- g1 converts phosphatidylinositol-4,5-bisphosphonate (PIP2) to diacyl glycerol (DAG) and inositol-1,4,5-triphosphate (IP 3) which stimulate Ca2+ release from intracellular stores. The Ca2+ flux triggers activation of protein kinase C (PKC) leading to MAP kinase activation of ERK-1/2 and subsequent expression of genes important for cellular proliferation. Note that signaling through VEGFR-2 does not activate ERK-1/2 via the Grb/Sos/Ras pathway as commonly seen with most other RTKs (Fig. 9.6). 2. Endothelial cell migration. The migration of endothelial cells through tissue requires the reorganization of actin filaments which is regulated by the GTP-binding proteins Rac and Rho. In endothelial cells, two pathways stem from VEGFR-2

Fig. 9.6 Schematic representation of VEGFR-2 signaling pathways. Binding of VEGF to the VEGFR-2 extracellular domain causes receptor homodimerization, resulting in autophosphorylation of the RTK domain, leading to signaling events that regulate endothelial cell proliferation, migration, survival, and permeability

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to activate this process. Phosphorylated Y951 binds VEGF receptor-associated protein (VRAP, also called T-cell specific adaptor protein (TSAd), Rlk, and Itkbinding protein (RIBP) or Lck adaptor (LAD)). VRAP recruits Src, leading to Rac/ Rho GTPase-driven actin reorganization required for cell migration. In addition to activating the MAP kinase pathway as described above, phosphorylation of Y1175 recruits the adaptor molecule SHB. Docking SHB to VEGFR-2 provides a scaffold for the assembly of focal adhesions, which are important for cell–extracellular matrix interactions, through the recruitment and phosphorylation of focal adhesion kinase (FAK) and also provides a scaffold for the activation of PI3K. Activated PI3K phosphorylates PIP2 to generate phosphatidylinositol-3,4,5-triphosphate (PIP3) which also leads to activation of Rac and Rho. In addition to Rac and Rho activation, phosphorylation of Y1214 recruits Nck to VEGFR-2, leading to subsequent activation of Cdc42, p38 MAPK, and Hsp27, to induce actin filament reorganization important for endothelial cell sensing and migration towards increasing VEGF gradients (Fig. 9.6). 3. Endothelial cell survival. While endothelial cells in most established blood vessels in adult vascular beds can persist in the absence of VEGF signaling, endothelial cells of newly formed vessels die in the absence of VEGF signaling. This VEGF-A-dependent endothelial cell survival is mediated through VEGFR-2 activation. As described above, phosphorylation of Y1175 of VEGFR-2 leads to PI3K phosphorylation of AKT (p-AKT) (Fig. 9.6) which inhibits the forkhead transcription factor FOXO1 and proapoptotic proteins such as BAD and caspase 9. 4. Vascular permeability. VEGF-A was originally isolated for its ability to induce vascular permeability and this property of VEGF is thought to be a valuable target to improve delivery of chemotherapeutics to cancer tissue in patients. Vascular permeability is regulated though phosphorylation of Y1175 of VEGFR-2 to induce phospholipase C-mediated cleavage of PIP2 to IP3 and DAG. IP3 releases Ca2+ from intracellular stores to activate eNOS to produce NO and induce endothelial cell permeability (Fig. 9.6).

9.2.1.9

VEGFR-3

VEGFR-3 (Flt-4) signaling was previously thought to primarily regulate lymphangiogenesis with its ligands VEGF-C and VEGF-D; however, recent evidence suggests that VEGFR-3 plays an important role in angiogenesis as well. Like VEGFR-2, VEGFR-3 is also an RTK. The extracellular domain of VEGFR-3 is composed of six immunoglobulin domains with the second and third domain being covalently linked solely through disulfide bonds of cysteine side-chains (Fig. 9.7). Upon binding to VEGF-C or VEGF-D, VEGFR-3 dimerizes to phosphorylate Tyrosine residues in the intracellular domain. Of these, Y1063, Y1068, Y1230, Y1231, and Y1337 are important for the catalytic function of the receptor. Y1063 activates CRK/MKK4/JNK signaling pathway to activate c-JUN and promote cell survival. Phosphorylation of Y1230 and Y1231 activates the classical PI3K and

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Fig. 9.7 Schematic representation of VEGFR-3 signaling pathways

MAPK/ERK pathways that are important for cellular proliferation and migration as described for VEGFR-2 signaling above. While VEGFR-3 is a master regulator of lymphangiogenesis, it has recently been found to regulate angiogenic sprouting. In a migrating vascular plexus, VEGFR-3 is expressed by tip cells at the leading edge of the plexus where its activation in endothelial cells induces angiogenic sprouting. Interestingly, VEGFR-3 has been found to be down-regulated by Notch, which is expressed in stalk cells as described below, providing insight into how VEGFR-3 expression is regulated to achieve hierarchical organization of the migrating vascular plexus.

9.2.2

miR-126

MicroRNAs are small genetic elements that are transcribed and processed to regulate expression of target genes through binding to the 3¢ untranslated region of the target gene mRNA (Fig. 9.8). This emerging class of molecules has very recently been identified as key mediators of angiogenic signaling. Endothelial cells exclusively express the microRNA miR-126 and this microRNA plays a crucial role in modulating VEGF signaling. miR-126 regulates the expression of p85b and Spred1, both of which are crucial regulators of VEGF signaling (Fig. 9.8). p85b is a regulatory subunit of the PI3K complex which plays a key role in endothelial cell migration as described above. miR-126 represses expression of p85b to reduce inhibition of PI3K signaling driven by VEGF/VEGFR-2 signaling. Similarly,

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Fig. 9.8 Regulation of angiogenesis by the miR-126/Egfl7 locus. Mir-126 is expressed from the Egfl7 message as a pre-miRNA, processed, and exported out of the nucleus where it targets the 3¢ untranslated region (3¢UTR) of p85b and Spred1 to regulate expression

Spred1 is a regulator of MAPK/ERK signaling and miR-126 also reduces expression of Spred1 to reduce inhibition of this pathway. Interestingly, miR-126 sits within an intron of Egfl7, a gene that has been previously implicated in angiogenesis (Fig. 9.8). Recently, knockout mouse models have helped determine that the regulation of angiogenesis by the Egfl7/miR-126 locus during development is mainly attributed to miR-126 regulation of p85b and Spred1 as described above rather than the protein encoded by Egfl7 host gene (Kuhnert et al. 2008).

9.2.3

Delta-like 4/Notch Signaling

Delta-like 4/Notch signaling controls the fine tuning of vascular plexus formation (Roca and Adams, 2007). There are four members of the Notch gene family which encode transmembrane proteins with unique extra- and intracellular components. The extracellular region contains 3-lin-12/Notch motifs and multiple EGF repeats while the intracellular region contains a RAM domain, multiple cdc10/ankyrin repeats, nuclear localization signal(s), and a protein instability domain. The Notch receptors all bind either Jagged 1, Jagged 2, delta-like (Dll) 1, Dll3 or Dll4, all of which contain extracellular regions with EGF-like repeats and a DSL motif. Of this family, Notch-1, Notch-4, Jagged-1, Dll1, and Dll4 are expressed by endothelial cells with Dll4 being exclusively expressed by endothelial cells. When Notch binds Dll4 – for example, on an adjacent endothelial cell – the intracellular domain of Notch is processed by gamma-secretase to releases the Notch intracellular domain (NICD) which translocates to the nucleus where it acts as a cofactor to activate or suppress the expression of target genes. Similarly, the interaction of Dll4 with Notch

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causes a reverse signaling where Dll4 is ubiquitinated and internalized; however, the function of this internalization on the endothelial cell remains to be investigated. During angiogenesis, Notch is expressed by stalk cells while Dll4 is expressed by tip cells. While the mechanism remains to be further studied, the role of Dll4 and Notch signaling is to dampen the endothelial cell response to VEGF. For example, Dll4/Notch signaling reduces the formation of filopodia so that the endothelial cell takes on a proliferative vascular tube forming stalk cell phenotype as opposed to a migratory mitogen-sensing tip cell phenotype. Thus, as tip cells in the vascular plexus migrate towards a VEGF stimulus (Fig. 9.9), they express Dll4 to bind Notch receptors on adjacent endothelial cells to signal for these adjacent endothelial cells to form stalk cells through down-regulation of genes important for vascular migration such as VEGFR-3 (as described above). This process allows for the vasculature to form a hierarchical structure in an organized fashion (Fig. 9.9).

9.2.4

Slit2/Robo4 Signaling

Slit2/Robo4 signaling has recently been identified as a negative regulator of VEGF signaling similar to Dll4/Notch signaling as described above. Roundabout proteins

Fig. 9.9 Regulation of endothelial cell phenotype by Dll4/Notch signaling. As endothelial cell sprouts migrate toward VEGF gradients, migratory tip cells express Dll4 that binds Notch receptors on adjacent endothelial cells. After binding Dll4, the Notch intracellular domain is cleaved and translocates to the nucleus to regulate the expression of target genes important for induction and maintenance of the proliferative endothelial stalk cell phenotype

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(Robo) are guidance receptors and to date four have been identified with Robo4 being specifically expressed by endothelial cells. When activated by Slit2, Robo4 signals to inhibit the effects of VEGF signaling in stalk cells of the growing vascular plexus. Thus, Slit2/Robo4 signaling appears to dampen the effects of VEGF signaling in endothelial stalk cells to prevent them from assuming the migratory tip cell phenotype – a process which is essential for forming an organized functional vascular network.

9.2.5

Fibroblast Growth Factor

Fibroblast growth factor (FGF) signaling contributes to the maintenance of vascular integrity and has also been shown to stimulate angiogenesis. The FGF family consists of 22 ligands and four FGF receptors (FGFRs). The FGFRs are RTKs that contain 2–3 immunoglobulin domains, a transmembrane domain, and two RTK domains. FGF/FGFR signaling is a broad-spectrum pathway that is active in many different cell types undergoing an array of processes, including angiogenesis and vascular homeostasis in endothelial cells. It is thought that FGF/FGFR signaling contributes to vascular homeostasis through maintenance of tight junctions and adherens junctions. Notably, FGF signaling during angiogenesis has gained increased attention as it can serve as an escape mechanism to allow pathological angiogenesis (such as tumor angiogenesis) to commence despite VEGF inhibition as discussed below.

9.3

Vascular Cell Adhesion

Endothelial and mural cell migration is a process that is dependent on cell adhesion. When quiescent endothelial cells are stimulated to undergo angiogenic migration, the endothelial cells must be able to degrade their surrounding extracellular matrix and, accordingly, modify their adhesion junctions so that they can begin to migrate. During this process, endothelial cells interact with the extracellular matrix and undergo cell–cell communication as the vascular tube emerges into a new region of tissue. One class of molecules that facilitate endothelial cell adhesion to the extracellular matrix and neighboring cells are the integrins.

9.3.1

Integrins

Integrins, which are heterodimeric transmembrane proteins composed of an a and a b subunit, undergo uni- or bi-directional signaling that is essential for cell–extracellular matrix and cell–cell interactions respectively. Specifcally, integrins avb3, avb5, a1b1, a2b1, a4b1, and a5b1 play important roles in both

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endothelial and pericyte adhesion through their interaction with components of the extracellular matrix (Table 9.1) (Silva et al. 2008). Integrin signaling mediates focal adhesion formation, a process that is essential for cell migration. Structurally, integrins are composed of an a and a b subunit which both span the plasma membrane. In the absence of a ligand, the a and b subunits interact in the cyctoplasmic domain to maintain a resting state. Upon ligand activation, integrins change their conformation to allow the a and b subunits to release from each other in the cytoplasmic domain through a process mediated by talin. The intracellular domain of activated ligands facilitates formation of focal adhesions with paxillin and FAK in addition to several other signaling events. For example, integrin signaling is thought to modulate the signaling of vascular RTKs. Integrin avb3 enhances endothelial cell biological response to VEGF-A and integrin a5b1, and is thought to synergize with Ang-1 signaling (discussed below). Similarly, PDGF receptors (PDGFRs) are thought to

Table 9.1 Integrins involved in angiogenesis Integrin Interacting Expression substrate Collagen Endothelial cells and a1b1 mural cells a2b1 a3b1

Laminin

a4b1

Fibronectin

a5b1

a6b1 a9b1 avb3 avb5 avb8 a7b1

Laminin Osteopontin, VCAM-1 Vitronectin

Endothelial cells and mural cells (primarily leukocytes) Endothelial cells and mural cells

Endothelial cells

Effect on angiogenesis Required for angiogenesis and mural cell adhesion Required for angiogenesis and mural cell adhesion; regulates VEGFR-1 signaling Required for angiogenesis and mural cell adhesion Required for angiogenesis and mural cell adhesion Required for angiogenesis and mural cell adhesion; binds Ang-1 and VEGFR-1; cross-talk with Tie2 None reported Regulates VEGF-driven angiogenesis Pro- and anti-angiogenic regulator; synergizes with VEGFR-2

Vitronectin, collagen, and laminin

Non-vascular

Interacts with TGF-b signaling to stabilize vasculature

Laminin

Mural cells

Regulates mural cell development; interacts with PDGF signaling

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interact with integrins to enhance cell proliferation, migration, and survival by localizing PDGFR and other signaling events at focal adhesions to initiate cross-talk from multiple signaling cascades. Genetic deletion of individual subunits of vascular-specific integrins results in embryonic lethality with pronounced angiogenic phenotypes, highlighting the importance of integrinmediated cellular adhesion and intracellular signaling during angiogenesis.

9.4

Mural Cell Recruitment and Vascular Maturation

Once the primitive vascular tube is formed by endothelial cells, mural cells (known as pericytes on capillaries, and as vSMC on arteries) are recruited to grow alongside the endothelial cells and stabilize the newly formed blood vessel (Fig. 9.2).

9.4.1

Platelet-Derived Growth Factor

PDGF signaling is required for mural cell stabilization of newly formed blood vessels (Armulik et al. 2005). PDGF-B is expressed as a propeptide that is further processed and secreted by endothelial cells. Further, PDGF-B contains a retention motif for binding heparan sulfate proteoglycans, a portion of the extracellular matrix, to allow accumulation of high levels of PDGF-B in close proximity to newly formed blood vessels. Mature PDGF-B forms a homodimer, termed PDGF-BB, through disulfide linkages and binds PDGFR beta (PDGFR-b), an RTK. The extracellular domain of PDGFR-b is composed of five immunoglobulin domains while the intracellular domain contains two tyrosine kinase motifs (Fig. 9.10). Upon binding to PDGF-BB, PDGFR-b undergoes homodimerization which results in autophosphorylation of tyrosine residues in the intracellular domain. Similar to the VEGF receptors described above, phosphorylation of tyrosine residues in the intracellular domain of PDGFR-b induces several classic signaling cascades including MAPK/ERK, PI3K/AKT, and PLC-g/PKC/Ca2+ (Fig. 9.10) (Andrae et al. 2008). Furthermore, PDGFR-b is thought to directly interact with integrins to form focal adhesions important for cell migration (Andrae et al. 2008). As angiogenesis commences, the endothelium of newly formed blood vessels secretes PDGF-BB which binds to the PDGFR-b of mural cells inducing their recruitment towards the newly formed vessels. Whether or not PDGF-BB/PDGFR-b signaling is required for maintenance of interactions between mural and endothelial cells in mature quiescent blood vessels is currently debated by experts in the field.

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Fig. 9.10 Endothelial cells recruit mural cells (smooth muscle cells on arteries and pericytes on capillaries and veins) to newly formed blood vessels though secretion of PDGF-BB. The PDGF-BB ligand binds to PDGFR-b on the surface of mural cells to induce the proliferation and migration pathways shown

9.4.2

Angiopoietin-1 (Ang-1)/Tie2 Signaling

Angiopoietin-1 (Ang-1)/Tie2 signaling plays an important role in angiogenesis, vascular remodeling, mural cell recruitment, and vascular homeostasis as absence of the genes encoding either Ang-1 or Tie2 results in embryonic lethality with angiogenic deficits (Armulik et al. 2005; Makinde and Agrawal 2008). Tie2 is an RTK expressed on the surface of endothelial cells (among other cell types including some pericytes). The extracellular domain of Tie2 contains three EGF repeats flanked by two immunoglobulin domains while the intracellular domain contains RTK domains (Fig. 9.11). Ang-1 is the primary agonist of Tie2 signaling. This 70 kD glycoprotein contains a carboxy-terminal fibrinogen-like domain, a central coiled domain, and a short amino-terminal. It is the carboxy terminal that is required for binding Tie2. Ang-1 forms a homotetrameric complex through disulfide bonding in order to bind to Tie2. Upon binding with Ang-1, Tie2 undergoes homodimerization, resulting in autophosphorylation of tyrosine residues in the RTK domain, leading to activation of several well-known signaling cascades similar to those of VEGFR and PDGFR signaling. Phosphorylated tyrosine 1101 of the Tie2 RTK domain binds the Src homology 2 (SH2) region of the p85 subunit of PI3K to activate AKT and subsequent endothelial cell survival and migratory

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Fig. 9.11 The angiopoietin/Tie2 signaling pathways regulate vascular maturation. Angiopoietin-1 signals through the Tie2 RTK on endothethial cells and pericytes to induce vascular quiescence. Angiopoeitin-2 is thought to be secreted by endothelial cells and antagonizes Tie2 through an autocrine mechanism to allow for endothelial cells to emerge from a quiescent state and undergo vascular sprouting

pathways. Tie2 signaling also leads to the induction of p21 and MAPK/ERK pathways which are also important for cell survival. Ang-1/Tie2 signaling is important for both endothelial cell migration and mural cell recruitment to newly formed blood vessels but has little effect on cell proliferation. Notably, Ang-1 induction of Tie2 signaling also leads to a reduction in vascular permeability. Interestingly, Tie2 has another ligand named angiopoietin-2 (Ang-2), which is thought to antagonize signaling. Both Ang-1 and Ang-2 bind separate regions of the Tie2 extracellular domain with equal affinity. In a stable resting vasculature, mural cells express and secrete Ang-1 to induce stabilization of adjacent endothelial cells via paracrine signaling through Tie2 on the endothelial cell surface. Similarly, autocrine Tie2 signaling in mural cells from mural cell-secreted Ang-1 also contributes to vascular stability. Once an angiogenesis stimulus begins, endothelial cells are thought to express Ang-2 to inhibit Tie2 signaling on endothelial cells and mural cells through autocrine and paracrine signaling, respectively. This inhibition of Tie2 signaling results in pericyte loss and subsequent endothelial cell sprouting and migration (Fig. 9.11).

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Ephrin-B2/EphB4 signaling

Ephrin-B2/EphB4 signaling regulates cell adhesion and is essential for vascular remodeling. EphB4 is an RTK that is expressed by endothelial cells and mural cells of veins while ephrin-B2 is a transmembrane protein with a small intracellular domain that is predominantly expressed by arterial endothelial cells. Interaction of EphB4 and ephrin-B2 induces a bidirectional signaling cascade to promote the association of Crk adaptor proteins with p130 (CAS) to induce the formation of focal adhesions. Both ephrin-B2 and EphB4 are equally crucial for vascular development.

9.4.4

TGF-b signaling

TGF-b signaling is required for the induction of mural cells around the first blood vessels during development and is also important for endothelial cell proliferation and differentiation. TGF-b is a secreted homodimeric protein that signals through multiple serine threonine kinase receptors including activin-receptor-like kinase 1 (Alk1), Alk5, and endoglin to activate the nuclear effector Smad5. Genetic loss of function of Alk1 or endoglin in humans is the cause of hereditary hemorrhagic telangiectasia (HHT) type 1 and 2, respectively. HHT is an autosomal dominant disorder affecting 1:8,000 individuals and is characterized by dilated, tortuous blood vessels with thin walls that bleed easily as well as vascular malformations in the brain, liver, or lung that cause serious complications such as hemorrhage, stroke, or abscess formation (Bobik 2006).

9.5 9.5.1

Angiogenesis in Disease Cancer

Cancer is highly dependent on angiogenesis as evidenced by numerous of preclinical studies and also recent findings in patients that angiogenesis inhibitors slow the progression of this disease (Ho and Kuo, 2007; Kerbel 2008). The field of angiogenesis began in 1971 when Judah Folkman, a pediatric surgeon, hypothesized that tumor growth was dependent on blood vessel growth. In the early 1980s two scientists independently isolated VEGF. Harold Dvorak isolated VEGF from ascities fluid and actually first named the protein vascular permeability factor while Napoleana Ferrara isolated VEGF from cow pituitary gland and described its ability to induce endothelial cell migration. In 2004, a function-blocking monoclonal antibody against VEGF, bevacizumab (Avastin), gained approval for clinical use in the treatment of colon cancer in combination with chemotherapy, with subsequent FDA approval in lung cancer. Since then, over 30 angiogenesis inhibitors have

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begun to make their way into the clinic and have been shown to be effective for treating multiple types of cancer. The majority of angiogenesis inhibitors target VEGF signaling due to the central role this pathway plays in tumor angiogenesis. There are multiple classes of VEGF inhibitors including: (1) function-blocking monoclonal antibodies against VEGF ligand (bevacizumab (Avastin) and ranibizumab), (2) decoy receptors (VEGFTrap), (3) function-blocking monoclonal antibodies against VEGFRs (IMC-1121b), (4) function-blocking aptamers against VEGF (pegaptinib), and (5) small-molecule inhibitors of the RTK activity of the VEGFRs (Sorafenib and Sunitinib (Sutent)). As mentioned above, bevacizumab (Avastin), the function-blocking antibody against VEGF, was the first angiogenesis inhibitor to earn FDA approval after it proved to increase survival of patients when combined with standard chemotherapy regimens. The small-molecule inhibitors of VEGF signaling, sorafenib (Nexavar) and sunitinib (Sutent) followed suit after demonstrating efficacy in cancer patients with FDA approval in renal cell carcinoma, hepatocellular carcinoma and gastrointestinal stromal tumors. Both of these small molecules are inhibitors of RTK signaling that compete for ATP-binding sites and have shown increased specificity for VEGFR-2 but also against numerous other kinases. Both drugs are now used for treating renal cell carcinoma which is a cancer that arises from cells in the kidney. Interestingly, renal cell carcinomas often have mutations in the VHL pathway, which normally regulates levels of VEGF expression as described above. This loss of the VHL pathway leads to high levels of VEGF production by renal cell carcinoma that contributes to the progression of the disease and this phenomenon perhaps explains why these two small-molecule inhibitors of VEGF signaling are so efficacious in this cancer. The other VEGF inhibitors mentioned above are currently in clinical trials. Tumor cells and tumor infiltrating cells – including fibroblasts and macrophages – secrete high levels of VEGF, especially under hypoxic conditions, leading to an explosive growth of new blood vessels that migrate into the tumor tissue from the surrounding normal vasculature. One of the key hallmarks of tumor angiogenesis is the abnormal tumor vessels it generates. Tumor blood vessels have a very bizarre morphology with loss of arterial–capillary–vein hierarchy, pericytes that are loosely associated with the endothelium, numerous endothelial sprouts, and a drastic variance in vessel diameter. Further, tumor blood vessels are immensely permeable, leading to high tumor interstitial pressure. These phenotypes are probably attributed to excessively high levels of VEGF, as inhibition of VEGF signaling in tumors leads to both a reduction in vascular density and also normalizes the tumor blood vessels that survive treatment. As patients are treated with VEGF inhibitors in combination with standard chemotherapy regimens, it has been proposed that the so-called “normalization” of the tumor vasculature enhances patient response to tumor therapy through lowering tumor interstitial pressure and increasing the amount of chemotherapeutic delivered to the tumor tissue. However, while this interpretation of clinical data is intriguing, it is currently difficult to assess the relative contributions of chemotherapeutic delivery to tumor tissue versus the anti-tumor efficacy of decreased tumor angiogenesis in human patients.

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While VEGF signaling has been shown to play a central role in tumor angiogenesis, there are limitations to using VEGF inhibitors in cancer patients. Hypertension and kidney disease are side-effects of bevacizumab (Avastin). Further, there are instances where tumors employ an escape pathway to develop resistance to VEGF inhibitors. For example, pre-clinical mouse tumor models treated with VEGF inhibitors for prolonged periods of time begin to increase expression of bFGF to induce angiogenesis. Such mechanisms imply that using a cocktail of inhibitors that target multiple angiogenic pathways would be a more effective approach to inhibiting angiogenesis. For example, the small-molecule inhibitor sorafenib may be promising as it inhibits VEGFR-1, VEGFR-2, VEGFR3, and PDGFR-b. Along these lines, several non-VEGF-related angiogenesis inhibitors have been developed and are undergoing clinical trials. Angiostatin and endostatin are endogenous inhibitors of angiogenesis formed by degradation of the extracellular matrix. Angiogstatin is a 38 kD proteolytic fragment of plasminogen, and endostatin is a 20 kD fragment of collagen XVIII. Both angiostatin and endostatin have demonstrated potent anti-tumor effects in pre-clinical models. Fumagillin (TNP-470), an inhibitor of methionine aminopeptidase, was originally isolated from the fungus Aspergillus fumigatus and shown to be crucial for endothelial cell proliferation and has shown efficacy in preclinical models. Thalidomide, best known for its removal from the market because of severe teratogenicity, has demonstrated the strongest non-VEGF-related anti-angiogenic clinical activity in disorders with abnormal angiogenesis such as refractory multiple myeloma, idiopathic myeloid metaplasia, and Kaposi’s sarcoma. Several agents that target integrins are currently in clinical trials as well. Currently, there are three classes of intergin inhibitors: (1) synthetic peptides (Cilengitide), (2) monoclonoal antibodies (Vitaxin), and (3) peptidomimietics (S247). To date, the most widely used integrin inhibitors have been Cilengitide, which targets avb3 and Vitaxin which targets avb5. However, these agents have performed poorly in clinical trials, suggesting that perhaps avb3 and avb5 are not the best integrins to target. Ongoing efforts are currently trying to develop inhibitors of other integrins involved in angiogenesis. In conclusion, the preclinical experimental evidence establishing that tumor growth is dependent on blood vessel growth has translated into new treatment modalities for providing a higher standard of care for cancer patients. This success has established anti-angiogenic therapy and opened new avenues to develop additional approaches to anti-angiogenic therapy with results superior to VEGF inhibition alone.

9.5.2

Macular Degeneration

Neovascular “wet” age-related macular degeneration (AMD) is a degenerative disease of the central region of the retina (the macula) that leads to loss of central

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vision. The disease results from inappropriate blood vessel growth from the choroidal vasculature in the retinal space. As VEGF is thought to be the primary inducer of this process, ranibizumab (Lucentis), a Fab fragment of an anti-VEGF monoclonal antibody and pegaptinib (an aptamer against VEGF165) were developed for the purpose of treating AMD. Both agents have proven to be successful in preventing the progression of AMD and gained FDA approval. Notably, ranibizumab (Lucentis) not only prevented vision loss but also improved vision in a significant number of patients suffering from AMD. Currently, the use of bevacizumab (Avastin) for the treatment of AMD is being evaluated as this drug is less costly than ranibizumab or pegaptinib (Ho and Kuo 2007). Similar promising AMD studies are underway with VEGF Trap.

9.5.3

Diabetic retinopathy

Diabetic retinopathy is a major cause of morbidity in patients with type 1 and 2 diabetes. In this disease, chronic elevation of blood glucose levels causes vascular changes that result in damage and ischemia to the underlying retinal tissue. As the disease progresses, multiple retinal cell types release VEGF in an abortive attempt to revascularize the ischemic region of the retina. This process creates a condition known as proliferative retinopathy where neovascularization and increased vascular permeability, both of which are induced by VEGF, further damage the retina. Clinical studies have begun to document regression of diabetes-related neovascularization following treatment with the VEGF inhibitors bevaczumab (Avastin), ranibizumab, or pegaptinib which are already approved by the FDA for use in treating cancer or AMD as described above.

9.6

Summary

Angiogenesis, which is crucial for development of multiple diseases, is governed by a very delicate balance of positive and negative regulators which regulate endothelial cell state between the quiescent, the migratory tip cell and the proliferative stalk cell phenotypes to form the immature vascular plexus composed solely of endothelial cells. This process is regulated by VEGF, angiopoietin, Dll4/Notch, and integrin signaling as described above. The newly formed vascular plexus then undergoes maturation through recruitment of mural cells utilizing a combination of PDGF and angiopoietin signaling to stabilize the vasculature. Our understanding of this process has led to the development of over 30 angiogenesis inhibitors that are either making their way through clinical trials or have already proven to be successful in treating vascular dependent diseases, and represent a promising new modality for treatment of these disorders.

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References Andrae J, Gallini R, Betsholtz C (2008) Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 22:1276–1312 Armulik A, Abramsson A, Betsholtz C (2005) Endothelial/pericyte interactions. Circ Res. 97:512–523 Bobik A (2006) Transforming growth factor-betas and vascular disorders. Arterioscler Thromb Vasc Biol. 26:1712–1720 Ferrara N, Gerber HP, LeCouter J (2003) The biology of VEGF and its receptors. Nat Med. 9:669–676 Ho QT, Kuo CJ (2007) Vascular endothelial growth factor: biology and therapeutic applications. Int J Biochem Cell Biol. 39:1349–1357 Holmes K, Roberts OL, Thomas AM, Cross MJ (2007) Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell Signal. 19:2003–2012 Kerbel RS (2008) Tumor angiogenesis. N Engl J Med. 358:2039–2049 Kuhnert F, Mancuso MR, Hampton J, Stankunas K, Asano T, Chen CZ, Kuo CJ (2008) Attribution of Angiogenesis Phenotypes of the Murine Egfl7 Locus to the microRNA miR-126. Development. 135:3989–3993 Makinde T, Agrawal DK (2008) Intra and extravascular transmembrane signalling of angiopoietin-1-Tie2 receptor in health and disease. J Cell Mol Med. 12:810–828 Olofsson B, Jeltsch M, Eriksson U, Alitalo K (1999) Current biology of VEGF-B and VEGF-C. Curr Opin Biotechnol. 10:528–535 Olofsson B, Pajusola K, Kaipainen A, von Euler G, Joukov V, Saksela O, Orpana A, Pettersson RF, Alitalo K, Eriksson U (1996) Vascular endothelial growth factor B, a novel growth factor for endothelial cells. Proc Natl Acad Sci U S A. 93:2576–2581 Otrock ZK, Makarem JA, Shamseddine AI (2007) Vascular endothelial growth factor family of ligands and receptors: review. Blood Cells Mol Dis. 38:258–268 Pugh CW, Ratcliffe PJ (2003) Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 9:677–684 Roca C, Adams RH (2007) Regulation of vascular morphogenesis by Notch signaling. Genes Dev. 21:2511–2524 Shibuya M (2006) Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol. 39:469–478 Shibuya M, Claesson-Welsh L (2006) Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res. 312:549–560 Shibuya M, Ito N, Claesson-Welsh L (1999) Structure and function of vascular endothelial growth factor receptor-1 and -2. Curr Top Microbiol Immunol. 237:59–83 Silva R, D’Amico G, Hodivala-Dilke KM, Reynolds LE (2008) Integrins: the keys to unlocking angiogenesis. Arterioscler Thromb Vasc Biol. 28:1703–1713 Tammela T, Enholm B, Alitalo K, Paavonen K (2005) The biology of vascular endothelial growth factors. Cardiovasc Res. 65:550–563

Part III

Signaling Platforms

Chapter 10

Spatial and Temporal Control of Cell Signaling by A-Kinase Anchoring Proteins F. Donelson Smith, Lorene K. Langeberg, and John D. Scott

10.1

Introduction

Protein phosphorylation is the principal means of reversibly controlling cell signaling events. Our current appreciation of the role of phosphorylation has grown out of early experiments by Fischer and Krebs (1955). They demonstrated that conversion of inactive muscle phosphorylase b into active phosphorylase a requires ATP and is catalyzed by a phosphorylase b kinase. They then showed that phosphorylase b kinase itself is controlled by a cAMP-stimulated serine kinase. These findings allowed Fischer, Krebs and colleagues to identify protein kinase A (PKA) and formulate the concept of a “kinase cascade” (Krebs et al. 1959). Subsequently, the field of protein kinase research has flourished, ever growing in importance with discoveries such as the identification of phosphotyrosine by Hunter and co-workers (Eckhart et al. 1979), the discovery of Src tyrosine kinase by Brugge, Ericsson and co-workers (Brugge et al. 1979), the seminal work of Pawson and colleagues on phosphotyrosine recognition motifs (Sadowski et al. 1986), and the crystallization and structural determination of the PKA catalytic subunit by Taylor and colleagues (Knighton et al. 1991). We now know that the ~500 members of the human kinome represent a superfamily of enzymes that participate in all aspects of cellular regulation (Manning et al. 2002). Protein kinase research is now focusing on how these enzymes are organized in relation to their effectors and substrates within the three dimensions of the cell. Interestingly, the study of PKA is shedding new light on the complexity of these protein–protein interactions. A-kinase anchoring proteins (AKAPs), a family of functionally related proteins that anchor PKA and other signaling enzymes, have emerged as important signal-organizing components for PKA cascades. These proteins

J.D. Scott (*), F.D. Smith and L.K. Langeberg Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, HSC Room K-336, Seattle, WA, 98195, USA e-mail: [email protected]

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_10, © Springer-Verlag Berlin Heidelberg 2010

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also serve as models for understanding how cell signaling is organized in time and space by anchoring and scaffolding proteins.

10.2

Early Appreciation of a Role for Compartmentalization

Early physiological experiments clearly showed that stimulation of cAMP synthesis by different agonists produces distinct outputs, even within the same tissue. These observations suggested that activation of particular G-protein-coupled receptors (GPCRs) favored the production of PKA pools located in different subcellular compartments. Conclusive evidence supporting this concept was not obtained until the early 1980s, when it was shown that adrenergic stimulation selectively activates PKA associated with the particulate fraction in cardiomyocytes, while stimulation with prostaglandin E1 (which induces different physiological effects) activates a cytosolic pool of PKA in the same cells (Buxton and Brunton 1983). These findings were consistent with earlier biochemical studies showing that the PKA holoenzyme, comprised of two regulatory subunits and two catalytic subunits, exists in two forms: a type I PKA holoenzyme, that was, at the time, thought to be cytoplasmic, and a type II PKA holoenzyme, which was considered to be exclusively particulate. However, direct evidence that the two PKA holoenzymes were retained in different subcellular compartments was lacking. The missing link was provided in 1982 by Theurkauf and Vallee, who demonstrated that type II PKA co-purifies with microtubules and that the regulatory RII subunit binds to the microtubule-associated protein MAP2 (Theurkauf and Vallee 1982). These data marked the first identification of an AKAP. Many more of these RII-binding proteins were detected by Lohmann and co-workers (1984) using an overlay technique to probe proteins immobilized on nitrocellulose membranes with the purified RII subunit. By the late 1980s, Rubin, Erlichman and co-workers (Leiser et al. 1986; Bregman et al. 1989) had used this technology to characterize a bovine brain protein, called P75, and its murine ortholog, P150 (these proteins are now known as AKAP75 (human AKAP79) and AKAP150, respectively. Scott and co-workers used this technique in the early 1990s to show that the RII subunit must be dimerized for interaction with AKAPs and that each anchoring protein contains a reciprocal binding sequence of 14–18 amino acids that form an amphipathic helix (Scott et al. 1990; Carr et al. 1991). The RII overlay assay was then adapted to screen phage cDNA libraries for the purpose of cloning novel AKAPs (Carr and Scott 1992). One of these proteins, initially called Ht31 but now known as AKAP-Lbc, contains a sequence of 18 amino acids that has been used as a peptide disruptor of RII–AKAP interactions inside cells (Carr et al. 1992a,b). The action of this peptide as a disruptor of PKA anchoring was first demonstrated in studies showing that perfusion of Ht31 into cultured hippocampal neurons disrupts the location of PKA in relation to a key substrate, the a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)type glutamate receptor (Rosenmund et al. 1994). The functional consequence of displacing PKA from AMPA receptors was to decrease the responsiveness of the ion

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channel to synaptic signals (Rosenmund et al. 1994). At the same time, Catterall’s group (Johnson et al. 1994) used the Ht31 peptide to demonstrate that disruption of PKA anchoring uncouples cAMP-dependent regulation of the L-type Ca2+ channel. In 1997, a cell-soluble version of Ht31 was generated by conjugating a steroyl group to the N terminus (Vijayaraghavan et al. 1997). This peptide and other Ht31 derivatives are now widely used to establish whether anchored pools of PKA participate in various cAMP signaling events (Burns-Hamuro et al. 2003; Alto et al. 2003). Elegant structural studies from Jennings and colleagues (Newlon et al. 1999, 2001) have shown that a hydrophobic face on Ht31 fits into a groove created by the N-terminal regions of the RII dimer, forming a nanomolar-affinity complex. Related work from Taylor’s group (Huang et al. 1997a,b) has shown that a subset of dual-specificity AKAPs can also interact with the RI subunit. Thus, by the late 1990s, two important biochemical properties of AKAPs were emerging: first, AKAPs bind the R subunit dimer through a well-conserved amphipathic a-helical motif; and second, each anchoring protein is targeted to a unique localization in a given cell type by an identifiable targeting motif (Tasken and Aandahl 2004).

10.3

AKAP Signaling Complexes

Another biological role of AKAPs became apparent when it was discovered that AKAP79 (bovine AKAP75 and rodent AKAP150) bound the protein phosphatase PP2B in addition to anchoring PKA (Coghlan et al. 1995). This finding was especially important, as it suggested that signals controlling phosphorylation and dephosphorylation of a single substrate can pass through the same AKAP signaling complex. This notion of multivalent AKAPs continued to evolve when subsequent studies showed that AKAP79 also binds protein kinase C (PKC), thereby providing a means to integrate cAMP with Ca2+ and phospholipid signals at the same subcellular locus (Klauck et al. 1996). Further work has demonstrated that Gravin (AKAP250) and AKAP-Lbc colocalize PKA with PKC, whereas AKAP220 and AKAP149 place PKA in close proximity to the type 1 protein phosphatase PP1 (Nauert et al. 1997; Carnegie et al. 2004; Schillace and Scott 1999; Steen et al. 2000). There is now reason to believe that most, if not all, anchoring proteins bring PKA together with other protein kinases, protein phosphatases and other signaling enzymes. In the following sections, we discuss the role of these AKAP signaling complexes in the synchronization of compartmentalized signal transduction events.

10.4

AKAPs and Phosphodiesterases: Compartmentalization of cAMP Action

Control of cAMP flux requires the coordinated action of two enzyme classes: adenylyl cyclases that synthesize cAMP from ATP (occurring primarily at the plasma membrane), and intracellularly compartmentalized pools of phosphodiesterases

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(PDEs), which locally metabolize cAMP into 5¢-AMP. The Houslay and Conti laboratories have identified several PDE-binding proteins that target distinct isozymes to specific cellular microenvironments (Houslay and Adams 2003). A prototypical example is PDE4D3, which is differentially localized via interactions with scaffolding proteins such as myomegalin and b-arrestin (Yarwood et al. 1999; Perry et al. 2002; Verde et al. 2001). Furthermore, many AKAPs cluster PKA with PDEs to terminate cAMP signals as they diffuse into the cell (Dodge et al. 2001; Baillie et al. 2005). In cardiomyocytes, the muscle-selective AKAP (mAKAP) assembles a negative-feedback loop containing PKA and PDE4D3 (Dodge et al. 2001) (Fig. 10.1a). PKA-mediated phosphorylation of Ser13 in PDE4D3 augments mAKAP binding (Carlisle-Michel et al. 2004), whereas phosphorylation of Ser54 enhances the catalytic efficiency of the enzyme to favor cAMP metabolism (Sette and Conti 1996). These effects are counterbalanced by extracellular signal-regulated kinases (ERKs) that phosphorylate PDE4D3 on Ser579 to suppress PDE activity (Hoffmann et al. 1999). This latter phosphorylation event might be catalyzed by ERK5, which is also a component of the mAKAP complex (Dodge-Kafka et al. 2005; Michel et al. 2005). This configuration not only ensures bidirectional control of PDE4D3 activity but also has been postulated to generate local fluctuations in cAMP and concomitant pulses of PKA activity (Fig. 10.1a). Work from Tasken and colleagues (2001) suggests that AKAP450 performs an analogous function at centrosomes, where it organizes a signaling complex of PKA, PDE4D3 and the protein phosphatases PP1 and PP2A (Fig. 10.1b). Likewise, AKAP110 targets the PDE4A isoform to the acrosome of sperm, whereas

Fig. 10.1 AKAPs that bring together PKA and PDEs. (a) mAKAP tethers PKA and PDE4D3 at the nuclear membrane to regulate the local flux of cAMP. An increase in local cAMP activates PKA, which phosphorylates PDE4D3 to increase its catalytic activity and to enhance its association with mAKAP. The activity of PDE4D3 eventually reduces cAMP back to basal concentrations and shuts off the kinase. PDE4D3 also acts as a scaffold to bring ERK5 (and its upstream activating kinase MEK5) into the complex. ERK5 negatively regulates PDE4D3 activity by phosphorylating it on distinct sites. This arrangement enables mAKAP and PDE4D3 to act as integrators of cAMP and mitogenic signaling pathways. (b) AKAP450 has a similar function at centrosomes, where it anchors PKA, PDE4D3 and two phosphatases. (c) Members of the MTG family are localized to the Golgi, where they target PKA and the PDE7A isoform

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AKAP149 and AKAP121, as well as the Nervy and myeloid translocation gene (MTG) anchoring proteins, respectively, target the PDE4A and PDE7A isoforms to various subcellular locations (Asirvatham et al. 2004; Bajpai et al. 2006) (Fig. 10.1c). These AKAP–PKA–PDE units respond to upstream signals that emanate from GPCR and adenylyl cyclase networks, creating a complex and continually changing signaling environment in which cAMP concentrations are distributed unevenly in the cell. These findings underscore the pleiotropic nature of this important second messenger and highlight the central role of AKAPs in the customized regulation of cAMP signaling.

10.5

Phosphorylation in AKAP Complexes

Soon after AKAPs were discovered, it was postulated that they that would be the preferred substrates for their associated kinases (Coghlan et al. 1993). Although experimental evidence supporting this prediction has been slow in coming, three recent examples suggest that it might be correct. First, the AKAP Yotiao associates with various ion channels, including the KCNQ1 subunit of K+ channels responsible for IKS currents that shape the duration of cardiac action potentials in response to b-adrenergic agonists (Westphal et al. 1999) (Fig. 10.2a). Inherited mutations in KCNQ1 subunits have been linked to long QT syndrome (LQTS), a disease characterized by cardiac arrhythmias and sudden death (Kass and Moss 2003). Intriguing work from Kass and co-workers (Marx et al. 2002; Chen et al. 2005) shows that the anchored PKA phosphorylates Ser43 in Yotiao to enhance cAMP-dependent activation of IKS. Although the precise mechanism is not clear, it seems that a disruption of interactions between the AKAP and the KCNQ1 subunit might be responsible for the impaired b-adrenergic regulation of IKS detected in some individuals with KCNQ1 mutations. Second, Gravin (also known as AKAP250) is another anchoring protein that binds PKA and PKC. Work from Malbon and colleagues (Shih et al. 1999; Tao et al. 2003) suggests that Gravin is a substrate for its own associated kinases and that phosphorylation facilitates the association of this AKAP with agonist-occupied b-adrenergic receptors. Third, AKAP-Lbc is a multifunctional PKA- and PKC-anchoring protein that acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rho and synchronizes the activation of a third protein kinase called protein kinase D (PKD) (Diviani et al. 2001; Klussmann et al. 2001; Carnegie et al. 2004). Two recent studies show that PKA-mediated phosphorylation of Ser1565 in AKAP-Lbc generates a binding site for 14-3-3 proteins, which suppress the Rho-GEF activity of AKAPLbc (Fig. 10.2b) (Diviani et al. 2004; Jin et al. 2004). The synergistic actions of anchored PKA and PKC also contribute to activation of PKD. Phosphorylation by PKCh primes PKD for activation, whereas PKA-mediated phosphorylation of Ser2737 in AKAP-Lbc releases PKD from the activation complex (Carnegie et al. 2004) (Fig. 10.2b).

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Fig. 10.2 Phosphorylation of AKAPs regulates their function. (a) Yotiao couples PKA and PP1 to KCNQ1 subunits that are part of the channel responsible for IKS currents. b2-adrenergic signaling activates PKA, which then phosphorylates the channel and Yotiao, leading to cAMP-dependent activation of the channel. Yotiao seems to act as both a scaffold for coordinating local phosphorylation or dephosphorylation events and an accessory subunit that shapes channel output. (b) AKAP-Lbc is a Rho-GEF that also anchors PKA, PKC and PKD. Phosphorylation of AKAPLbc by anchored PKA regulates the activation of PKD via PKC and inhibits Rho-GEF activity by inducing 14-3-3 binding

These examples of highly localized phosphorylation events that occur in AKAP signaling complexes highlight the utility of kinase anchoring as a means to restrict the substrate accessibility of broad-spectrum enzymes such as PKA and PKC.

10.6

Combinatorial Assembly of Distinct AKAP Signaling Complexes

A basic premise of AKAP action is that signaling specificity is achieved through targeting various enzymes towards selected substrates. This use of distinct enzyme combinations provides a way in which to expand the repertoire of cellular events that can be modulated by a given AKAP. In 2005, Hoshi and co-workers (2005) demonstrated this concept with the finding that AKAP79/150 coordinates different enzyme combinations to modulate the activity of two distinct neuronal ion channels: AMPA-type glutamate receptors and KCNQ2 potassium channels (Fig. 10.3a,b) (Hoshi et al. 2005). These studies investigated the mechanism of agonist-induced rundown of AMPA currents, a process that is known to involve AKAP79/150 (Fig. 10.3a). PKA-mediated phosphorylation of the AMPA channel stabilizes the current (Banke et al. 2000) and this process is opposed by PP2B-mediated dephosphorylation of the channel (Tavalin et al. 2002).

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a

b

c M-Channel

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Fig. 10.3 Combinatorial assembly of distinct enzyme complexes on AKAP79. This AKAP is coupled to AMPA glutamate receptors, KCNQ2 channel complexes and b-adrenergic receptors, where it can anchor PKA, PKC and PP2B. Shown are the configurations of the AKAP79 complex that regulates synaptic AMPA receptor function (a), the AKAP79 complex that functions in the muscarinic suppression of M currents in SCG neurons (b), and the AKAP79 complex that is associated with b-adrenergic receptors (c). Different combinations of AKAP79-binding partners are used with each substrate

By combining RNA interference of the endogenous protein in neurons and replacement with AKAP79/150 forms unable to anchor selected binding partners, Hoshi and co-workers (2005) showed that PP2B anchoring is primarily responsible for AMPA channel rundown. The same strategy applied to cervical ganglion neurons (SCGs) demonstrated that AKAP79/150 and PKC are involved in modulating M current (a K+ conductance that negatively regulates neuronal excitability) (Hoshi et al. 2005). These data point to a fascinating situation. In hippocampal neurons, AKAP79/150 coordinates PKA- and PP2B-mediated modulation of AMPA currents, but any AKAP79/150-associated PKC remains inactive in this process. By contrast, AKAP79/150 enables PKC to facilitate M-current regulation in SCG neurons, whereas PKA and PP2B seem to be non-essential (Fig. 10.3b). Interestingly, AKAP79/150 can also bind to b-adrenergic receptors (Fraser et al. 2000; Cong et al. 2001; Lynch et al. 2005). Potentially, this binding permits the assembly of a third AKAP79/150 signaling complex in which PKA contributes to phosphorylation-dependent downregulation of the b-adrenergic receptors (Fig. 10.3c). Although the contextual cues that drive the preferential assembly of these three different AKAP79/150 complexes are unclear, one possibility is that the initial binding event between the anchoring protein and its target substrate promotes a sequence of conformational changes that directs recruitment of the next binding partners. For example, the association of AKAP79/150 with the KCNQ2 channel might provide a configuration that retains the membrane tethering and anchoring of PKC. Similarly, the formation of a ternary complex containing AMPA channels, the MAGUK adaptor protein and AKAP79/150 might have to be established before PKA and PP2B can be orientated towards the channel. Other factors, however, such as the co-translational assembly of protein complexes via localized protein synthesis, or species-specific or cell-type-specific expression of particular binding partners

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might also influence the composition of these “context-dependent” signaling networks (Fig. 10.3) (Chen and Kass 2005). Another way in which to vary the “context-dependent” modulation of signaling networks might be to induce time-dependent changes in AKAP complexes. For example, pathophysiological changes in the mAKAP complex have been recently linked to some forms of heart failure. Ryanodine receptors (RyRs) are intracellular Ca2+ release channels that are present in multiprotein signaling complexes at the sarcoplasmic reticulum in muscle cells to mediate excitation–contraction coupling. Upon b-adrenergic stimulation, anchored PKA phosphorylates the RyR to sensitize the channel to activation by an increase in Ca2+. Much work suggests that chronic changes in the mAKAP–RyR1 complex, including loss of anchored PDE4D3 and hyperactivation of the anchored PKA, correlate with the onset of “leaky” channels found in some models of exercise-induced cardiac arrhythmias and heart failure (Marx et al. 2001; Lehnart et al. 2005). Whether these changes in the mAKAP complex can be detected routinely in individuals affected with some types of heart disease, or whether this protein represents a viable therapeutic target for intervention, remains to be seen. Nonetheless, these findings yet again support an active role for AKAPs in synchronization of physiologically relevant signaling events.

10.7

Time: A Critical Dimension in Signaling

Although we have identified most, if not all, of the proteins that make up the cAMP signal transduction cascade, we still face the challenge of resolving the mechanics of their action in real time. Fluorescent probes that report the activation dynamics of cAMP effector proteins such as PKA, cyclic-nucleotide-gated (CNG) ion channels and Epac-GEFs have become the methods of choice to visualize the dynamics of cAMP signaling inside cells. Pioneering work by Roger Tsien’s laboratory led to the development of a PKA-based probe that could monitor cAMP production by the loss of fluorescence resonance energy transfer (FRET) between recombinant regulatory (R) and catalytic (C) subunits conjugated with fluorescein and rhodamine dyes, respectively (Fig. 10.4a) (Adams et al. 1991). A decade later, Zaccolo and Pozzan (2002) improved this technique by creating a genetically encoded PKA reporter that recorded FRET between a yellow fluorescent protein (YFP)-conjugated C subunit and a cyan fluorescent protein (CFP)conjugated R subunit (Fig. 10.4c). This reporter was successfully used to measure microdomains of cAMP along sarcomeric Z lines in cardiomyocytes in response to adrenergic stimulation. Zaccolo, Pozzan and co-workers (Mongillo et al. 2004) have shown that AKAPs are involved in anchoring the kinase to these regions and that compartmentalized pools of PDE3 and PDE4 suppress signals located in these cAMP microdomains. Other investigators have created biosensors based on the properties of CNG channels and Epacs. Ca2+ influx through CNG ion channels is stimulated in response to an increase in cAMP. When coupled with the Ca2+-sensitive indicator

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Fig. 10.4 Evolution of real-time biosensors for activation of cAMP and PKA. The past 15 years have seen the steady development of cAMP biosensors. (a) In 1991, fluorescently labeled regulatory (R) and catalytic (C) subunits were microinjected into cells for monitoring intermolecular FRET. (b) By 2001, activation of cAMP could be detected by imaging the Ca2+ flux through CNG ion channels that are highly responsive to cAMP. Inset, data show that isoproterenol stimulation of HEK293 cells causes rapid accumulation of cAMP, as measured by changes in the signal from the Ca2+ indicator dye Fura-2. This dye has different absorbance wavelengths depending on whether Ca2+ is bound, allowing ratiometric measurement of Ca2+ concentrations. (c) In 2002, a genetically encoded reporter of PKA activation was developed that detects a loss of intermolecular FRET on dissociation of a CFP-conjugated R subunit from a YFP-conjugated C subunit. (d) In 2004, a FRET-based biosensor directly responsive to cAMP, ICUE1 (indicator of cAMP using Epac), was developed in which the cAMP-binding region of Epac1 is labeled with both enhanced CFP (ECFP) and citrine. Inset, data show that FRET decreases in response to a rise in cAMP in (continued)

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Fura-2 and PDE inhibitors, mutants of cAMP-selective CNG channels function as real-time biosensors of cAMP accumulation at the plasma membrane (Fig. 10.4b) (Fagan et al. 1999; Rich et al. 2000, 2001). Likewise, the cAMP-binding domain of the Epac-GEF has been used to generate fluorescent cAMP biosensors that detect compartmentalized accumulation of the second messenger (Fig. 10.4d) (Nikolaev et al. 2004; DiPilato et al. 2004). FRET-based reporters of PKA activity provide an alternative to measuring cAMP concentrations. AKAR2 is a chimeric protein consisting of CFP, a consensus PKA substrate sequence, a Forkhead Homology (FHA) domain that binds phosphoamino acids, and the YFP variant citrine. PKA-mediated phosphorylation of the PKA consensus site engages the FHA domain to enhance FRET between the fluorescent moieties (Zhang et al. 2001, 2005) (Fig. 10.4e). This reporter also samples local phosphatase activity, because FRET decay is dependent on dephosphorylation of the PKA site and relaxation of the molecule. AKAR2 reporters have been recently used to examine two aspects of PKA activation dynamics. First, chronic insulin treatment delays activation of PKA through b-adrenergic receptors in differentiated 3T3-L1 adipocytes. Work by Zhang and co-workers (2005) suggests that in chronic hyperinsulinemia complexes of the b-adrenergic receptor and AKAP are decompartmentalized in relation to the cAMP synthesis machinery. Second, Dodge-Kafka and colleagues (2005) have used a modified AKAR2 reporter to show that recruitment of PKA and a PDE into the FRET reporter complex generates localized pulses of cAMP that are shorter in duration than when PDE is not present (Fig. 10.4e and 10.4f). Thus, various cAMP-responsive events that have differing durations and are responsive to distinct thresholds of cAMP might emanate from the same microdomain. This possibility would be particularly relevant to mAKAP signaling complexes that contain three functionally distinct cAMP-dependent enzymes (PKA, PDE4D3 and Epac1): PKA is responsive to nanomolar concentrations of cAMP and would become active early in a second messenger response; by contrast, the activities of PDE4D3 (Michaelis constant, Km = 1–4 mM) and Epac1 (dissociation constant, Kd = 4 mM) would commence only when cAMP accumulated to micromolar concentrations (Dodge-Kafka et al. 2005). Conversely, inactivation of PDE4D3 and Epac1 would precede reformation of the PKA holoenzyme as the cAMP concentrations declined. Fig. 10.4 (continued) HeLa cells; this translates to a higher ECFP/citrine emission ratio. (e) FRET reporters that monitor PKA phosphorylation. First developed by the Tsien group in 2001 (77), the AKAR reporters use phosphorylation-dependent intramolecular interactions as an index of PKA activation. More recent versions of this reporter contain the FRET donor and acceptors (ECFP and citrine), a consensus PKA site and an FHA domain (top) On phosphorylation by PKA, the FHA domain binds to the substrate phosphothreonine and FRET increases. This reporter has been modified to study signaling by anchored PKA and the role of PDEs by incorporating an RIIbinding peptide (PKA BD), as in AKAR–PKA (bottom left), or an RII-binding peptide and a PDE4D3-binding domain (PDE4 BD; bottom right) Coupling of PKA to the AKAR reporter leads to long-lasting PKA activity and FRET (left) The duration of this signal is attenuated when PDE4D3 is bound to the reporter (right)

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Concluding Remarks

We anticipate that these cutting-edge imaging technologies will be central to obtaining precise definitions of the spatial and temporal patterns of anchored kinase function in the next decade. The information gained could be used in conjunction with computational models of signal transduction to predict the effects of perturbing the system. Ultimately, a balance of both approaches might contribute to the development of therapeutics that target “signaling diseases” such as heart failure, asthma, diabetes and cancer. Therefore, pharmacological manipulation of kinase signaling in space and time will ultimately give us more control over where and when things happen in the cell. Acknowledgments We thank past and present members of the Scott Laboratory for helpful discussions. J.D.S. was supported in part by a grant from the National Institutes of Health (DK54441).

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cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ Res 95:67–75 Nauert JB, Klauck TM, Langeberg LK, Scott JD (1997) Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein. Curr Biol 7:52–62 Newlon MG, Roy M, Morikis D, Hausken ZE, Coghlan V, Scott JD, Jennings PA (1999) The molecular basis for protein kinase A anchoring revealed by solution NMR. Nat Struct Biol 6:222–227 Newlon MG, Roy M, Morikis D, Carr DW, Westphal R, Scott JD, Jennings PA (2001) A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes. EMBO J 20:1651–1662 Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218 Perry SJ, Baillie GS, Kohout TA, McPhee I, Magiera MM, Ang KL, Miller WE, McLean AJ, Conti M, Houslay MD, Lefkowitz RJ (2002) Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins. Science 298:834–836 Rich TC, Fagan KA, Nakata H, Schaack J, Cooper DM, Karpen JW (2000) Cyclic nucleotidegated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion. J Gen Physiol 116:147–161 Rich TC, Tse TE, Rohan JG, Schaack J, Karpen JW (2001) In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors. J Gen Physiol 118:63–78 Rosenmund C, Carr DW, Bergeson SE, Nilaver G, Scott JD, Westbrook GL (1994) Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 368:853–856 Sadowski I, Stone JC, Pawson T (1986) A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps. Mol Cell Biol 6:4396–4408 Schillace RV, Scott JD (1999) Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220. Curr Biol 9:321–324 Scott JD, Stofko RE, McDonald JR, Comer JD, Vitalis EA, Mangili JA (1990) Type II regulatory subunit dimerization determines the subcellular localization of the cAMP-dependent protein kinase. J Biol Chem 265:21561–21566 Sette C, Conti M (1996) Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation. J Biol Chem 271:16526–16534 Shih M, Lin F, Scott JD, Wang HY, Malbon CC (1999) Dynamic complexes of beta2-adrenergic receptors with protein kinases and phosphatases and the role of gravin. J Biol Chem 274:1588–1595 Steen RL, Martins SB, Tasken K, Collas P (2000) Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly. J Cell Biol 150:1251–1262 Tao J, Wang HY, Malbon CC (2003) Protein kinase A regulates AKAP250 (gravin) scaffold binding to the beta2-adrenergic receptor. EMBO J 22:6419–6429 Tasken KA, Collas P, Kemmner WA, Witczak O, Conti M, Tasken K (2001) Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area. J Biol Chem 276:21999–22002 Tasken K, Aandahl EM (2004) Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev 84:137–167 Tavalin SJ, Colledge M, Hell JW, Langeberg LK, Huganir RL, Scott JD (2002) Regulation of GluR1 by the A-kinase anchoring protein 79 (AKAP79) signaling complex shares properties with long-term depression. J Neurosci 22:3044–3051 Theurkauf WE, Vallee RB (1982) Molecular characterization of the cAMP-dependent protein kinase bound to microtubule-associated protein 2. J Biol Chem 257:3284–3290

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Chapter 11

Mitochondria, a Platform for Diverse Signaling Pathways Astrid C. Schauss and Heidi M. McBridee

11.1

Introduction

Guided by nearly 50 years of elegant in vitro biochemical experiments, it has become clear that mitochondria fulfill a number of essential functions in the cell, from oxidative phosphorylation to the regulation of cell death. The metabolic processes within mitochondria were thought to be regulated primarily by the availability of substrates, including pyruvate, free phosphates, and ADP. However, the last decade has seen a revision of these ideas. Emerging evidence indicates that complex cells like neurons and muscle fibers require that the positioning and shape of mitochondria be tightly controlled. Intuitively, the regulation of mitochondrial positioning in cells suggests that the machinery driving mitochondrial movement and function must be integrated within cellular signaling cascades. In addition, the internal architecture of mitochondrial cristae reflects the metabolic state of mitochondria (Mannella 2006). This internal structure is highly plastic and dynamic shifts in morphology coincide with a number of physiological events, such as transitions between different respiratory states and cristae remodeling during apoptosis (Zick et al. 2009). The fact that the plasticity of mitochondria is tightly coupled to its function provides an additional opportunity for regulation that will allow the organelle to be highly responsive to the cellular conditions. The molecular basis of mitochondrial dynamics and its links to intracellular signaling has been the subject of intense investigation over the past 10 years. These factors and their relationship to mitochondrial function will be described in more detail in the following section.

H.M. McBridee (*) and A.C. Schauss University of Ottawa Heart Institute, 40 Ruskin St, Ottawa, ON, Canada, K1Y 4W7 e-mail: [email protected]

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The Fusion Machinery

Mitochondrial fusion was initially demonstrated to occur in all cell types. The generation of a mouse from a heterokaryon carrying two distinct populations of mitochondria, each carrying unique mtDNA mutations, provided proof that mitochondrial fusion occurred between these two populations (Nakada et al. 2001; Ono et al. 2001). The fusion between mitochondria carrying different mutations resulted in a functional rescue, where the intact genes from each mitochondria complemented the deficiency of the mutant mtDNA. From these early studies, it was clear that mitochondrial fusion was an ongoing process in physiology. Fusion of mitochondria is essential for the maintenance of the mitochondrial genome because it appears to buffer mitochondrial damage and promote respiration (Detmer and Chan 2007). The machinery that regulates mitochondrial fusion is beginning to be understood, and the signaling cascades that impinge on these pathways are the subject of current investigation. At a mechanistic level, mitochondrial fusion requires the coordinated intermixing of both the outer and inner mitochondrial membranes, a more complex event compared to the more familiar fusion events described for vesicle transport (Fig. 11.1). The core machinery that drives mitochondrial fusion is also unique, without the use of SNARE family members or their related machinery (Hoppins et al. 2007). Instead, the central components for the mitochondrial fusion machinery in mammals includes at least three GTPases. Two mitofusin homologs, Mfn1 and Mfn2, are anchored in the mitochondrial outer membrane with a third GTPase called Opa1 (Optical atrophy 1) located in the intermembrane space. Mfn1 was shown to tether mitochondria by forming coiled-coil interactions with other Mfn1 proteins on the opposing mitochondrial membrane (Koshiba et al. 2004). The formation of protein interactions in trans is functionally reminiscent of the antiparallel coiled-coil complexes formed by SNAREs within other intracellular fusion events (Wickner and Schekman 2008). Mfn2 is almost 60% identical to Mfn1, however, evidence indicates that it has an overlapping yet distinct function in the regulation of mitochondrial fusion (Chen et al. 2007; Ishihara et al. 2004; Neuspiel et al. 2005). In humans, point mutations in Mfn2 cause Charcot–Marie–Tooth (CMT) subtype 2A, a disease characterized by degeneration of the longest motor and sensory nerves, which enervate the hands and feet (Zuchner et al. 2004). The essential nature of mitochondrial fusion is evident from deletion studies in mouse models where the loss of either Mfn1 or Mfn2 results in midgestational lethality (Chen et al. 2003). In addition to Mfn1 and Mfn2, the third dynamin-related GTPase, Opa1, is essential for mitochondrial inner membrane fusion and is implicated in cristae remodeling (Hoppins et al. 2007). Mutations in Opa1 cause a disease that results in degeneration of retinal ganglion cells and loss of visual acuity (Alexander et al. 2000; Delettre et al. 2000). Opa1 is present in eight mRNA splice forms that can undergo proteolytic processing within the mitochondria, producing a complex mixture of long and short isoforms (Akepati et al. 2008; Satoh et al. 2003). Both long and short Opa1 splice variants are important for mitochondrial fusion activity (Zick et al. 2009). The proteases responsible for cleaving Opa1 have been intensely

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Budding Fission

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Fig. 11.1 Mitochondrial dynamics is defined by the balance of motility, fission and fusion. The top image illustrates how mitochondria may be anchored within the cell through actin cytoskeletal elements, with long-range movement facilitated by kinesins and dyneins along microtubule tracks. Constriction around the center of a mitochondrial tubule leads to the formation of smaller fragments, and fusion between two organelles allows the mixing of their contents. Mitochondria have also been shown to produce small vesicles that pinch from the sides of the organelle. The mitochondrial inner membrane cristae are also highly dynamic, with a number of newly identified proteins playing roles in cristae remodeling. The inset contains images of two HeLa cells labeled with a mitochondrial matrix marker showing either a highly fused reticulum (left side) or a fragmented phenotype (right side)

investigated, since their identity could shed light on the regulation of mitochondrial fusion. It appears that the primary protease is an intermembrane space AAA protease called Yme1L; however the matrix AAA proteases, prohibitins and the rhomboid protease PARL contribute to this process as well. The degeneration of the long Opa1 isoforms are stimulated upon the loss of electrochemical potential and during apoptosis. This might be a mechanism to block damaged mitochondria from fusing back into the reticulum (Hoppins et al. 2007). The regulation of mitochondrial outer membrane fusion depends not only on protein complexes but also on changes in the lipid composition of the mitochondrial outer membrane. In mammals, a mitochondrial-anchored phospholipase D protein, MitoPLD, has been shown to be required for mitochondrial fusion (Choi et al. 2006). MitoPLD hydrolyses cardiolipin to phosphatidic acid (PA), a fusogenic lipid (Schlame 2008). The role of PA in mitochondrial fusion is still not understood, but

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it seems to function after Mfn1/2-dependent tethering since its silencing leads to docked mitochondria that cannot fuse (Choi et al. 2006). It is clear that the current list of proteins that function in the regulation of mitochondrial fusion is incomplete and much needs to be done before we will understand the mechanisms that govern this complex process.

11.1.2

The Fission Machinery

Mitochondrial fission is an essential aspect of organelle biogenesis, required for the transmission of mitochondria to the daughter cells during mitosis. The drive to understand this process was key to the development of this new field of mitochondrial dynamics (Yaffe 1999). Yeast geneticists developed screens to identify proteins required for mitochondrial distribution and genome maintenance in dividing cells, with almost all of the players identified to date emerging from these powerful approaches (Altmann and Westermann 2005; Dimmer et al. 2002; McConnell et al. 1990). From these studies it became apparent that the steady-state morphology of the mitochondria is the result of the regulated balance between mitochondrial fusion and mitochondrial fission (Fig. 11.1). This balance is shifted in a number of cellular conditions. For example there is an increase in mitochondrial fragmentation at the onset of mitosis, presumably to facilitate the partitioning of the mitochondrial reticulum between cells (Taguchi et al. 2007). In addition, mitochondria also fragment during apoptosis, a process that appears to be functionally implicated in the progression of the death program (Suen et al. 2008). The signaling pathways that trigger these morphological transitions will be described more fully in the following sections, but first we will describe the machinery that regulates mitochondrial fission. The first major component identified to play a role in mitochondrial fission is the dynamin-related protein Dnm1p in yeast or Drp1 in mammals (Shaw and Nunnari 2002). This GTPase is recruited from the cytosol onto discrete foci on the mitochondria, some of which mark sites of future fission events. Drp1 is thought to act as a mechanoenzyme by forming a spiral around the mitochondrial tubule that constricts upon GTP hydrolysis and by that facilitates the scission of the mitochondria (Ingerman et al. 2005). A second mitochondria fission protein, hFis1, is a small integral outer membrane protein that is involved in the recruitment of Dnm1p to the mitochondria, and is required for the assembly of Drp1 into functional oligomers (Hoppins et al. 2007). In the yeast model, two linkers between Dnm1 and Fis1 were found to be essential, Mdv1 and Caf4 (Westermann 2008). Both share high similarity and partly overlapping functions. The orthologs of these proteins in mammals have not yet been identified. Recently a new mitochondrial fission factor Mff was isolated from a genome-wide RNAi screen for proteins that alter mitochondrial dynamics in Drosophila cells (Gandre-Babbe and van der Bliek 2008). This is another outer membrane, tail-anchored protein that shares many functional features with Fis1. Interestingly Drp1, Fis1 and Mff are all required for peroxisomal fission, suggesting that these two organelles may share common regulatory mechanisms in organelle

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biogenesis. The links between mitochondria and peroxisomes were recently made even stronger with the identification of a unique vesicular transport pathway that delivers mitochondrial cargo directly to the peroxisomes (Neuspiel et al. 2008). The formation of these vesicles from the mitochondria does not require Drp1, indicating that a separate fission mechanism may exist. To date only one mitochondrial protein has been identified as a cargo for these vesicles, which is a mitochondrial-anchored protein ligase called MAPL. The function of MAPL in the peroxisomes is unclear, although it may play a role in regulating peroxisomal morphology since its overexpression stimulates mitochondrial fission (Neuspiel et al. 2008). Further work is required to dissect the functional implications of these novel transport routes.

11.1.3

Motility and Mitochondrial Positioning

The third determinant of mitochondrial morphology and distribution is the regulation of mitochondrial motility along cytoskeletal tracks. The ability to move from one area of the cell to another allows an increase in the local production of energy, but can also provide local calcium sinks to buffer spikes in calcium and other ion fluctuations. More recent data points to an additional role as a unique signaling platform, where intermediates in known signaling cascades are anchored within the mitochondrial outer membrane (McBride et al. 2006). This emerging area of research suggests that the local positioning of the mitochondria relative to the plasma membrane or other organelles may be important for the propagation of signaling cascades. This hints that the machinery that moves the mitochondria must also be tightly regulated. Mitochondria are mainly transported along microtubules in both directions using kinesin and dynein motors (Frederick and Shaw 2007). The mechanisms of recruitment of these motors to mitochondria are unclear, but at least two proteins have been found to serve as adaptor complexes that anchor kinesin-1 to mitochondria. Miro is a mitochondrial outer membrane GTPase with a calcium-binding motif (Soubannier and McBride 2009), suggesting that the movement of mitochondria is regulated by both GTPase switch mechanisms and calcium. Miro is found in a complex with a cytosolic, coiled-coil containing protein called Milton, which functions as an organelle-specific kinesin light chain (Glater et al. 2006). These adaptor proteins may link the mitochondria to the cytoskeleton; however, it is unclear how these processes are regulated. The direction of mitochondrial movement may depend on its energetic state since it has been shown in neurons that mitochondria with a high membrane potential preferentially migrate from the cell body to the distal portion, whereas mitochondria with low membrane potential progress in the opposite direction (Miller and Sheetz 2004). The selective delivery of the “fittest” mitochondria to the active nerve terminals would have important functional implications and might explain why so many neurodegenerative diseases are linked to dysfunctions in mitochondrial dynamics. The shape and motility of mitochondria has also been shown to have implications for cellular development. For example, the initiation and generation of dendritic

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spines requires that mitochondria be in a fragmented state, presumably to be able to migrate across the narrow junctions into the spine (Fig. 11.2). When mitochondrial fission was stimulated by overexpressing the fission protein Drp1, mitochondria become more fragmented and mobile. This results in an increased number of mitochondria within the dendrite as well as an increased density of dendritic spines and synapses. Conversely, in hippocampal neurons expressing mutant Drp1, mitochondria are fused and remain primarily within the soma, resulting in very few mitochondria migrating into the dendrite (Li et al. 2004). Similarly the loss of Miro in Drosophila neurons not only leads to accumulation of mitochondria within the neuronal cell bodies, but the absence of mitochondria from the neuromuscular junctions leaves the larva with impaired movement (Guo et al. 2005). These studies

ATP 2+ Ca -flux

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Fig. 11.2 Spatial distribution of mitochondria in neurons is critical for development and synaptic function. This illustration of a neuron shows the cell body with dendrites and dendritic spines in the top inset, and the synaptic junction at the tip of an axon in the lower inset. Mitochondrial fragments have been shown to be requisite for the formation of dendritic spines, with long, tubular mitochondria leading to a reduction in spine number. The regulated movement of mitochondria with high electrochemical potential to the synaptic terminals leads to an enrichment of functional mitochondria. These organelles help facilitate synaptic transmission through the local production of ATP and in their capacity to buffer intracellular calcium

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highlight the importance of mitochondrial positioning in the developing organism and help to provide model systems to study the mechanisms that regulate these processes in time and space.

11.1.4

The Functional Requirement for Mitochondrial Plasticity

The shape and position of the mitochondria can obviously allow the local production of ATP within the cell; however, it also appears that the dynamic properties of the mitochondria are also essential for their ability to function as the cellular engine. It was shown that under conditions of high glucose where mitochondria must catabolize pyruvate very quickly, mitochondria were seen to undergo Drp1dependent fragmentation within the first 10–15 mins after glucose addition (Yu et al. 2006). This process was reversible, with the reformation of the fused reticulum occurring between 30–60 mins following the treatment. This transient fragmentation was critical for the breakdown of glucose since the inhibition of fission blocked pyruvate uptake into the mitochondria and the transient generation of ROS (reactive oxygen species). This example emphasizes the control of mitochondrial metabolism by the morphological state of the organelle (Yu et al. 2006). During the development of certain tissues, the mitochondrial network undergoes dramatic remodeling events as well. Electron microscopy studies have revealed a transition from rod-like mitochondria in embryonic rat myocardiocytes to interconnected reticuli in the cardiac muscle of adult animals (Skulachev 2001). Strikingly, these timed mitochondrial remodeling events correlate with a higher expression level of Mfn2 during differentiation of myoblasts (Bach et al. 2003). The metabolic requirement for mitochondrial plasticity is likely to be primarily due to the dynamic remodeling of the cristae junctions (Zick et al. 2009). Alterations in cristae morphology have been associated with altered metabolism, since the highly invaginated membrane controls the flow of metabolites and their access to the electron transport chain (Mannella 2006). One main player in cristae remodeling is the fusion protein Opa1, a GTPase whose oligomerization has been shown to control the formation of the cristae junction (Frezza et al. 2006). Opa1induced changes in the mitochondrial morphology and cristae structure have been shown to be essential for the progression of apoptosis (Frezza et al. 2006). The requirement for mitochondrial plasticity in apoptosis was first established with the observation that mitochondrial fragmentation was important for the efficient release of intermembrane space proteins like cytochrome c (Frank et al. 2001). Later, electron microscopy and functional studies demonstrated that cristae junctions are also disassembled during cell death, which allows the release of proapoptotic proteins like cytochrome c into the cytosol (Scorrano et al. 2002). The newly established dual role for Opa1 in controlling cristae architecture and its requirement for mitochondrial fusion highlight the interconnected nature of the machinery that governs morphology and function, both in steady state and during cell death (Fig. 11.3).

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Fig. 11.3 Morphological transitions are critical for the progression of apoptosis. Upon the activation of cell death, the recruitment of pro-apoptotic proteins like Bax/Bak to the mitochondrial membrane triggers the stable recruitment of Drp1. This leads to the stimulation of mitochondrial fission, an inhibition of the Mfn2 fusion machinery and the remodeling of the cristae. The permeabilization of the outer membrane then facilitates the release of the intermembrane space protein cytochrome c, which activates the apoptosome and drives the final stages of cell death

As expected from the embryonic lethality of mutants in mitochondrial fusion, mammalian cells with impaired mitochondrial fusion grow very slowly and have reduced respiration activity and partly reduced membrane potential (Chen et al. 2005). From yeast cells to primary neurons, the lack of mitochondrial fusion leads to the loss of mtDNA, further demonstrating the essential nature of mitochondrial plasticity (Chen et al. 2007; Hermann et al. 1998). Why fusion is required for the inheritance of the mitochondrial genome is so far not understood, but it has been proposed that the intermixing of matrix contents provides additional protection against mitochondrial ROS and nitrous oxide. It has been estimated that 1–5% of the oxygen consumed during oxidative phosphorylation is converted to ROS as a dangerous but unavoidable by-product of respiration. This buffering granted by mitochondrial fusion may help to prevent the accumulation of mutations in the

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mitochondrial genome. Fusion of mitochondria counteracts the manifestation of respiratory deficiencies because it can allow complementation of mtDNA gene products in heteroplasmic cells that have accumulated different somatic mutations (Nakada et al. 2001). In addition to impairment of respiration, mutations in mtDNA can have a second, more insidious consequence. Mitochondrial DNA mutations that reduce the accuracy of electron transfer increase the likelihood of ROS production and further mtDNA lesions, leading to an amplification of damage. This scenario is the basis for the hypothesis that mitochondrial dysfunction plays a critical role in aging. It predicts that an accumulation of mtDNA mutations lead to a progressive decline in respiratory function over time and eventually culminate in age-associated pathologies and death (Balaban et al. 2005). In a healthy cell, excessive harmful ROS and nitrous oxides are decomposed by protective antioxidant machinery within the mitochondria and peroxisomes. It is important to note that these potentially damaging species also play important positive roles in propagating cellular signals (Pouyssegur and Mechta-Grigoriou 2006), so the relationship between mitochondrial metabolism, morphology and signaling all seem to utilize mitochondrial metabolites as second messengers.

11.1.5

Mitochondrial Dynamics in Quality Control

Given that mitochondria are constantly producing dangerous intermediates as a result of the electron transport chain activity, the removal of oxidized or damaged proteins and lipids is of critical importance. Mitochondrial protein turnover is known to occur through three independent mechanisms. First there are proteases that reside both in the matrix and the intermembrane space that degrade unfolded proteins into small peptides that are exported from the organelle and further degraded within the proteasome (Tatsuta and Langer 2008). Second, some mitochondrial outer membrane tail-anchored proteins have been shown to be degraded by the proteasome directly (Neutzner et al. 2007), although the mechanism for their extraction from the membrane is unclear. No specific machinery has yet been identified to facilitate this process, although it is likely that a system similar to the endoplasmic reticulum-associated degradation (ERAD) may exist (Nakatsukasa and Brodsky 2008). Third, whole mitochondria are known to be degraded within the autophagosome (Scherz-Shouval and Elazar 2007). This pathway has attracted much attention recently since it has been shown that autophagic removal of mitochondria occurs under steady-state conditions. Supporting this hypothesis, recent studies have revealed that some mitochondrial fission events result in the separation of fusion-incompetent fragments that do not recover their electrochemical potential (Twig et al. 2008). These organelles do not become re-incorporated into the reticulum and are instead selectively targeted to the autophagosome. A partial explanation of this may be that the mitochondrial fragment that loses its electrochemical potential is likely to become fusion-incompetent through the potential-dependent cleavage of the fusion GTPase Opa1 (Duvezin-Caubet et al. 2006). Mitochondrial fusion

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requires the presence of both the uncleaved and cleaved forms of Opa1, and the cleavage of Opa1 was shown to be stimulated when mitochondria lose their potential (Griparic et al. 2007; Song et al. 2007). Therefore, the potential-sensitive cleavage event of Opa1 may underlie the fusion incompetence and subsequent transport to the autophagosome (Twig et al. 2008). The emerging question now becomes whether these fragments contain damaged mitochondrial cargo and if this cargo was specifically sorted into one tip of the mitochondria prior to the fission event. In an exciting twist, very recent studies have shown that the Parkinson’s disease gene Parkin is recruited to damaged mitochondria and is required for their targeting to the autophagosome (Narendra et al. 2008). Parkin is a ubiquitin E3 ligase and therefore it is suggested that it may tag potentially damaged, unfolded or oxidized proteins to be sorted into mitochondrial fragments destined for degradation (McBride 2008). Since Parkinson’s disease is characterized by the selective loss of dopaminergic neurons through an apoptotic mechanism, it is currently thought that the trigger for this death signal is from the accumulation of dysfunctional mitochondria. Along with Parkin, at least 4 additional genes related to this disease have a mitochondrial function, including the outer membrane kinase PINK1, the GTPase/kinase LRRK2, the intermembrane space protease Omi/HtrA2 (Plun-Favreau et al. 2007) and the ROS scavenger DJ-1 (Schapira 2008). These recent studies with Parkin lead to the emerging idea that the removal of damaged mitochondria is highly selective and that the machinery that governs their shape, including Drp1 and Opa1, may also contribute to the selectivity of this process (Lemasters 2005). From all of these studies it becomes clear that our previous view of mitochondria as an isolated and autonomous endosymbiont has been profoundly altered. In this chapter the molecular machinery for mitochondrial fusion, fission and positioning as well as their implication in metabolism, autophagy and cell death were introduced, bringing together different fields of mitochondrial research. Mitochondria can now be considered as a community that is highly integrated and interconnected to fulfill its role in a unified manner. In the next section we will discuss how the mitochondrion functions as a signaling platform that links cellular signaling cascades to functional changes.

11.2

Mitochondrion as a Signaling Platform

A number of unexpected discoveries have recently placed the mitochondrion as a critical player in many intracellular signaling cascades (Soubannier and McBride 2009). It has become evident that some recently identified mitochondrial proteins, anchored to the outer membrane, actively recruit cytosolic signaling intermediates to the mitochondrial surface. This is a recurrent theme found in pathways related to immunology, nutrient sensing and cell cycle. In addition, there is an increasing awareness of the important role played by mitochondrial kinases, phosphatases, ubiquitination, sumoylation, acetylation and lipid microdomains, in linking cellular demands to mitochondrial performance. These discoveries are leading us into unexplored areas of mitochondrial research and will be discussed in this section.

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Post-translational Modifications Regulate Mitochondrial Function

The regulation of mitochondrial function through phosphorylation was initially described 40 years ago, when it was found that phosphorylation of the pyruvate dehydrogenase complex inhibited the conversion of pyruvate to acetyl-coA (Linn et al. 1969). The kinase responsible for this, pyruvate dehydrogenase kinase 1-4, was later shown to be primarily regulated through transcription (Holness and Sugden 2003). For example, conditions of cellular starvation upregulated expression of the kinase, leading to the reduction of PDH activity when pyruvate levels were low. Since these early studies, the mitochondrial phosphoproteome has been characterized, with modified targets identified in almost all functional categories (Hopper et al. 2006; Lee et al. 2007; Reinders et al. 2007). Interestingly, the phosphoproteome of mitochondria appears to change upon calcium signaling, highlighting the dynamic and transient nature of these post-translational modifications (Hopper et al. 2006). The challenge now has been to identify the kinases and phosphatases that modulate these mitochondrial proteins. Some, like PDK, are resident mitochondrial enzymes, while others may be recruited to mitochondria from cytosol only upon certain stimuli. In fact, many important signaling proteins have been localized to mitochondria in a very dynamic manner, including p53, ERK, Jnk, Akt, and Src (McBride et al. 2006). In general, it is now clear that mitochondria intersect with many signaling pathways through the canonical mechanisms of phosphorylation. In addition to phosphorylation, mitochondrial functions are also modulated through other modifications, including SUMOylation, acetylation and ubiquitination. The ubiquitination of some mitochondrial outer membrane proteins has been shown to lead to their degradation by the proteasome. In some instances, this degradation process appears to provide a highly selective means for protein turnover, for example in the removal of the fusion GTPase Fzo1p from yeast mitochondria during mating (Neutzner and Youle 2005). The role of mitochondrial acetylation was initially discovered due to the presence of three distinct sirtuin NAD-dependent deacetylase enzymes, SirT3, SirT4, and SirT5 within the mitochondrial matrix. Sirt3 has been shown to deacetylate and thereby activate a central metabolic regulator in the mitochondrial matrix, glutamate dehydrogenase, and furthermore activate isocitrate dehydrogenase 2, an enzyme that promotes regeneration of antioxidants and catalyzes a key regulation point of the citric acid cycle. Sirt5, in contrast, deacetylates cytochrome c, which has a central function in oxidative metabolism as well as apoptosis initiation (Schlicker et al. 2008). Given the NAD dependence of the sirtuins, it has been proposed that they function as sensors for metabolic intermediates and can fine-tune the activity of the enzymes within the TCA cycle and electron transport chain (Ahn et al. 2008). The presence of multiple NADdependent deacetylases and the large number of unidentified substrates makes it likely that acetylation will prove to regulate many more processes than we currently appreciate. In terms of mitochondrial dynamics, it was recently shown that the fission GTPase Drp1 is a target for phosphorylation, ubiquitination and SUMOylation

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(Cerveny et al. 2007). The phosphorylation of Drp1 was shown to either stimulate or inhibit its fission activity, depending on the site of phosphorylation (Han et al. 2008). Some of the identified signals for this phosphorylation are calcium and cAMP, a molecule that can sense the energy status of the cell. Besides phosphorylation, Drp1 is also regulated by ubiquitination. In general, ubiquitination represents a cellular regulatory mechanism for modifying proteins designated for degradation or quality control. The mitochondrial E3 ubiquitin ligase MARCH V/MITOL was recently demonstrated to support fission by facilitating the recruitment of Drp1 to sites of mitochondrial division. Silencing of MARCH V leads to an inhibition of mitochondrial fission and highly elongated mitochondria, which suggests a stronger impact on the activity of the fission than the fusion complex (Karbowski et al. 2007). A third post-translational modification called SUMOylation was also found to regulate Drp1 activity (Harder et al. 2004; Wasiak et al. 2007; Zunino et al. 2007). Like ubiquitin, SUMO proteins are covalently conjugated to their targets, and it has recently been shown that they can also form mixed chains with all three SUMO proteins linked to the same substrate (Tatham et al. 2008). This highly dynamic and reversible conjugation process leads to changes in conformation and associated protein interactions, which often alters the subcellular localization of the substrate. In the case of Drp1, SUMOylation promotes its fission activity and stabilizes the protein against degradation. Importantly, biochemical analysis has shown that there are many mitochondrial substrates for SUMOylation (Harder et al. 2004; Zunino et al. 2007), so there remains a great deal to learn about how this modification is used in the regulation of mitochondrial morphology and function.

11.2.2

Mitochondria as Signaling Intermediates

The evidence that the mitochondria are regulated through the established paradigms of post-translational modifications provides us with new insights into how this organelle may respond so accurately to the cellular condition. It would suggest that potential signaling cascades that are initiated at a cell surface receptor may be propagated through the mitochondria in order to inform this organelle of any environmental change. Interestingly, new data pushes this idea further than had been envisioned. There are at least two examples where the mitochondria play a truly central role in signal transduction cascades that we will discuss. The mammalian target of rapamycin (mTOR) is a kinase found in a complex called mTORC1 or mTORC2 and functions downstream of the central metabolic signaling switche, Akt (also called protein kinase B) (Sarbassov et al. 2005). The activation of the mTORC1 complex requires a complex cascade where activated Akt phosphorylates GAP proteins which inactivates them, leading to the activation of the small GTPase Rheb. GTP-bound Rheb binds to mTORC1, which leads to its activation. The cellular targets of mTORC1 are to promote cellular growth, and proliferation. This requires the influx of amino acids, and the shift within the mitochondria to produce intermediates in protein and nucleotide synthesis, like oxaloacetate and a-ketoglutarate

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(DeBerardinis et al. 2008). Given this direct requirement for altered mitochondrial function upon Akt activation, there should be some mechanistic requirement for signaling to transduce through this organelle. Indeed, it was recently shown that the inactive mTORC1 complex was retained on the mitochondrial surface through its binding to the peptidyl-prolyl isomerase FKBP38, which is anchored within the outer membrane (Bai et al. 2007). The complex on the mitochondria was released into the cytosol upon the binding of GTP-bound Rheb. Whether the mitochondrial residence of mTORC1 provides any metabolic cues has not yet been determined, but it is certainly intriguing that this important signaling complex is restricted to the mitochondria. One of the most extensively studied linkages between mitochondrial function and signaling is through the cAMP transduction cascades. The production of cAMP at the plasma membrane occurs upon activation of receptors, including G protein coupled receptors, which trigger the action of adenylate cyclase. There are also soluble adenlyate cyclase enzymes that are regulated by calcium and bicarbonate. The subcellular and tissue distribution of the soluble adenylate cyclase is a determining factor in their regulation. As a second messenger, cAMP binds and activates the regulatory subunit of protein kinase A, whose localization and function in the cell is regulated by the A-kinase anchoring proteins, AKAPs. AKAP121 is a mitochondrial protein that contains an RNA-binding motif that mediates binding to a number of nuclear-encoded mitochondrial messages. The association with the import machinery is thought to provide a tight coupling between translation and import of mitochondrial proteins (Carlucci et al. 2008). Interestingly, the AKAP121 complex contains PKA and mRNA, but it has also been shown to contain the phosphodiesterase 4A, the serine/ threonine phosphatase PP1 and the Src-associated tyrosine phosphatase PTPD1 (Cardone et al. 2004; De Rasmo et al. 2008; Livigni et al. 2006). More work needs to be done in order to understand the molecular targets of these phosphatases and how their function may be modulated by these cAMP-dependent complexes.

11.2.3

Apoptosis and Mitochondrial Fragmentation

The apoptotic machinery functions as a sentinel for cellular transformation, triggering the cell in question to commit suicide for the greater good. As expected, defective apoptotic processes have been implicated in a variety of human diseases, including cancer. From a molecular perspective, the pathways of apoptosis lead to the activation of caspases that orchestrate the non-inflammatory demolition of the cell. Whether the death signal is initiated through receptors at the cell surface, or through internal cellular stressors, changes in mitochondrial membrane permeability are essential for the progression of cell death. A series of related proteins of the Bcl-2 family reside within the mitochondrial outer membrane where they play a protective, anti-apoptotic role (Youle and Strasser 2008). The tail-anchored proteins Bcl-2 and Bcl-xL are also localized in the endoplasmic reticulum where they have a protective function (HeathEngel et al. 2008). The protective effects of the Bcl-2 family are neutralized upon binding to the pro-apoptotic proteins like Bax or Bak, all of which are cytosolic and

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become recruited to the mitochondria upon the induction of conformational changes (Leber et al. 2007). The pro-apoptotic proteins Bax and Bak have also been shown to play a key role in the formation and opening of what has been termed the “permeability transition pore.” The precise biochemical nature of this pore has been the subject of intense investigation and its composition continues to be the subject of some debate (Chipuk and Green 2008). Regardless of its composition, it is clear that the function of the apoptotic pore is to form a large channel through which many intermembrane space proteins, including cytochrome c, are released into the cytosol. The opening of the PTP is also linked to the loss of mitochondrial membrane potential and the uptake of calcium from the cytoplasm. Upon release from the mitochondria, cytochrome c activates the assembly of the large, multiprotein complex called the “apoptosome,” which leads to the cleavage of many cellular proteins, activation of DNAses and the destruction of the cellular architecture (Riedl and Salvesen 2007). One of the most exciting new discoveries in the field of apoptosis is the observation that the morphology of the mitochondria plays a central role in the execution of the death program (reviewed in Suen et al. 2008). A number of phenomenon have been reported that indicate a tight coupling between the apoptotic and mitochondrial morphology machinery (Fig. 11.3). First, the mitochondria become highly fragmented and lose motility as an early step in the death program, before cytochrome c is released. This fragmentation coincides with a block in mitochondrial fusion and a Bax/Bak-dependent stable recruitment of Drp1 to the mitochondrial membrane (Karbowski et al. 2004; Wasiak et al. 2007). Interestingly, Drp1 is recruited to sites that colocalize with Bax and Mfn2, consistent with a direct relationship between the apoptotic and fission/fusion machinery. The fragmentation of mitochondria is a requisite for the efficient progression of the death program since the inhibition of Drp1 activity, or activation of the fusion machinery, significantly delays apoptosis (Suen et al. 2008). The importance of ongoing mitochondrial fusion in the protection of cells against death signals became most evident when the fusion GTPase Mfn2 was conditionally silenced in the cerebellum. Upon loss of Mfn2, the Purkinje cells effectively disappeared, leading to motor defects and death in the mice by the 17th day after birth (Chen et al. 2007). The precise cause of Purkinje cell death was unclear, but the results provide strong evidence that mitochondrial fusion is protective (Chen et al. 2007). Consistent with these links between mitochondrial morphology and apoptosis, direct interactions between the pro-apoptotic machinery, including Bax and Bak, have been seen with Mfn2 (Brooks et al. 2007; Karbowski et al. 2006). Future work is needed to understand how the morphology machinery may be modulated during the apoptotic program. This emerging area of study highlights how the function of the mitochondria is intimately coupled to cellular signaling cascades.

11.3

Conclusions

This chapter has outlined a number of different aspects of mitochondrial function, from an ancient endosymbiont to a highly dynamic participant in intracellular decisions. There are many critical aspects of mitochondrial function that have not been

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touched upon. However, the central theme has been developed with an aim to provide an appreciation for the complexity of the central metabolic engine of our cells. As we learn more about the integration of mitochondria within the cell, we are becoming aware of fundamentally new and unexpected behaviors of this organelle. These include the intricacies of mitochondrial quality control, with direct relevance to neurodegenerative diseases like Parkinson’s disease (McBride 2008), the importance of mitochondrial shape-shifting proteins in cell death (Suen et al. 2008), and the flexibility of the inner cristae membranes and its importance to the activity of the respiratory chain (Zick et al. 2009). Given that mitochondrial dysfunction is a hallmark feature of so many human disease conditions, it is imperative that we continue to develop a better understanding of this organelle and its signaling capacity. In this way we can hope to manipulate these pathways through the design of drugs with defined molecular targets to reverse cellular damage and enhance mitochondrial function.

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Chapter 12

Mitogen-Activated Protein Kinases and Their Scaffolding Proteins Danny N. Dhanasekaran and E. Premkumar Reddy

12.1

Introduction

Cells respond to diverse intracellular and extracellular cues by evoking appropriate responses that are mediated by a complex array of signaling networks involving distinct ligands, receptors, G proteins, small GTPases, kinases, and transcription factors. Of these signaling components, the mitogen-activated protein kinases (MAPKs) play a major role in transmitting signals from membrane receptors to specific effector molecules in pathways involving cell proliferation, differentiation, oncogenesis, migration, and apoptosis (Davis 2000; Chang and Karin 2001; Morrison and Davis 2003; Huang et al. 2004; Qi and Elion 2005; Goldsmith and Dhansekaran 2007; Dhanasekaran and Johnson 2007). Typically, signaling by the three-tier MAPK module is initiated by specific growth or stress stimuli with the activation of an upstream MAP kinase kinase kinase kinase (MAP4K) by a small GTPase belonging to the Ras- or Rho-family. Activated MAP4K, in turn stimulates the cognate three-tier MAPK module via the sequential phosphorylation of constituent MAP3K, MAP2K, and MAPK. The downstream MAPK is activated by the phosphorylation of a threonine and a tyrosine residue of the TPY motif by the dualspecificity MAP2K. The MAPK then translocates from the cytosol to the nucleus where it regulates the activity of target transcription factors through phosphorylation (Raman et al. 2007; Meloche and Pouysségur 2007; Turjanski et al. 2007).

D.N. Dhanasekaran (*) OU Cancer Institute, The University of Oklahoma Health Sciences Center, 975 NE 10th Street, Oklahoma City, OK, 73104, USA e-mail: [email protected] E.P. Reddy Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA, 19140, USA

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_12, © Springer-Verlag Berlin Heidelberg 2010

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Of the different MAPK modules that have been identified, the ones involving extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38MAPK are well characterized (Dhanasekaran and Reddy 1998; Volmat and Pouyssegur 2001; Chang and Karin 2001; Raman et al. 2007; Dhanasekaran et al. 2007). In many instances, despite their activation by the same or similar upstream signaling events, these MAPK modules regulate distinctly different cellular responses in a cell-type and/or context-specific manner. Earlier studies with the budding yeast Saccharomyces cerevisiae and more recent studies with mammalian cells have identified the role of scaffolding proteins in providing a molecular mechanism for such context-specific signaling by MAPKs.

12.2

Prototypic MAPK Modules of Yeast

Our understanding of the signaling by three-tier MAPK signaling modules and their interacting scaffolding proteins was mainly derived from the mating pheromone signaling pathway of S. cerevisiae. In the haploid cells of S. cerevisiae, the Ste2 receptor, activated by the pheromone a-factor, stimulates a heterotrimeric G protein (GPa1/Ste4/Ste18) by catalyzing the exchange of guanine nucleotides in the a-subunit, Gpa1 (Fig. 12.1). The GTP-bound a-subunit and the bg-subunit (Ste4 and Ste18) disassociate and the disassociated bg-subunit interacts with the scaffolding protein Ste5. This interaction leads to the translocation of Ste5 to the plasma membrane and its interaction with Ste20. Subsequently, Ste20 activates the

Fig. 12.1 An archetypal MAPK scaffold involving Ste5 in Saccharomyces cerevisiae. Scaffolding proteins such as Ste5 of S. cerevisiae is involved in assembling the three-tier kinases consisting of MAP3K, MAP2K, and MAPK into a signaling module. In this canonical MAPK scaffold, Ste5 links the heterotrimeric G protein, Gpa1/ste4/ste18, specifically the bg-subunit (Ste4/Ste18), to the Ste11, Ste7, and Fus3 kinase module in response to the activation of the GPCR Ste2/3 by the mating pheromones

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three-tier MAPK module defined by Ste11, a MAP3K, Ste7, a MAP2K, and Fus3, a MAPK (Elion 2001; Elion et al. 2005; Dohlman and Slessareva 2006). Phosphorylated Fus3 translocates to the nucleus where it stimulates the activation of transcription factor Ste12 and consequent activation Ste12-dependent genes. In addition to pheromone signaling, several other cellular responses in S. cerevisiae, such as vegetative growth, invasive growth, starvation, and osmolarity responses, are mediated by signaling pathways regulated by MAPKs (Elion et al. 2005; Qi and Elion 2005). Analyses of these signaling pathways indicate that the context-specific signaling by these constituent kinases rely on the scaffolding proteins that channel the signals specifically and rapidly to the intended target without any crosstalk. This is clearly manifested in the activation of the MAPK, Kss1, which is activated during vegetative growth or starvation (Elion 2001). Although both Fus3 and Kss1 kinases share the upstream kinases STE20, Ste11, and Ste7, the activation of one pathway by a specific stimulus does not lead to the activation of the other, presumably due to the insulatory role of scaffolding proteins such as Ste5, Spa2, and Sph1 in their respective MAPK modules. In some instances, the constituent kinases themselves can act as scaffolding proteins. During the osmo-adaptive pathway of S. cerevisiae, PBS2, a MAP2K in this pathway, can provide a scaffolding role by binding to the upstream MAP3Ks as well as the downstream MAPK (Posas et al. 1998). Thus, the studies with S. cerevisiae have unraveled the fundamental mechanisms through which MAPKs and their scaffolding proteins can interact to elicit multiple signaling responses.

12.3

Mammalian MAPK Signaling Modules

In mammalian cells, nine distinct MAPK modules have been identified: ERK1/2, ERK3, ERK4, ERK5, ERK6/p38MAPKg, ERK7, ERK8, JNK1/2/3, and p38MAPK a/b/g/d (Uhlik et al. 2004; Bogoyevitch and Court 2004; Zarubin and Han 2005; Coulombe and Meloche 2006; Bogoyevitch 2006; Dhanasekaran and Johnson 2007). The pathways regulated by ERK1/2, JNK1/2/3, and p38MAPKa/b/g/d modules have been well characterized (Whitmarsh 2006; Kolch 2006; McKay and Morrison 2003, 2007; Raman et al. 2007) and are primarily involved in the regulation of cell growth, survival, apoptosis, and oncogenesis. These MAPKs and their scaffolding proteins are discussed here.

12.3.1

ERK Signaling Pathway

ERK1/2 signaling pathways regulate several aspects of cell growth including cell proliferation, survival, and motility. Typically, ERK activation by growth factorstimulated receptor tyrosine kinase involves the recruitment of specific effector molecules such as SOS, a Ras-guanine nucleotide exchange factor that catalyzes the exchange of guanine nucleotides in Ras (McKay and Morrison 2007). The

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Fig. 12.2 ERKs and their scaffolding proteins. The ERK signaling module consists of Raf, MEKs, and ERKs as MAP3K, MAP2K, and MAPKs, respectively. Scaffold protein KSR links signaling from membrane receptors to ERK signaling modules. MP1 and p14 complex can interact with MORG1 or RTK signaling to direct the signal to the MKK1–ERK1 signaling module. MORG1 is involved in linking specific ligands such as those of GPCRs to ERK1/2 module. The scaffolding roles of paxillin and b-arrestin are more involved in directing the signaling outputs from ERK module to specific cytosolic compartments (see text for details)

GTP-bound Ras facilitates the translocation of the Ser/Thr kinase Raf, a MAP3K, to the plasma membrane for activation through phosphorylation. The activated Raf stimulates the dual specificity MAP2Ks, MKK1 and MKK2, through the phosphorylation at Ser-217/ 218 and Ser-221, respectively. MKK1 and MKK2 in turn, activate ERK1 and ERK2 through phosphorylation of specific threonine and tyrosine residues at the TPY motif. Phosphorylated ERKs translocate to the nucleus where they activate transcription factors such as TCFs promoting cell proliferation. Three isoforms of Raf, namely A-Raf, B-Raf, and C-Raf, have been identified (Craig et al. 2008). While C-Raf is ubiquitously expressed, A-Raf and B-Raf are expressed in a tissue-specific manner. A-Raf is selective in the activation of MKK1, whereas B-Raf and C-Raf can activate both MKK1 and MKK2 (Craig et al. 2008). Several ERK-specific scaffolding proteins such as kinase suppressor of Ras signaling (KSR) (Therrien et al. 1995; Kornfeld et al. 1995; Sundaram and Han 1995; Clapéron and Therrien 2007), MEK partner 1 (MP1) (Schaeffer et al. 1998), mitogen-activated protein kinase organizer 1 (MORG1) (Vomastek et al. 2004), paxillin (Ishibe et al. 2003), b-arrestin (Lefkowitz and Shenoy 2005), and MEKK1 (Karandikar et al. 2000) have been identified as playing a crucial role in the context-specific signaling of ERK1/2 (Fig. 12.2).

12.3.1.1

KSR

KSR was identified as a regulator of Ras-signaling pathway in Drosophila, Caenorhabditis elegans, and mammalian cells (Therrien et al. 1995; Kornfeld et al. 1995; Sundaram and Han 1995). Mammalian KSR is a 105-kDa protein that interacts with Raf, MEK, and ERK. Two isoforms of KSR, namely KSR1 and KSR2, have

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been identified, of which only KSR1 is well characterized (Muller et al. 2000; Ohmachi et al. 2002; Channavajhala et al. 2003). Coexpression studies have indicated that KSR1 forms a signaling complex consisting of Raf, MEK1/2, ERK1/2 (Morrison 2001; Roy and Therrien 2002), 14-3-3 (Xing et al. 1997; Cacace et al. 1999), and Gbg-subunits (Bell et al. 1999), along with other molecular chaperones such as heat shock proteins HSP68, HSP70, and HSP90 (Stewart et al. 1999). Studies from different laboratories have defined the mechanism by which KSR1 finely regulates ERK signaling pathway (Roy and Therrien 2002; Morrison 2001; Morrison and Davis 2003; Raabe and Rapp 2003; Ory et al. 2003; Ory and Morrison 2004; Matheny et al. 2004; Matheny and White 2005; Kolch 2005; Clapéron and Therrien 2007). In quiescent cells, an inactive ternary complex containing KSR1, MKK1/2, and PP2A is retained in the cytosol by 14-3-3 and Ras-sensitive E3 ubiquitin ligase (IMP). Upon growth factor stimulation, Ras stimulates the translocation of KSR1–MKK1/2 complex to the membrane by promoting the degradation of IMP and dephosphorylation of KSR1 and Raf at specific serine residues involved in 14-3-3 interaction via PP2A. In the plasma membrane, MKK1/2-complexed KSR-1 further recruits the upstream Raf and downstream ERK1/2 kinases, thereby potentiating ERK-activation. 12.3.1.2

MP1, p14, and MORG1

The 14-kDa MP1, identified as a MEK1-interacting protein in a yeast two-hybrid analysis specifically interacts with MKK1 and ERK1 but not with Raf, MKK2, or ERK2 (Schaeffer et al. 1998). Overexpression studies have shown that MP1 potentiates the activation of ERK1 (Schaeffer et al. 1998; Sharma et al. 2005) and exists as a complex with the MKK1-ERK1 module along with a 14-kDa protein known as p14 (Teis et al. 2002, 2006). Silencing either MP1 or p14 attenuates MKK1ERK1 signaling, primarily in late endosomes. It has also been shown that MP1-p14 scaffold is specifically involved in fibronectin-stimulated, but not, platelet-derived growth factor (PDGF)-stimulated ERK response of REF52 fibroblasts, thereby suggesting a context-specific role in ERK signaling pathways (Pullikuth et al. 2005). Although MP1-p14 heterodimer does not interact with Raf, potentially it can tether Raf into the scaffold through its interaction with MORG1, a 35-kDA molecular weight protein that can associate with A-Raf, B-Raf, MEK1, MEK2, ERK1, and ERK2 (Vomastek et al. 2004). MORG1 was isolated as a binding partner of MP1 in a yeast two-hybrid screen. Expression of MORG1 enhances FBS-stimulated ERK response whereas silencing MORG1 attenuates such a response (Vomastek et al. 2004). It is interesting to note that MORG1 enhances lysophosphatidic acid or phorbol ester-mediated activation ERK and not that of EGF, suggesting a role in context-specific signaling of ERK module (Vomastek et al. 2004). 12.3.1.3

Paxillin

Paxillin is a 68-kDa protein, which is present at focal adhesions in association with other focal adhesion specific proteins (Brown and Turner 2004). Studies focused on

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defining the mechanism underlying human growth factor (HGF)-stimulated cell spreading in mIMCD-3 cells have indicated that paxillin is constitutively associated with MKK1/2 in these cells (Ishibe et al. 2003). HGF stimulates the scaffolding activity of paxillin by inducing the Src-mediated phosphorylation of paxillin at tyrosine-118, upon which the phosphorylated paxillin recruits inactive ERK. Subsequently, activated Raf associates with paxillin and stimulates the tethered MKK1/2 and ERK. Activated ERK can now phosphorylate the focal adhesion complex proteins to promote the recruitment of focal adhesion kinase (FAK) to the focal adhesion complexes. Thus, paxillin organizes a signaling hub at focal adhesions to promote cell spreading via ERK signaling.

12.3.1.4

b-Arrestin

In addition to their role in GPCR internalization, b-arrestins have been shown to interact with the components of the ERK signaling pathway (Lefkowitz and Shenoy 2005). While b-arrestins interact with GRK-phosphorylated GPCRs to promote their endocytosis, b-arrestin-scaffolded ERK modules are also co-internalized along with the GPCRs. By bringing together GPCRs and ERK modules into close proximity in endocytic vesicles, b-arrestins appear to promote the activation of the ERK module by the internalized GPCRs. Interestingly, the ERK signaling facilitated by b-arrestin seems to be restricted to cytosolic signaling events. In addition to these scaffolding proteins, proteins such as CNK1 (Ziogas et al. 2005), CNK2 (Bumeister et al. 2004), IQGAP1 (Roy et al. 2004, 2005), and 14-3-3 (Van der Hoeven et al. 2000) have been shown to be involved in context-specific response. While CNK1 is involved in Src-mediated activation of Raf, CNK2 is specifically involved in NGF-mediated activation of ERK signaling pathway (Bumeister et al. 2004). Likewise, IQGAP1, a putative CDC42/Rac-GAP that can interact with both MEK1 and ERK1, is involved in EGF-stimulated ERK signaling pathway (Roy et al. 2004, 2005) whereas 14-3-3 protein has been shown to tether PKC signaling to Raf (Van der Hoeven et al. 2000). Emerging evidence indicates that these “partial scaffolds” can play a modulatory role in ERK signaling pathways (Morrison and Davis 2003; Kolch 2005).

12.3.2

JNK Signaling Pathway

Jun-N-terminal kinase (JNK) is involved in the phosphorylation of the N-terminal Ser-63 and Ser-73 residues of c-Jun (Dérijard et al. 1994). Diverse growth factors, environmental stimuli, and cytotoxic, as well as genotoxic, stress signals activate JNK signaling pathways. The initial event leading to the activation of JNK primarily involves the activation of Rac or CDC42, members of the Rho family of GTPases (Coso et al. 1995; Minden et al. 1995). In addition to Rac and CDC 42, Rho has also been shown to be involved in the activation of JNK through a PAK-independent pathway (Teramoto et al. 1996). The GTP-bound Rac or CDC42 binds and stimulates

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Fig. 12.3 JNKs and their scaffolding proteins. The MAP3Ks of the three-tier JNK module consists of a large number of kinases such as MEKKs, MLKs, DLK, and ASK1. Only a few representative MAP3Ks are depicted here. MKK4 and MKK7 form the MAP2Ks of this module that can activate the different isoforms of JNKs. The major scaffolding proteins involved in the spatiotemporal organization of JNK modules are JIP1, JIP2, JSAP-1, and JLP. These scaffolding proteins link JNK modules to specific extracellular as well as intracellular stimuli and play a critical role in context-specific signaling of JNK signaling modules (see text for details)

the 65 ±68-kDa p21-activated Ser/Thr kinase (PAK), a MAP4K. PAK in turn, stimulates the downstream MAP3Ks such as MEKK1 via phosphorylation. The stimulated MAP3Ks activate MKK4 or MKK7 through Ser/Thr phosphorylation, following which the MKK4 and MKK7 activate the JNKs through phosphorylation at Thr-183 and Tyr-185 of the TPY motif (Dérijard et al. 1995). JNKs, thus activated, translocate to the nucleus to phosphorylate c-Jun and other transcription factors such as ATF2 and Elk1 (Raman et al. 2007; Dhansekaran and Johnson 2007). To date, ten different alternatively-spliced isoforms of JNK derived from three distinct genes, JNK1, JNK2, and JNK3, have been identified (Johnson and Nakamura 2007). While both MKK4 and MKK7 can activate JNK, MKK7 is highly specific to JNK whereas MKK4 can activate both JNK and p38MAPK (Dhanasekaran and Reddy 1998; Raman et al. 2007). A multitude of MAP3Ks such as MEKK1, MEKK4, MEKK5, ASK1, tumor progression locus-1 (Tpl1), MLK2, and TGFbactivated kinase-1 (TAK1) that can activate MKK4 or MKK7 have been characterized (Cuevas et al. 2007). Stimuli- or context-specific signaling of the JNK module is mediated by specific JNK-specific scaffolding proteins (Fig. 12.3). These scaffolding proteins are JIP1, JIP2, JIP3/JSAP1, JLP, SPAG9, JIP4, and POSH (Tapon et al. 1998; Whitmarsh et al 1998, 2001; Ito et al. 1999; Yasuda et al. 1999; Lee et al. 2000; Jagadish et al. 2005; Kelkar et al. 2005; Dhanasekaran et al. 2007). 12.3.2.1

JIP1

JIP1 was identified as a JNK-interacting protein in a yeast two-hybrid screening analysis (Dickens et al. 1997). It was also isolated independently as a cellular factor enhancing the function of the Glut2 gene promoter in pancreatic cells (Bonny et al.

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1998). Two splice variants of JIP1, namely JIP1a and JIP1b, have been identified. Both JIP1a and JIP1b contain an amino terminal JNK binding domain (JBD), a SRC homology-3 (SH3) domain, and a phosphotyrosine binding (PTB) domain. JIP1b contains 47 additional amino acids at the carboxyl-terminus, forming an additional carboxy-terminal PTB domain. Most of the functional studies have been characterized using JIP1b (henceforth referred as JIP1). JIP1 has been shown to enhance the activation of JNK via the MAP3K MLK3 and the MAP2K MKK7 (Whitmarsh et al. 1998; Nihalani et al. 2001). It has also been shown that JIP1 can interact with MKP7 phosphatase to attenuate the activation of JNK (Willoughby et al. 2003). Thus, it appears that JIP1 can play a dynamic role in JNK signaling by promoting its activation as well as attenuation. JIP1 has also been shown to associate with p190-RhoGEF (Meyer et al. 1999). Rho can stimulate the JNK module (Teramoto et al. 1996), suggesting the possibility that JIP-1 tethers Rho-GEF to it. Although JIP can interact with both JNK1 and JNK2 as well as several MAP3Ks such as MEKK3, MLK3, DLK, and HPK1 (Whitmarsh et al. 1998; Yasuda et al. 1999; Nihalani et al. 2001) its interaction with the MAP2K is restricted to MKK7 (Whitmarsh et al. 1998; Yasuda et al. 1999; Nihalani et al. 2001). JIP1b, the alternative spliced form of JIP1a, appears to play a role in type I diabetes. Type I diabetes results from the destruction of pancreatic b cells (Mauricio and Mandrup-Poulsen 1998). In these cells, it has been observed that IL1 increases JNK1 and consequent pancreatic cell apoptosis via the downregulation of JIP1/IB1 (Nikulina et al. 2003; Haefliger et al. 2003). Thus, JIP1 acts as an inhibitor of JNK during this process (Bonny et al. 2000; Haefliger et al. 2003). JIP1b also plays a critical role in Alzheimer’s b-amyloid precursor protein (APP) signaling to JNK, which is involved in Alzheimer’s disease progression (Matsuda et al. 2001; Scheinfield et al. 2002; Taru et al. 2002). It has been shown that JIP1b is required for the interaction between APP and the JNK module involved in this pathological process (Scheinfeld et al. 2002; Inomata et al. 2003; Muresan and Muresan 2005). It is worth noting here that JIP2, which is analogous to JIP1 in domain architecture, interacts with the components of the JNK module in a similar fashion (Yasuda et al. 1999; Negri et al. 2000; Schoorlemmer and Goldfarb 2002).

12.3.2.2

JSAP1

JSAP scaffold protein was identified as a binding partner for JNK3 in a yeast two-hybrid screening analysis (Ito et al. 1999). It was also indentified as a JNK1interacting protein and named JIP3 (Kelkar et al. 2000). JSAP1/JIP3 lacks the SH3 domain of JIPs and instead contains a leucine zipper domain. JSAP1 interacts with the MAP3Ks such as MEKK1 (Ito et al. 1999), MLK3 (Kelkar et al. 2000) and activator of S phase kinase (ASK1) (Matsuura et al. 2002). JSAP1 can also interact with both MKK4 and MKK7 (Ito et al. 1999, Kelkar et al. 2000). Although all of the isoforms of JNK can interact with JSAP1, JNK3 shows more affinity for JSAP1 than JNK1 or JNK2 (Kelkar et al. 2000). To date, four alternatively-spliced isoforms of JSAP1 namely, JSAP1a, JSAP1b, JSAP1c, JSAP1d containing 1305-,

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1314-, 1337-, and 1336-amino acids, respectively, have been identified (Ito et al. 2000; Kelkar et al. 2000). Ectopic expression of JSAP1 stimulates the activation of JNK3 (Kuboki et al. 2000; Matsuura et al. 2002). In addition, JSAP1 strongly inhibits the activation of the ERKs through its sequestering interactions with Raf-1 and MEK1 (Ito et al. 1999; Kuboki et al. 2000). The ability of JSAP1 to regulate JNK and ERK pathways in an opposing fashion may have an important physiological significance in cellular contexts in which an increased JNK activity along with a decreased ERK activation is required (Kuboki et al. 2000). It has also been shown that JSAP1 links JNK modules to other critical signaling molecules such as toll-like receptor (TLR) and FAK in a context-specific manner. JSAP1 is involved in linking TLR signaling to JNK activation during lipopolysaccharides (LPS)-mediated activation of TLR (Matsuguchi et al. 2003). Likewise, JSAP1 brings together FAK and the JNK module to promote fibronectin-stimulated cell migration (Takino et al. 2005) in which JSAP1-mediated activation of JNK leads to the activation of associated FAK and subsequent signaling events involved in cell migration.

12.3.2.3

JLP

JLP was identified in a screen for Myc/Max-interacting proteins using Max as a probe in a lgt11 expression library (Lee et al. 2002). Similar to JSAP1, JLP contains leucine zipper domains. Furthermore, JLP also contains three SH2 and SH3 binding sites. In addition to the transcription factors Myc and Max, JLP interacts with MEKK3, MKK4, p38MAPKa and JNK1 (Lee et al. 2002). The interacting sites of JNK1 as well as p38MAPKa have been mapped to two distinct domains of JLP, spanning amino acids 1–110 and 210–398, respectively. This suggests that JLP can tether both JNK and p38MAPK so that signals can be transmitted to either or both of these MAPKs from the upstream MAP3K and MAP2Ks. The observation that MKK4, the MAP2K recruited by JLP, can stimulate both JNK and p38MAPK is consistent with the findings that JLP potentiates the activation of both JNK (Lee et al. 2002; Nguyen et al. 2005; Kashef et al. 2005, 2006) and p38MAPK (Takaesu et al. 2006; Kang et al. 2008). JLP has also been shown to tether other signaling molecules to the JNK module. It has been shown that JLP links the a-subunit of G12 (Ga12) or G13 (Ga13) to the activation of JNK signaling module (Kashef et al. 2005, 2006). It has also been shown that JLP enhances the complex formation between Ga13 and ASK1 in promoting the stability of ASK1 (Kutuzov et al. 2007). Although this study does not focus on the scaffolding role of JLP in the activation of JNK, it has provided evidence that JLP tethers Ga13 to ASK1, a MAP3K involved in the activation of JNK. In addition to these interactions, JLP has also been shown to interact with kinesin motor proteins through kinesin light chain 1 (KLC1) (Nguyen et al. 2005). Through these interactions, JLP plays a critical role in the spatiotemporal regulation of JNK signaling.

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Two different splice variants of JLP, namely JNK-interacting protein-4 (JIP4) (Kelkar et al. 2005) and sperm-associated antigen 9 (SPAG9) (Jagadish et al. 2005) have also been identified. Unlike the ubiquitous expression of JLP, JIP4 shows a strong expression in testis, brain, kidney and liver along with a weaker expression in heart (Kelkar et al. 2005). While JIP4 has been shown to interact with MEKK3, MKK4 and JNK, it appears to be more involved in the activation of p38MAPK involving MKK3 and MKK6 (Kelkar et al. 2005). Expression of the 84-kDa splice variant SPAG9 appears to be restricted to the haploid spermatid cells during spermatogenesis in the macaque, baboon, and human species (Jagadish et al. 2005). Although the MAP3Ks and MAP2Ks that interact with SPAG9 are yet to be identified, it has been demonstrated that SPAG9 interacts with higher-binding affinity to JNK2 and JNK3 compared to JNK1. The functional significance of SPAG9 and its associated JNK signaling pathway in spermatid development and/or function remains to be established.

12.3.2.4

POSH

Plenty of SH3 (POSH) protein was identified as a Rac1-interacting protein in a yeast two-hybrid screen (Tabon et al. 1998). In addition, POSH associates with multiple components of the JNK pathway including MLKs, MKK4, MKK7, JNK1 and JNK2, potentiating the activation of JNK and promoting the NGFwithdrawal-mediated apoptosis of PC12 cells (Xu et al. 2003). Thus, POSH appears to function as a scaffolding protein in linking Rac1 and the downstream JNK module in an apoptotic pathway. It has been observed that POSH associates with JIP1 and the POSH–JIP1 complex is involved in inducing apoptosis (Kukekov et al. 2006). Expression of dominant negative POSH or silencing POSH using siRNA abrogated the apoptotic response. In addition to these proteins, MEKK1 and b-arrestin have been shown to play a scaffolding role in JNK signaling pathways (Lefkowitz and Shenoy 2005; Dhanasekaran et al. 2007; Raman et al. 2007). It has been observed that MEKK1 binds to MKK4 and to JNK1 thus assembling a three-tier kinase module without any additional scaffold proteins (Xu and Cobb 1997; Uhlik et al. 2004; Takekawa et al. 2005). It has also been documented that b-arrestin-2 plays a scaffolding role in the JNK signaling pathway by tethering the components of JNK along with the internalized GPCRs in the endosomes (Lefkowitz and Shenoy 2005).

12.3.3

p38MAPK Signaling Pathway

The p38 MAPK was initially identified as a 38-kDa kinase that was rapidly phosphorylated upon exposure of cells of monocytic lineage to the endotoxin, lipopolysaccharides (Han et al. 1993). A search for the MAP2K involved in

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Fig. 12.4 p38MAPKs and their scaffolding proteins. Diverse MAP3Ks such as MEKK3, ASK1, MEKK4/MTK1, TAK1, MLK3, GCK, and HPK1 can activate p38MAPK via MKK3, MKK4, or MKK6. Major scaffolding proteins that are involved in the organization of the p38MAPK signaling module are OSM, JIP2, and JLP (see text for details)

the activation of p38MAPK identified MKK3 (Dérijard et al. 1995) and MKK6 (Stein et al. 1996; Moriguchi et al. 1996) that can activate p38MAPK and MKK4 that can activate both p38MAPK and JNK (Dérijard et al. 1995). These MAP2Ks activate p38MAPK through the phosphorylation of Thr-180 and Tyr-residue-182 in the T-X-Y sequence motif (TGY). To date, four distinct forms of p38MAPK (p38MAPKa, p38MAPKb, p38MAPKg and p38MAPKd) have been cloned. Of these isoforms, MKK3 preferentially activates p38MAPKa, p38MAPKg and p38MAPKd whereas MKK6 activates all of them (Cuenda and Rousseau 2007). Both MKK3 and MKK6 exist in several alternatively spliced isoforms with tissue-specific patterns of expression (Han et al. 1996). Diverse MAP3Ks including ASK1, MEKK4/MTK1, TAK1, MLK3, germinal center kinase (GCK), and hematopoietic protein kinase-1 (HPK1) activate p38MAPK via MKK3, MKK4, or MKK6 (Raman et al. 2007). To date, three distinct proteins have been shown to play a scaffolding role in p38MAPK signaling (Fig. 12.4); they are osmosensing scaffold for MEKK1 (OSM), JIP2, and JLP (Uhlik et al. 2003; Kelkar et al. 2005; Lee et al. 2000; Takaesu et al. 2006).

12.3.3.1

OSM

OSM has been identified as linking the components of the p38MAPK module during osmotic stress (Uhlik et al. 2003). OSM can interact with Rac, MEKK3, MKK3, and p38MAPK. Overexpression of OSM has been shown to enhance MEKK3-mediated phosphorylation of MKK3. It has been shown to be required for hyperosmotic stress-induced activation of p38MAPK, as the RNAi-mediated silencing of OSM completely inhibited hyperosmolarity-induced activation of

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p38MAPK. In contrast, silencing of OSM had only a modest inhibition on anisomycin-induced activation of p38MAPK, thereby suggesting a context-specific scaffolding role for OSM. It appears that OSM exists in a stable complex with MEKK3 in the cytosol and recruits MEKK3 along with MKK3-p38MAPK to Racenriched, actin-mediated membrane ruffles, where it facilitates the activation of p38MAPK in response to hyperosmolarity (Uhlik et al. 2003).

12.3.3.2

JIPs and JLP

It has been established that JNK-interacting proteins such as JIP2, JSAP1, JIP4, and JLP can also interact with p38MAPK. Co-immunoprecipitation studies with JIP2 have shown that JIP2 associates with mixed lineage kinase-3 (MLK3), MKK3 and p38MAPKa in addition to Tiam1, a Rac-specific guanine nucleotide exchange factor (Buchsbaum et al. 2002). Overexpression of JIP2 along with Tiam-1, enhanced the complex formation and activation of the p38MAPK (Buchsbaum et al. 2002). Thus, it appears that JIP2 can provide a functional scaffold for the p38MAPK signaling module starting with the activation of Rac (Buschsbaum et al. 2002). In addition, it has also been shown that IB2, a splice variant of JIP2, can associate with fibroblast growth factor homologous factor-2 (FHF2) in order to link and activate a p38MAPK signaling module consisting of MLK3, MKK3 and p38MAPKd (Schoorlemmer and Goldfarb 2002). JIP4 has also been shown to be involved in ASK1-stimulated activity of p38MAPK, specifically p38MAPKa and p38MAPKb via MKK3 and MKK6 (Kelkar et al. 2005). Consistent with the findings that JLP can interact with p38MAPK (Lee et al. 2000), JLP has been observed to play a scaffolding role in the p38MAPK signaling pathway (Takaesu et al. 2006). It has been observed that JLP plays a critical scaffolding role for p38MAPK in Cdo-mediated myogenic differentiation. Cdo is a receptor-like transmembrane protein that promotes myogenesis. A yeast two-hybrid screen identified the interaction between Cdo and JLP (Takaesu et al. 2006). Further analysis of this interaction indicated that the Cdo-associated JLP could activate p38MAPK, and stimulated the expression of myogenic regulatory factor MyoD, which is required for myoblast differentiation. In addition, an in vivo molecular complex involving endogenous Cdo, JLP and p38MAPK can be observed during myoblast differentiation (Takaesu et al. 2006; Kang et al. 2008). Recently, it has also been shown that JLP plays a role in cytosolic retention of p38MAPK involved in Ga13mediated cell migration (Gantulga et al. 2008).

12.4

Conclusions

As discussed in this chapter, studies with the MAP kinases and their scaffold proteins have indicated that these proteins play critical roles in diverse signaling pathways involved in the regulation of cell proliferation, migration, differentiation, and apoptosis. Although the upstream as well as downstream signaling molecules of

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a specific MAPK are often shared, the fidelity of signaling, with minimal crosstalk, is maintained by the respective scaffolding proteins. As discussed here, the scaffolding proteins play a crucial role in spatiotemporal and context-specific signaling of the MAPKs. It should also be noted that many of these scaffolds also associate with proteins that are involved in attenuating MAPK signaling. In some instances, these signal terminators may be specific MAPK phosphatases as in the JIP and b-arrestin scaffolds (Willoghby et al. 2003; Willoghby and Collins 2006). In other instances the scaffolds may contain intrinsic ubiquitin ligase activity defined by the PHD domain, as in the case MEKK1, to switch or modulate JNK and ERK signaling (Lu et al. 2002; Witowsky and Johnson 2003; Xia et al. 2007). Alternatively, they may associate with proteins with ubiquitin ligase activity as in the cases of KSR (Matheny and White 2005). Thus, the MAPK signaling modules and their scaffolding proteins modulate the amplitude, switching kinetics, as well as the duration of signaling in diverse physiological contexts. Acknowledgments Studies presented in authors’ laboratories were supported by the National Institutes of Health Grants CA123233 (to D.N.D) and CA109820 (to E.P.R).

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Chapter 13

Molecular and Functional Determinants of Ca2+ Signaling Microdomains Indu S. Ambudkar, Hwei L. Ong, and Brij B. Singh

13.1

Background

Calcium ions play a central role in the regulation of essential cellular activity, including secretion, muscle contraction and other processes such as cell proliferation and cell death. The mechanisms responsible for generating calcium signals are very diverse. Cells regulate the diversity in Ca2+ signals by spatiotemporal organization of the molecular entities involved in generating, propagating, and sensing the specific signals. Recent progress in understanding the complexities of calcium signaling has depended largely on the development of unique imaging technology as well as molecular biology tools that allow relatively fast identification of molecular components involved in this process as well as the regulatory component. Basically, the cell has access to two sources of calcium: an infinite supply of external calcium and a more finite internal store sequestered within the endoplasmic reticulum (ER). Coordinated functions of uptake and release mechanisms regulate the [Ca2+] within regions in the cell such as the cytosol, ER and nucleus. Of these, cytosolic Ca2+ signals are of particular importance in the regulation of cell function. Ca2+ concentration in this compartment is tightly controlled; typically around 100 nM in resting cells and up to a couple of mM following stimulation, with the exact elevation dependent on the cell type and the function to be regulated. This increase in cytosolic Ca2+ is the initial signal that is decoded by various effector molecules, and then transduced and utilized for regulation of cell function. Thus, the spatiotemporal characteristics of this signal are carefully controlled and modulated by the cell based on immediate needs. Notably, several diseases have been associated with disruption in Ca2+ signaling mechanisms, including neurological diseases. I.S. Ambudkar (*) and H.L. Ong Secretory Physiology Section, MPTB, NIDCR, NIH, Bethesda, MD, 20892, USA e-mail: [email protected] B.B. Singh Department of Biochemistry and Molecular Biology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, 58201

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Two main processes allow generation of a Ca2+ signal in the cytosol: Ca2+ release from intracellular Ca2+ stores and Ca2+ entry from the extracellular milieu. In the case of a highly specialized Ca2+ entry mechanism, store-operated calcium entry (SOCE), the ER and plasma membrane work in concert to govern cytosolic and ER Ca2+ signals. In this chapter we will primarily discuss the molecular basis that determines the organization and regulation of Ca2+ entry signals associated with SOCE channels.

13.2

Compartmentalization of Store-Operated Calcium Entry

The physiological function of many tissues is regulated by cytosolic calcium increase mediated by a unique Ca2+ influx pathway which is initiated upon the release of Ca2+ from the internal stores. The key triggering event is the depletion of or decrease in the [Ca2+] in the ER lumen ([Ca2+]ER). SOCE is mediated via the activation of specific plasma membrane channels, termed store-operated calcium (SOC) channels (Putney et al. 2001), which are ubiquitously expressed in all cell types. SOCE not only ensures refilling of the intracellular Ca2+ stores but also provides sustained elevation of cytosolic [Ca2+] ([Ca2+]i) which is required for the prolonged activation of numerous cellular functions, including fluid secretion and T lymphocyte activation. Physiologically, SOCE is activated by agonist-stimulated generation of inositol-1,4,5-trisphosphate (IP3), and IP3-mediated release of Ca2+ from ER-Ca2+ stores via IP3R. The resulting decrease in [Ca2+]ER triggers SOCE. Understanding the mechanism by which the status of Ca2+ stores in the ER is transmitted to the PM, to activate or inactivate SOC channels, has presented a major challenge to investigators in this field. Among the many models proposed to describe this elusive pathway, three have received most attention (Putney et al. 2001): (1) one of the first hypotheses to be proposed was the conformational coupling hypothesis which suggests that the ER signal is relayed to the SOC channel via a direct interaction of the channel with IP3R in the ER; this has been recently modified with the discovery of STIM1 as the regulatory protein in ER that interacts with and activates the plasma membrane channel (further discussed below); (2) the diffusible factor hypothesis suggests that a factor is released or generated in response to internal Ca2+ store depletion; and (3) the secretion coupling model proposes regulated recruitment of channels by fusion of intracellular vesicles into the plasma membrane. A major roadblock towards understanding this mechanism thus far had been the lack of knowledge regarding the channels or accessory regulatory components. Furthermore, the channel characteristics are quite diverse in different cell types. The first SOC channel activity to be characterized, calciumrelease-activated calcium (CRAC) channel, mediates a highly Ca2+-selective calcium current (ICRAC) detected in T-lymphocytes and rat basophilic leukemia cell line (Parekh and Penner 1997; Parekh and Putney 2005). Based on the methodology and criteria used for measuring and identifying CRAC channel function, SOC channels with different biophysical characteristics were subsequently identified in various cell types (Ambudkar 2006; Liu et al. 2004; Parekh and Putney 2005). Members of transient receptor potential canonical (TRPC) subfamily of cation

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channels had been previously proposed as critical components of SOC channels. However, studies within the last 2–3 years have identified new components of SOCE that provide insight into the possible mechanism by which this process is regulated and account for the diversity in SOC channels in different tissues. Although the exact composition of all SOC channels still continues to be a matter of contention in the field, there is general agreement that specific, spatially determined, interaction(s) between ER protein(s) and plasma membrane channel supports SOCE. It has been suggested that ER in the subplasma membrane region is most likely to be coupled to SOCE and that depletion of local Ca2+ stores in this region of the ER is sufficient to activate SOCE. Further evidence for close proximity of ER to the plasma membrane was provided by data showing that Ca2+ entering the cell to refill intracellular stores is rapidly pumped into the ER by the high capacity sarcoendoplasmic reticulum Ca2+ (SERCA) pumps allowing minimal diffusion of the cation in the subplasma membrane region. Such studies lead to the proposal that there is close apposition of the ER and plasma membrane at the site of SOCE (Ambudkar 2004; Berridge 2004; Betsuyaku et al. 2001; Liu et al. 1998; Parekh 2003; Putney 1990). It is now well established that SOCE is a highly compartmentalized process that occurs within very specific, likely functionally relevant, microdomains in the cell. There is sufficient evidence to suggest that the assembly of the channels, the upstream effector proteins, as well as downstream sensor proteins together in a signaling complex determine the generation and decoding of a particular Ca2+ signal. In fact, the architecture of such Ca2+ signaling microdomains may facilitate direct physical or functional coupling between molecular components that are involved in the activation and/or inactivation of Ca2+ entry channels. Spatial control of the Ca2+ signal is achieved by assembly of Ca2+ signaling proteins in multiprotein complexes that are localized in distinct regions of cells via specific scaffolding proteins (Ambudkar et al. 2006; Berridge et al. 2003; Kiselyov et al. 2003; Li et al. 2004). Typical proteins in such a complex include membrane receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins), phospholipase, calcium channels, plasma membrane Ca2+ (PMCA) pump and regulatory proteins in the ER (e.g., IP3R, SERCA). A good example of this is observed in polarized epithelial cells where initiation of internal Ca2+ release occurs in the apical region and this can be correlated with the relative enrichment of Ca2+ signaling proteins in this region of the cell, including phospholipase C (PLC), G protein a subunits such as Gaq and Ga11, and IP3Rs (Ambudkar et al. 2006; Bandyopadhyay et al. 2005; Kiselyov et al. 2003; Li et al. 2004; Muallem and Wilkie 1999). Biochemical evidence provided by immunoprecipitation data show that ER proteins like IP3R and SERCA can be immunoprecipitated with plasma membrane proteins, again supporting the concept that close associations between the plasma membrane and ER exist within cells and have a role in regulation of Ca2+ entry. Importantly, the PMCA pump, mitochondria and the SERCA pump have been localized functionally to the cellular region where Ca2+ entry occurs (Liu and Ambudkar 2001; Liu et al. 1998; Parekh 2003). These components contribute to fast removal of Ca2+ from this region and prevent Ca2+-dependent feedback inhibition of Ca2+ entry (Liu et al. 1998; Parekh 2003). Thus, it was proposed that Ca2+ entry channels may form a complex with these Ca2+ signaling

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proteins. The Drosophila Trp channel provides a well-characterized prototype of such a signaling complex (Minke and Cook 2002; Montell 2001). Similar to this, mammalian TRPC channels are also assembled in a multiprotein signaling complex (Ambudkar et al. 2004; Brazer et al. 2003; Lockwich et al. 2000, 2001). Both protein and lipid components contribute to the function and regulation of Ca2+ signaling (Ambudkar 2004; Brazer et al. 2003; Kiselyov et al. 2005; Lockwich et al. 2000, 2001). Cytoskeletal and scaffolding proteins, such as HOMER, receptor for activated C-kinase 1 (RACK1) and SAP90 (synapse-associated protein with a molecular weight of 90 kDa), have an important role in the assembly of Ca2+ signaling complexes (Patterson et al. 2004; Worley et al. 2007). Plasma membrane lipids also crucially impact Ca2+ signaling by regulating the function and trafficking of plasma membrane proteins in addition to being substrates for plasma membrane-localized enzymes (e.g., PtdIns(4,5)P2, which is a substrate for receptor-regulated PLC). These lipids also regulate vesicular trafficking and can have more direct effects on channel function per se (Hardie 2003). Furthermore, distinct plasma membrane lipid domains known as lipid raft domains (LRDs), including caveolar LRDs (LRDs containing the cholesterol-binding protein, caveolin-1 (Cav-1)) provide a platform for the assembly of Ca2+ signaling complexes (Ambudkar et al. 2004; Isshiki and Anderson 2003; Okamoto et al. 1998; Simons and Toomre 2000). Ca2+ signaling events such as receptor-mediated turnover of PtdIns(4,5)P2 have also been localized to plasma membrane caveolar microdomains (Ambudkar 2004; Ambudkar et al. 2004; Brazer et al. 2003; Lockwich et al. 2000, 2001), and several Ca2+ influx-regulated processes, such as the generation of cAMP and the activation of nitric oxide synthase (NOS), have been associated with caveolae (Cooper 2003; Fleming and Busse 2003). Consistent with these findings, SOCE has been shown to occur within caveolar LRDs and is dependent on lipid raft integrity as disruption of the lipid rafts severely affected SOCE (Ambudkar 2004; Ambudkar et al. 2004; Isshiki and Anderson 2003; Lockwich et al. 2000).

13.3

Plasma Membrane Microdomains Involved in SOCE

Lipid rafts are planar domains in the outer leaflet of the lipid bilayer of the plasma membrane and are found in virtually every eukaryotic cell. These domains are enriched in lipids such as sphingolipids, cholesterol, plasmenylethanolamine, and arachidonic acid, as well as key protein components of Ca2+ signaling. The presence of these lipids provides them with a unique liquid-ordered state that is distinct from the surrounding disordered phospholipid bilayer (Brown and London 1998). One property of lipid rafts is that they are resistant to low temperature detergent solubilization with nonionic detergents. Caveolae are a subclass of lipid rafts, with similar lipid composition (as lipid rafts), but containing a family of integral membrane proteins, the caveolins and cavins (Hill et al. 2008; Patel et al. 2008). These two proteins serve as the major structural components of caveolae and their multimerization determines the flask-shaped invaginated morphology of these domains in the plasma membrane (Hill et al. 2008; Razani et al. 2002). Caveolins have a scaffolding region that is involved in binding to the signaling proteins. This binding

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results in the assembly and regulation of these target proteins that interact with caveolin via conserved caveolin-interacting domains. Importantly, such binding allows co-localization of functional proteins within LRDs which increases the probability of specific and quick protein–protein interaction between them that is critical in the regulation and compartmentalization of the Ca2+ entry signal. Lipid rafts and caveolae have been shown to play an important role in the regulation of various cellular functions including organization of cell signaling machinery such as receptor tyrosine kinases and G protein-coupled receptors (GPCRs), establishment of cell polarity, cholesterol transport, potocytosis, and endocytosis (Okamoto et al. 1998; Patel et al. 2008; Simons and Toomre 2000). Since key protein and nonprotein components of Ca2+ signaling cascades, such as PIP2, Gaq/11, muscarinic receptor, PMCA, and IP3R-like protein, have been shown to be localized in caveolae (Patel et al. 2008), and Ca2+ signaling events such as receptormediated turnover of PIP2 have also been localized to caveolar microdomains in the plasma membrane, it was hypothesized that the caveolae play an intrinsic role in regulating Ca2+ influx and possibly in retention and/or trafficking of the calcium channels. The first clue that caveolins could influence calcium homeostasis was observed in muscle cells where the authors identified that the sarcoplasmic reticulum was immediately underneath the plasma membrane and was in close relationship to the caveolae (Gabella 1971). Furthermore, other investigators soon reported that caveolae could effectively increase [Ca2+]i, which may activate the contractile apparatus to produce a sustained vasoconstriction (Diculescu 1980). X-ray spectra showed that calcium-peak (corresponding to increases in [Ca2+]i) can be found within two different cellular compartments: in small invaginations of the sarcolemma, i.e., the caveolae system, and in the intrafibrillar sarcoplasmic reticulum (Meyer et al. 1982). Similarly, histochemical methods also confirmed that calcium was found in the lumina of caveolae, indicating that indeed caveolae is critical for calcium signaling (Suzuki and Sugi 1989). The final proof came when immunogold EM of cryosections revealed that PMCA pumps as well as IP3-regulated calcium channels were localized in caveolae of many nonexcitable cells, e.g., endothelial cells (Fujimoto 1993; Fujimoto et al. 1992). Importantly, lipid raft places two membranes (ER and plasma membrane) that are crucial for regulating calcium in close proximity to each other. Although these initial studies provided clues that caveolae are important for calcium signaling, not much research was performed to understand the mechanism or role of caveolae in calcium influx per se. Importantly, it was originally speculated that IP3R may be a plasmalemmal Ca2+ channel and that perhaps a single caveolae may be equipped with machinery for both Ca2+ influx and extrusion. Another study by Anderson’s group showed that agonist-stimulated Ca2+ signals originated in specific areas of the plasma membrane that were enriched in Cav-1 (Isshiki et al. 1998), which could be due to the crosstalk between ER and caveolae especially when IP3R agonists generated Ca2+ waves. Besides these observations, many other proteins that are important in calcium signaling have been identified within caveolae and/or LRDs, including G protein coupled receptors, PLC PKC, and many more (as reviewed in (Fujimoto et al. 1998). Furthermore, caveolins have been shown to either inhibit the activity of signaling proteins by interaction of the caveolin scaffolding domain or promote

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signaling via enhanced receptor–effector coupling (Feron and Balligand 2006). Thus, the present suggestion is that caveolae function as containers for the Ca2+ signaling machinery, including those involved in SOCE (Ambudkar et al. 2006; Isshiki et al. 2002).

13.4

TRPC Channel Involvement in SOCE

The transient receptor potential (TRP) proteins constitute a superfamily of cation channels which display diverse properties, mode of regulation, and physiological functions (Minke and Cook 2002; Montell 2005). They are activated by sensory signals such as stretch, osmolarity, and temperature; by various chemical ligands, stimulation of cell surface G protein- or tyrosine kinase-coupled receptors by neurotransmitters, growth factors, and hormones. TRP proteins generate channels by homomeric or heteromeric interactions between members of the same subfamily (Montell 2005; Schaefer 2005). Further, TRP proteins interact with accessory proteins which not only regulate gating of the channels but also determine their localization and plasma membrane expression (Ambudkar 2006; Montell 2005). This segregation of TRP channel regulation within functionally specific microdomains in the cell can generate spatially and temporally controlled [Ca2+]i signals. Thus, the functional organization of TRP channel complex dictates not only their regulation by extracellular stimuli but also serves as a platform to coordinate specific downstream cellular functions that are regulated as a consequence of their activation. The organization of TRP channel complexes is consistent with the compartmentalization of Ca2+ entry signaling discussed above. While there is general consensus that TRP canonical (TRPC) channels are activated downstream of agonist-stimulated PIP2 hydrolysis, there is considerable conflict regarding their exact mode of activation arising from the fact that almost all TRPCs have been shown to be activated by both store-dependent and -independent mechanisms (Montell 2005; Parekh and Putney 2005). There is convincing evidence for the contribution of TRPC1 and TRPC4 to SOC channels. TRPC3, TRPC6, and TRPC7 are generally thought to be store-independent, but here again there is some discrepancy. While a plethora of channels can be generated by heteromeric interaction between TRPC proteins, to date none has reproduced the characteristics of the CRAC channel. Channels generated by TRPCs appear to be relatively nonselective in nature. Therefore, TRPCs are considered not to contribute to CRAC channels. The channels formed by TRPCs have generally been referred to as SOCs instead. In the last two years, critical CRAC channel components have been identified which provide new insights into the function and assembly of TRPC-containing SOC channels (further discussed below). TRPC1, the first mammalian TRPC protein to be identified, is widely expressed in neuronal as well as non-neuronal tissues including muscle (Ambudkar 2007; Montell 2005; Rychkov and Barritt 2007; Zhu et al. 1995). Based on all the studies reported until now, data demonstrating endogenous TRPC1 as a SOC component

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have been most consistent. TRPC1 has been reported to contribute to SOCE in a variety of cell types, including salivary gland, keratinocytes, platelets, smooth, skeletal, and cardiac muscle, DT40, HEK293, neuronal, intestinal, and endothelial cells (Beech 2005; Cai et al. 2006; Dietrich et al. 2006; Fiorio Pla et al. 2005; Liu et al. 2000; Mehta et al. 2003; Mori et al. 2002; Rao et al. 2006; Tiruppathi et al. 2006; Vandebrouck et al. 2002; Zagranichnaya et al. 2005). TRPC1 forms diverse SOCs ranging from relatively selective to nonselective (Ca2+ versus Na+) (Brough et al. 2001; Liu et al. 2005). Such diversity is proposed to be a result of differences in the composition of the SOC channels generated by homomeric or heteromeric interactions of TRPC1 with other TRPC monomers. Although there is no conclusive evidence so far of a native homomeric TRPC1 channel, selective interactions of TRPC1 with other TRPCs, e.g., TRPC4/TRPC5 or TRPC3/TRPC7, have been reported (Cioffi et al. 2003; Liu et al. 2005; Strubing et al. 2003; Tiruppathi et al. 2006; Xu et al. 2006b; Zagranichnaya et al. 2005; Zeng et al. 2004). TRPC1– TRPC1 multimers are generated by interaction of the N-terminal coiled–coiled domain interactions (Engelke et al. 2002), while TRPC1–TRPC3 heteromers are formed by interaction of the first ankyrin repeat region in TRPC1 with an as yet unknown region in the TRPC3 N-terminus (Liu et al. 2005). Of these, only interactions with TRPC4, TRPC3 or TRPC7 have been linked with generation of SOC channels. Heteromeric TRPC3 + TRPC1 (Liu et al. 2005; Wu et al. 2004); TRPC1 + TRPC4 (Cioffi et al. 2003) and TRPC1 + TRPC3 + TRPC7 (Liu et al. 2005) channels are involved in SOCE. These studies provide a molecular basis for the previously described diversity in SOC channels.

13.4.1

Calcium Influx, TRPC Channel Complex, and Lipid Rafts

Since previous studies demonstrated that (1) SOCE occurred within LRD and (2) TRPC1 was a critical component of SOC channels involved in mediating Ca2+ entry, it was hypothesized that LRD have a role in the assembly or regulation of these channels. The first study providing evidence for this was reported by Lockwich et al. (2000) which showed that TRPC1 was compartmentalized in both lipid rafts and nonlipid rafts compartments (Lockwich et al. 2000). Importantly, disruption of LRDs not only disrupted agonist regulation of signaling of TRPC1 but also significantly decreased Ca2+ influx. These results have been confirmed in many cell types and with several other TRPC channels as well. It is now well established that TRPC1 channels reside in LRD and disruption of these domains decreases SOCE (Bergdahl et al. 2003; Brazer et al. 2003; Brownlow et al. 2004; Lockwich et al. 2000). Further, TRPC1 is assembled in a multiprotein complex within LRDs that includes key Ca2+ signaling proteins, e.g., GPCRs, IP3R, CaM, PMCA and Gaq/11. Importantly, Cav-1 binding domains have been identified in TRPC1 in both the N- and C-terminal regions (Brazer et al. 2003; Kwiatek et al. 2006; Tiruppathi et al. 2006).

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The presence of TRPC1 in caveolar LRDs is an important determinant of its function as a SOC channel. Disruption of caveolae by methyl-b-cyclodextrin significantly inhibits SOCE (Bergdahl et al. 2003; Brownlow et al. 2004; Lockwich et al. 2000). While LRDs could have more direct effect on channel function per se, these studies suggested that regulation of TRPC1, probably even the functional interactions of the channel with ER proteins involved in activation of SOCE, was mediated by the assembly and organization of the channel within these domains (Brazer et al. 2003; Lockwich et al. 2001; Uehara 2005). Importantly, TRPC1 was shown to coimmunoprecipitate with Cav-1, one of the three caveolin isoforms (Bergdahl et al. 2003; Lockwich et al. 2000). This was consistent with the wellestablished role of caveolae in the function of pulmonary artery endothelial cells (PAEC) (Alvarez et al. 2005; Bergdahl et al. 2003; Cioffi et al. 2005; Jho et al. 2005). Further studies revealed that TRPC1 and Cav-1 physically interact with each other via an N-terminal region of TRPC1 (amino acids (aa) 271–349) which contains a caveolin-binding motif between aa 322 and 349. Deletion of this binding domain in TRPC1 led to suppression of SOCE and altered routing of TRPC1 to the plasma membrane (Brazer et al. 2003). Thus, this region was proposed to be an important determinant of TRPC1 localization in the plasma membrane and its function. In a number of instances, Cav-1 interacts with and regulates the activity of associated signaling proteins (e.g., GPCRs; endothelial nitric-oxide synthase, Src) via a 20-amino acid segment (aa 82–101) in the caveolin scaffolding domain (CSD). In these cases, interaction with the CSD of Cav-1 either maintains the signaling protein in an inactive state until a stimulus is presented or terminates signal transmission after activation (Drab et al. 2001; Murthy and Makhlouf 2000). Kwiatek et al. (2006) have demonstrated that introduction of a competitive-binding CSD peptide attenuates SOCE in human PAEC. They also identified an additional 9-amino acid domain in the C terminus (aa 781–789) of TRPC1 that serves as a CSD binding sequence. Together these studies suggest Cav-1 may interact with TRPC1 via multiple domains, e.g., the N-terminal caveolin-binding motif (Brazer et al. 2003) and the C-terminal CSD (Kwiatek et al. 2006). These interactions presently appear to determine the localization of TRPC1 in the plasma membrane and possibly its regulation by store depletion-mediated signals. At the time of this writing, the exact role(s) of Cav-1 in TRPC1 function remains to be conclusively established. More recently, intriguing data have been provided from studies using Cav-1 deficient (Cav-1–/–) mice which confirm most of the previous findings. Loss of Cav-1 and caveolae reduced Ca2+ sparks and Ca2+ activity in muscle cells which could be accounted for by decreased TRPC1 routing to the LRDs (Murata et al. 2007). Cav-1 deficiency impaired Ca2+ entry but not IP3 production in the lung endothelial cells, resulting in decreased in ACh-induced prostacyclin release. In these cells, SOCE is mediated via TRPC4 + TRPC1 heteromeric channels and plasma membrane localization of TRPC1 is dependent on its interaction with TRPC4. Interestingly, localization of TRPC4 in the plasma membrane was disrupted in Cav-1–/– cells, as was its regulation via association with IP3R in the ER.

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This resulted in a corresponding disruption of TRPC1 localization as well. Most importantly, the abnormal phenotype in calcium handling in Cav-1–/– mice was restored to physiological levels by genetic reconstitution of Cav-1 back into endothelial cells. These results propose a firm genetic basis for Cav-1 in the assembly of TRPC channels at the plasma membrane and the regulation of SOCE. Together these findings firmly support the proposal that interaction of TRPC1 (Nor C-terminus) with Cav-1 plays a critical role in the functional expression of TRPC1 protein in the plasma membrane. Furthermore cholesterol per se, or at least the integrity of the cholesterol-rich caveolae, could have additional more direct effects on the channel function and/or regulation. Interestingly, B cell activation which is primarily mediated by CRAC channels has been also shown to be initiated in the rafts and was accompanied by the release of Ca2+ from the IP3R gated stores (Guo et al. 2000)

13.5

New Players and Recent Developments in SOCE

In the past three years, the field of Ca2+ signaling has been energized by the discovery of two essential components of SOCE. Stromal interacting molecule 1 (STIM1) was identified by two laboratories (Liou et al. 2005; Roos et al. 2005) by using a high throughput siRNA screen, as a protein that is required for SOCE. STIM1 is a single transmembrane domain protein with an EF-hand domain at its N-terminal end that is localized within the ER lumen. It was proposed that STIM1 functions as a sensor for [Ca2+]ER (Smyth et al. 2006; Soboloff et al. 2006). Indeed mutations in the EF-hand domain induced constitutive activation of SOCE (Liou et al. 2005; Zhang et al. 2005). The four-transmembrane protein, Orai1, was discovered using a siRNA screen similar to that used for STIM1 in an attempt to identify CRAC channel components (Vig et al. 2006b). It was further confirmed by genetic linkage studies in severe combined immunodeficiency patients (Feske et al. 2006). The identification of Orai1 provided a second boost of excitement to the calcium signaling field when functional studies suggested that Orai1 may be the pore-forming subunit of CRAC channel. Mutations in Orai1 were associated with defective ICRAC in T lymphocytes isolated from SCID patients (Feske et al. 2006; Smyth et al. 2006). Co-expression of Orai1 and STIM1 induced large increases in SOCE and ICRAC, and mutations in the negatively charged aa residues in the transmembrane domains altered the Ca2+ permeability of the CRAC channel (Peinelt et al. 2006; Prakriya et al. 2006; Vig et al. 2006a; Yeromin et al. 2006). A key question on how a protein present diffusely in the ER can activate plasma membrane channels was addressed by the exciting demonstration that depletion of [Ca2+]ER causes relocation of STIM1 from ER into clusters in peripheral ER–plasma membrane junctions (sites in the cell where the ER and PM are in close proximity to each other). This was subsequently proved by studies using a combination of Fluorescence Resonance Energy Transfer (FRET), Total Internal Reflection Flourescence Microscopy (TIRFM), and biochemical assays which showed that Orai1 and STIM1 were co-localized and that Orai1 was recruited

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to STIM1 puncta following stimulation. The sites of STIM1 and Orai1 clustering in the ER–plasma membrane junctions have been identified to be sites at which SOCE occurs (Luik et al. 2006; Ong et al. 2007b; Xu et al. 2006a). More importantly, STIM1 was shown to be a common component of all SOC channels. In fact, a review by Worley et al. (2007) suggested that SOCs should be defined not only as “store depletion-dependent” channels but rather as STIM1-dependent channels (Worley et al. 2007). In novel studies, STIM1 was also shown to interact with TRPC1-SOC channels (Huang et al. 2006; Lopez et al. 2006; Ong et al. 2007a) and regulate its function in a manner similar to that seen with Orai1. Even more surprising was the finding that functional Orai1 is essential for TRPC1- and TRPC3-generated SOC channels (Cheng et al. 2008; Liao et al. 2007; Muik et al. 2008; Ong et al. 2007a, 2007b). Further, the three proteins, TRPC1, STIM1 and Orai1, are recruited into a complex following stimulation (Ong et al. 2007a). Thus, the site of STIM1 puncta would determine not only its interaction with Orai1 but also with TRPC1. As discussed above, targeting of TRPC1 to the plasma membrane and its function is dependent on lipid rafts (Ambudkar et al. 2006; Brazer et al. 2003; Lockwich et al. 2000). Further, TRPC1 is associated with protein components from ER and plasma membrane within these domains. Thus, it was reasonable to hypothesize that LRDs would have a role in demarcating the site of SOCE and, therefore, the location of peripheral STIM1 puncta. Indeed recent studies reported by Pani et al. (2008) (Pani et al. 2008) and others (Alicia et al. 2008; Jardin et al. 2008) support this hypothesis, and show that STIM1 and TRPC1 partition into caveolar lipid rafts upon stimulation. More importantly, integrity of these domains is required for proper interactions between these proteins, which appear to take place within LRD, and consequently for activation of TRPC1 by STIM1. Based on these data, it has been proposed that LRDs have an important role in anchoring STIM1 puncta in the ER–PM junctional region of the cell. Thus, consistent with previous suggestions, LRDs appear to provide the platform for assembly of a STIM1-regulated TRPC1SOC channel. Since endogenous Orai1 is required for the generation of a TRPC1 + STIM1 channel, we propose that Orai1 is also localized within the same microdomain. However, this will need to be validated in future studies. The structure of STIM1 has been closely scrutinized in several studies to elucidate the regions that are important for its function and association with SOC channels. Huang et al. (2006) reported that the ERM domain of STIM mediates its association with TRPC1, 2 and 4, but not TRPC3, 6 and 7. Furthermore, the C-terminus of STIM1 has been proposed to activate TRPC1 channels in HEK293 cells (Huang et al. 2006). There is a lysine-rich region in the tail of STIM1 C-terminus, which has been the focus of several studies to further understand the activation of SOC channels by STIM1. Deletion of this region and substitution of the lysines with alanine or glutamine resulted in loss of both SOCE and the Ca2+dependent translocalization of NFAT to the nucleus (Huang et al. 2006). Furthermore, deleting the lysine-rich region also prevented the recruitment of STIM1 to the ER-plasma membrane junctions but did not affect the oligomerization of STIM1 (Liou et al. 2007). This lysine-rich region, also known as a polybasic motif, has

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been shown to function as a positively charged plasma membrane-targeting sequence that binds to the negatively charged phospholipids in the plasma membrane (Heo et al. 2006; Liou et al. 2007). Depletion of the plasma membrane phospholipids significantly decreased the association of small GTPases with the plasma membrane (Heo et al. 2006). Therefore, it is possible that the lysine-rich region in the C-terminus of STIM1 may serve to anchor STIM1 into the plasma membrane and facilitate its association with other proteins in the Ca2+ signaling complex. It would be interesting to see how manipulations of the lysine-rich region of STIM1 would also affect its clustering in the ER–plasma membrane junctions and its association with SOC channels. It is also important to consider the IP3R in this context. IP3R has long been regarded as a central protein in Ca2+ signaling and regulation of SOC channels (Berridge et al. 2003; Hisatsune and Mikoshiba 2005; Kiselyov et al. 2005; Putney et al. 2001). Several studies have now reported that IP3Rs also form clusters in the cell periphery in response to cell stimulation (Hisatsune and Mikoshiba 2005; Tojyo et al. 2008). It is also possible that IP3Rs are recruited to a plasma membrane–SOC channel complex, together with STIM1 following store depletion as shown in platelets (Lopez et al. 2006). However, whether IP3Rs are associated with the STIM1/TRPC1/Orai1 complex and what exactly its function is in the regulation of sustained agonist-activated SOCE will need to be determined.

13.6

Conclusion and Future Directions

To summarize, compartmentalization of calcium signaling proteins within specific cellular regions allows spatial control of the Ca2+ signals generated. Cells use such organization to differentiate between diverse Ca2+ signals that can arise under physiological conditions where multiple signaling systems are simultaneously activated. The problem the cell has to tackle is that each of these signaling systems might target a different cellular function. Thus, compartmentalization of the Ca2+ signaling proteins allows rapid and specific decoding of these various signals. It is now well accepted that downstream sensors and effector proteins are scaffolded close to the generation of the signal, i.e., in close proximity to the site of Ca2+ entry. As discussed above, LRDs provide a platform for the assembly of one such Ca2+ signaling complex, the SOC channel complex. Other channel complexes utilize scaffolding proteins such as those with specific protein interacting domains; e.g., INAD, HOMER, NHERF, PICK1, SAP, etc. Most often these scaffolding proteins form multimers thus providing multiple binding sites in close proximity for signaling proteins. In most cases cytoskeletal interactions with such scaffolding proteins provide an anchor for the complex in cells. In the case of the TRPC1–SOC channels complex, there is now strong evidence that LRDs are involved in assembly and targeting of the channel and also in dynamic recruitment of regulatory proteins, and gating of the channel. Further studies will be required to determine the exact molecular regions of the proteins involved in the formation of the signaling

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complex as well as the dynamic aspects of the complex following stimulation of the cells. High resolution imaging combined with bioinformatics/proteomics as well as molecular modeling should provide further understanding of the mechanism of SOCE. Special attention needs to be focused on delineating the steps involved in protein–protein interactions that determine targeting and localization from those involved in actual gating of the channel. Despite the remaining challenges in the field, recent developments in the field of SOCE have provided exciting impetus for further work, in addition to providing clues to a problem that had remained unsolved for more than two decades.

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Part IV

Nuclear Receptors / Transcription

Chapter 14

Eukaryotic Gene Transcription Jennifer H. Gromek and Arik Dvir

14.1

Overview

The genetic information of most organisms is kept in their DNA, a long, doublestranded polymer that is made of a very large number of building blocks called deoxy-ribo-nucleotides (hence, DNA). Only a small portion of DNA, less than 5%, codes for the ~25,000 genes found in humans. The linear nucleotide sequence in the genes is a template for making RNA, a single-stranded version of DNA in which the building blocks are ribo-nucleotides. The process of making the RNA is called transcription and is catalyzed in the cell by large proteins called RNA polymerases. There are three major types of RNAs, including ribosomal RNAs (rRNAs), messenger RNAs (mRNAs) and transfer RNAs (tRNAs). rRNAs are components of ribosomes, the complexes that carry out the synthesis of proteins in the cytoplasm. mRNAs are templates for the assembly of polypeptide chains from single amino acid building blocks, destined to become functional proteins. tRNAs are adapter molecules that translate the information in mRNA into a specific amino acid sequence. We know of three major types of RNA polymerases in eukaryotic cells: RNA polymerase I (RNAP I) synthesizes rRNA in the nucleolus, whereas both RNA polymerase II (RNAP II) and RNA polymerase III (RNAP III) synthesize RNAs in the nucleoplasm. RNAP II is the enzyme responsible for transcribing heterogeneous nuclear RNA (hnRNA), which is the precursor to mRNA. In this review we will be concerned only with transcription of mRNA coding genes. Gene transcription by RNAP II, which is the first critical step in the pathway to create cellular proteins, is a complex and highly regulated process. In additional to intracellular networks that regulate cell cycle and metabolism through gene transcription, signal transduction networks are almost always capable of modulating patterns of gene expression, and, by that, elicit long-term cellular responses.

A. Dvir (*) and J.H. Gromek Department of Biological Science, Oakland University, Rochester, MI, 48309, USA e-mail: [email protected]

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_14, © Springer-Verlag Berlin Heidelberg 2010

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To transcribe a gene, RNAP II needs to physically interact with the Promoter, a specific region on double-stranded DNA (dsDNA) located at the beginning of the gene sequence. Following this, a physical separation between the two strands of the DNA double helix is needed, so that one of the strands, called the template strand, can be used to create the RNA transcript. Ribonucleoside-triphosphates (NTPs) are used as precursors by polymerase, which strings them together into a single-strand mRNA product. The polymerase moves linearly and directionally along the template DNA, which can be quite long, and traverses hundreds or even thousands of bases. When the transcribing RNAP II reaches a short sequence on the template named a Terminator sequence, it is stimulated to come off the template and terminate transcription. The mRNA product is then subject to additional modifications, including 5¢-capping, 3¢-polyadenylation, often times splicing, and, finally, released to travel towards the cell cytoplasm or the endoplasmic reticulum. The major phases of the transcription process can be described as (1) recruitment, (2) transcription initiation and promoter escape, (3) transcript elongation, and (4) transcript termination. Any of these major phases in the transcription cycle is dependent on a multitude of protein factors in addition to RNAP II, and these factors lend themselves to regulation by a large variety of cellular control mechanisms. Therefore, the entire process of gene transcription is subject to regulation and modulation, practically at any step of the way. We will review the major steps of gene transcription and what is known about some of the major macromolecular players that take part in these steps, as well as how these might lend themselves to mechanisms of regulation and control.

14.2

Recruitment of the Transcription Machinery

Recruitment refers to the combined process that directs RNAP II to the promoter site of the gene so that it can begin transcribing. Considering that human DNA contains a total of some 3 x 109 base-pairs, a single gene encompasses less than 10−6 of that. Since eukaryotic DNA is packaged fairly tightly by its chromatin structure, there is a serious challenge of promoter accessibility and the polymerase has to be efficiently targeted to the promoter before transcription can begin. In addition, even when the promoter region of a specific gene is made accessible for RNAP II binding, the polymerase still relies on additional protein cofactors because it cannot recognize and bind the promoter by itself.

14.2.1

Nucleosome Modification

In eukaryotic cells, chromatin, the combination of DNA, RNA, and proteins which make up chromosomes, is fundamental to the organization of the genome. A repeated unit of chromatin is called a nucleosome. The assemblage of nucleosomes

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into chromatin is tightly managed to maintain appropriate levels of DNA transcription, replication, recombination and repair. Within the nucleosome, 146 base-pairs of DNA are wrapped around a histone octamer, consisting of two each of histones H2A, H2B, H3 and H4, assuring the inactivity of eukaryotic genes, except for those whose transcription is brought about by a positive regulatory mechanism. In DNA packaging, histone H1 serves as the linker between nucleosomes and stabilizes the DNA on the nucleosome core by binding at the point where the DNA enters and exits the core, thus forming a unit called the chromatosome. As the DNA is assembled into chromatin, the nucleosomes serve as general gene repressors, by preventing the binding of transcriptional activators and basal transcription factors. Chromatin is, therefore, associated with transcriptional repression, which must be “remodeled” to allow transcription factors access to DNA (Gelato and Fischle 2008). The most significant aspect of chromatin remodeling is the process by which the histones at the nucleosome site are modified in order to loosen their grip on the DNA. An active promoter region is associated with an altered form of the nucleosome, which includes histone acetylation, phosphorylation, ubiquitination, and methylation (Schneider and Grosschedl, 2007). The histone “tail,” amino- and carboxy-terminal domains that extend from the body of the core particle serve as sites for interaction with regulatory enzymes and proteins, including chromatin-modifying complexes. 14.2.1.1

Histone Acetylation/Deacetylation

Acetylation and deacetylation of histones is the primary target for transcriptional control by gene-specific regulatory proteins referred to as coactivators and corepressors. Histone acetylation is a modification that neutralizes the positive charge of the target lysine. Coactivators, such as Gcn5/PCAF, CBP/p300, and SRC-1, possess the enzymatic activity of histone acetyltransferase (HAT), which targets histone H3 and H4 tails (Bhaumik et al. 2007). On the other hand, corepressors recruit deacetylases, enzymes that reverse the acetylation activity, to promoter regions of the nucleosome (Hildmann et al. 2007). Increases in histone acetylation can affect both chromatin structure and function. Hyperacetylation reduces the ability of nucleosomes to fold and create a “closed” structure, thereby creating an “opened” conformation that is functionally linked to transcription (Kornberg and Lorch 1999; Bhaumik et al. 2007). In fact, a number of transcriptional coactivators and components of RNAP II machinery, including Elongator and FACT (Shilatifard and Conaway 2003), possess HAT activity. Additionally, phosphorylation of histone tails, seen at specific sites during cell division, may play an important transcription regulatory role leading to gene activation (Bhaumik et al. 2007). 14.2.1.2

ATP-Dependent Chromatin Remodeling Factors

Chromatin remodeling complexes have been identified that use the energy of ATP hydrolysis to accomplish remodeling. Based on their domain structures, there are four well-characterized families of mammalian chromatin-remodeling ATPases.

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These include: the SWI/SNF (switching defective/sucrose nonfermenting) family, the ISWI (imitation SWI) family, the NuRD (nucleosome remodeling and deacetylation)/Mi-2/CHD (chromodomain, helicase, DNA binding) family, and the INO80 (inositol requiring 80) family (Cairns 2007; Wang et al. 2007). All ATP-dependent chromatin-remodeling complexes can lead to transcriptional activation and repression, and the precise outcome of their action is dependent on the particular chromatin. The SWI/SNF remodeling complexes primarily disorganize and reorganize nucleosome positioning to promote accessibility for transcription-factor binding and gene activation. However, SWI/SNF also promotes gene repression under certain conditions (Cairns 2007). ISWI remodeling complexes mainly organize nucleosome positioning to induce repression, but are also involved in transcriptional activation and transcriptional elongation (Wang et al. 2007). NuRD/Mi-2/CHD remodeling complexes repress transcription in the nucleus, but can also activate the transcription of rRNA in the nucleolus (Cairns 2007; Wang et al. 2007). Additionally, the INO80 remodeling complexes appear to have both activating and repressive effects for specific sets of genes (Wang et al. 2007; Conaway and Conaway 2008). Detailed mechanisms of transcriptional regulation by these ATPase chromatin remodelers remain to be elucidated.

14.2.2

Promoter Interactions

14.2.2.1

The Core Class II Promoter

The gene promoter is part of the gene structure on a DNA molecule to which transcriptional machinery physically binds by direct protein–DNA interactions. This region directs transcription activity, and is the most common regulatory element in this process across the genome. Genes transcribed by RNAP II are generally referred to as class II genes. Their promoter region is located upstream of the transcribed sequence and includes DNA sequences specifically recognized by RNAP II and auxiliary proteins, as well as the physical start point for transcribing the gene (the transcription start site or the first base on the template strand that becomes transcribed into mRNA). The minimal or core class II promoter is required and sufficient for binding of RNAP II to the transcription start site and for initiation of transcription (Dvir 2003; Hahn 2004). This promoter consists of a TATA box, having the consensus sequence TATAA, ~25 base-pairs upstream of the initiator sequence element (Inr) (Thomas and Chiang 2006). Inr is a short nucleotide sequence contained between positions –3 and + 5 relative to the transcription start site. In addition to the TATA box and initiator, flanking sequences both upstream and downstream are also necessary, spanning from ~ –40 to + 30 relative to the transcription start site (Dvir 2003). In addition, several other core promoter elements have been identified in specific promoters, including DPE, BRE, MTE, DCE, and CpG islands (Hahn 2004; Thomas and Chiang 2006). RNAP II by itself is unable to locate its binding site at the promoter and, therefore, relies on a small complement of general transcription factors (Thomas and

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Chiang 2006). Most notable is the general transcription factor TFIID, which includes a subunit called the TATA-binding protein (TBP). TFIID binds at the TATA box, which induces a strong bend in the double helix, and paves the way for the formation of a specific protein–protein binding cascade, bringing the general transcription factors TFIIB and TFIIF to the promoter site. This is followed by the binding of two other general transcription factors, TFIIE and TFIIH, and finally RNAP II. These interactions bring the catalytic site of the polymerase in close proximity with the transcription start site. This preinitiation complex, containing RNAP II and five general transcription factors, is a minimal, but potent transcription complex that can carry promoter-specific transcription quite well using cellfree model systems. RNAP II itself is a large protein with 12 subunits and a molecular weight of > 500 kDa. The largest RNAP II subunit called Rpb1 contains a unique heptapeptide repeat sequence at its carboxy-terminus (CTD): Tyr1, Ser2, Pro3, Thr4, Ser5, Pro6, and Ser7, which is repeated up to 52 times. These seven amino acid repeats contain many hydroxyl groups making the Rpb1-CTD hydrophilic with many sites for phosphorylation. Only RNAP II whose Rpb1-CTD is non- or hypophosphorylated binds the promoter and initiates transcription (Phatnani and Greenleaf 2006). Once transcription starts, the Rpb1-CTD becomes highly phosphorylated on serine (ser2 and ser5) or threonine residues in the transcription initiation reaction. Following termination of transcription, a phosphatase recycles Rpb1-CTD to its unphosphorylated form.

14.2.2.2

Gene-specific Promoter Elements

While the basal transcription apparatus is sufficient to carry out transcription in some cell-free model systems, promoters require binding of additional transcription factors in the intact nucleus. The reasons for this are diverse. Some promoters have a weak (non-consensus) TATA box and initiator element, and the basal apparatus lacks the critical binding energy necessary for stable binding (Thomas and Chiang 2006). Also, in most promoters, additional proteins such as repressors or chromatin components tend to interfere with the direct binding of the polymerase. Such proteins need to be removed or displaced before gene-specific transcription factors can interact with their binding sites at the promoter. These transcription factors act positively and specifically to modulate transcription at the promoter by controlling the recruitment of chromatin-remodeling factors, by recruiting and correctly positioning the polymerase to the start site, and by modulating critical activities necessary for transcription initiation and subsequent steps (Gelato and Fischle 2008). Different promoters vary in the number of binding sites that they have for specific transcription factors, but most have a host of binding sites that span at the very least 250–300 base-pairs. The promoter includes binding sites for “housekeeping” transcription factors, which control transcription as required for regular maintenance and general metabolic activity of the cell, and also for signal-dependent transcription factors that would be triggered in response to hormonal or paracrine

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signals, modulating gene expression in the cell to meet the physiological need of the tissue, organ, or the organism. These promoter sites are most commonly located upstream of the transcription start site, but there are exceptions where some promoter elements are located downstream (Thomas and Chiang 2006). Other notable elements that are found in various class II promoters are DPE, MTE, DCE and BRE. The DPE (downstream promoter element) is typically found with an Inr. With the consensus sequence of A/G-G-A/T-C/T in positions + 28 to + 34, DPE interacts with transcription factors TAF6–TAF9. MTE (motif ten element) and DCE (downstream core element) are also situated downstream of the transcription start site. DCE contains three unique discontinuous subelements: SI, SII, and SIII. MTE functions with Inr to enhance RNAP II mediated transcription. An upstream promoter element called BRE (TFIIB recognition element) is a sevennucleotide sequence located upstream of the TATA box (Thomas et al. 2006).

14.3 14.3.1

Post-Recruitment Steps – MRNA Synthesis Transcription Initiation

When RNAP II is stably positioned at the transcription start site of the gene, the next step is transcription initiation, or the formation of the first phospho-di-ester bond (PDE bond) of nascent transcripts. Before this can take place, the doublestranded DNA template needs to unwind, allowing the two DNA strands to be separated from each other and form the “transcription bubble.” This is one of the most critical steps in eukaryotic mRNA synthesis because it requires ATP-dependent DNA-helicase activities that the polymerase does not possess. This activity is provided by the general transcription factor TFIIH: two of the nine polypeptide subunits of TFIIH are ATP-dependent DNA helicases, one that operates 5¢ to 3¢, and one that operates 3¢ to 5¢ (Thomas et al. 2006). TFIIH has been shown to bind downstream of the polymerase and provide the necessary unwinding action to allow for strand separation. Downstream DNA of 10–15 base-pairs ahead of the distal edge of the bound polymerase is necessary to allow TFIIH binding (Spangler et al. 2001; Dvir 2002). It is not clear why RNAP II requires a transcription factor that will catalyze promoter unwinding for it, as no other RNA polymerase, eukaryotic or bacterial, has this dependence. It has been suggested that the core promoter forms a tight loop around the RNAP II preinitiation complex, a loop that is reinforced by binding of the transcription factor TFIIF to DNA (Yan et al. 1999; Langelier et al. 2001). This looping produces additional resistance to unwinding that requires ATP and the unwinding action of the TFIIH helicase to overcome. Strand separation needs to occur before transcription initiation can take place, and thus the formation of the first PDE bond depends on ATP and TFIIH (Dvir 2003). This dependence on TFIIH provides another opportunity for transcription factors to control transcription, as will be described below. Another ATP- or

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GTP-dependent event at the onset of transcription is the phosphorylation of Rpb1-CTD. The exact role of this event has yet to be fully understood, but it has been suggested that it facilitates the removal of proteins that were initially bound to the subunit as part of the promoter recruitment activities. Phosphorylation can be catalyzed by another subunit of TFIIH, which contains a protein kinase activity, and possibly by other nuclear protein kinases as well. This phosphorylation activity has been shown to be dispensable for transcription in vitro, but the kinase subunit is essential for viability (Phatnani and Greenleaf 2006).

14.3.2

Promoter Escape

Following initiation, the polymerase moves linearly along the template in a proximal direction, elongating the nascent mRNA. During the assembly of the very first building blocks into the newly-formed (nascent) mRNA, the polymerase is prone to abortive initiation and promoter-proximal arrest and also depends on ATP and TFIIH for continued elongation. Arrest of transcription is a state where the transcription complex stalls, but still retains its ternary complex with the RNA transcript. If the early transcription complex is deprived of ATP, arrest will occur unless the transcript has reached a length of 14–15 nucleotides. This has been shown to be linked to the activity of the ATP-dependent DNA helicase subunits that reside in TFIIH (Dvir 2003). An abortive transcription cycle occurs when an early transcription complex releases its nascent RNA transcript, thereby terminating the transcription process altogether. Abortive transcription is documented to exist mainly at the stage when the mRNA is 3–10 nucleotides long. It is likely, but not proven, that ATP-dependent promoter escape, promoter-proximal arrest, and abortive initiation are mechanistically linked. Both abortive initiation and promoter-proximal arrest pose a kinetic impediment to transcription either by slowing down the rate of a single transcript production or by forming nonproductive transcription complexes that occupy the promoter and block binding of new complexes, thereby inhibiting further transcription cycles. This phase in transcription is referred to as Promoter Escape. It is expected that any interaction of a gene-specific transcription factor that will modulate the activity of TFIIH helicase subunits will affect the rate of transcription initiation and promoter escape. Similarly, any interaction that will change the stability of the early transcription complex or that will weaken the stability of the nascent RNA within the context of its own interaction with the polymerase will affect the success of transcript elongation during these very early steps in transcription. Structural studies of RNAP II have revealed that the length of mRNA enclosed within the polymerase structure is about 15 nucleotides (Kornberg 2007). This closely corresponds with the range of promoter escape as measured by the propensity for promoter-proximal arrest and abortive initiation. It is expected that when the RNA reaches this length it is more effectively stabilized by RNA–protein interactions.

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Elongation

During transcript elongation, a hinged portion of RNAP II stabilizes the elongation complex by acting as a sliding clamp that locks template DNA near the catalytic site. Three loops, referred to as the “rudder,” “lid,” and “zipper,” emanate from the clamp and may contribute to the clamp’s locking mechanism. In addition, RNAbinding grooves lock the sliding clamp into a closed position acting to further stabilize the elongation complex. A long “bridging helix” crosses the cleft near the catalytic Mg2+ ion. Bending this bridging helix could cause the helix to act as a ratchet and push on the base-pairs at the 3¢ end of the RNA–DNA hybrid. This may promote the translocation of RNAP II along the DNA by moving the RNA–DNA hybrid through the active site and switching from straight to bent conformations at each step of nucleotide addition (Shilatifard et al. 2003; Hahn 2004). Following initiation and promoter escape, the transcription complex also undergoes a structural change. This is evident by the smaller proximal footprint of the transcription complex and the cessation of the dependence on TFIIH and ATP (Dvir 2003). The transcription complex is in elongation mode and moving forward towards the end of the transcribed region of the gene. The stability and rate of transcription at this stage is greatly affected by interaction with a host of transcription factors that are generally called Elongation Factors. One of these factors is TFIIF, which also has a critical role in promoter binding and transcription initiation. Others are the elongation factors SII, elongin A, B, and C, and ELL. Most of these factors act by improving the processivity of nucleotide addition at the active site of the polymerase, thereby enhancing stability and increasing the rate of transcription (Shilatifard et al. 2003). Other factors associate with the CTD of the large subunit of RNAP II and affect elongation, a mechanism not yet understood, and additionally, some factors act as inhibitors to elongation.

14.3.4

Termination

Transcription by RNA polymerase II terminates over a terminator region, but it is not known what features define this region or how it affects termination. Much of the difficulty in resolving this issue is due to the fact that class II transcripts are processed at the 3¢ end with the addition of a poly(A) tail which replaces the true 3¢ end of the transcript. At this point during transcription, the CTD is dephosphorylated and polymerase dissociates from the template and from the transcript. The new transcript is processed at 5¢ end by capping, and at the 3¢ end by polyadenylation. The pre-mRNA is then spliced, removing introns, to produce an mRNA that is exported from the nucleus to the cytoplasm. During termination, the RNAP II elongation complex functions as a platform that coordinates mRNA processing (Gilmour and Ruopeng, 2007). Capping of RNAP II transcripts occurs once the transcripts reach 25–30 nucleotides in length. This process requires three enzymes: a RNA triphosphatase,

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which removes the g-phosphate at the 5¢ end of the nascent transcript; a guanylyltransferase, which transfers a GMP cap to the resulting diphosphate terminus of the nascent transcript; and a methyltransferase, which adds a methyl group to the N7 position of the GMP cap to form the transcript’s mature m7G(5¢)ppp(5¢) N cap structure (Soller 2006). Recent research demonstrates a mechanistic link between the RNAP II elongation complex and the mRNA capping machinery (Shilatifard et al. 2003). Both mammalian and yeast guanylyltransferases are recruited to transcribing RNAP II through the hyperphosphorylated CTD where they may bind tightly and specifically to CTD peptides. CTD phosphorylation by TFIIH may provide a signal that links capping to the synthesis of nascent transcripts by recruiting capping enzymes to RNAP II initiation and/or elongation complexes. In addition to its role in capping of nascent transcripts, the RNAP II CTD is linked to polyadenylation of pre-mRNAs (Shilatifard et al. 2003). Polyadenylation occurs in two steps: endonucleolytic cleavage of the mRNA precursor at a site downstream of the AAUAAA signal in the transcript and addition of 200 to 300 nucleotides of poly(A) to the newly generated transcript’s 3¢-OH terminus by poly(A) polymerase. A number of proteins are required for this process, including: cleavage/polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage CFI and CFII, and poly(A) polymerase. Both CPSF and CstF are capable of associating with RNAP II CTD. Unlike capping, polyadenylation does not depend on phosphorylation of the CTD, although the CTD is required for efficient 3¢ cleavage of transcripts prior to poly(A) addition. Yet another role linking the CTD with the RNA processing machinery is suggested by the observation that cotranscriptional splicing in cells requires a functioning hyperphosphorylated CTD (Shilatifard et al. 2003). Splicing is accomplished by the spliceosome, a complex formed by the snRNPs (small ribonucleoproteins). The snRNP proteins have been shown to interact with phosphorylated RNAP II and phosphorylated CTD peptides. Therefore, in addition to its involvement in capping and polyadenylation, it is likely that the CTD recruits components of the splicing machinery to the RNAP II elongation complex and promotes assembly of early splicing intermediates.

14.4

14.4.1

Major Enzymes and Molecular Complexes Involved in Activated Transcription RNA Polymerase II

Eukaryotic RNAP II is a large protein with 12 subunits and a molecular weight of > 500 kDa. Four elements called the core, clamp, shelf and jaw lobe, which all move relative to each other, make up RNAP II and are very similar in structure and function to their counterparts in bacteria (Hahn 2004). The core element forms the

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active center of RNAP II. The proposed structure for the polymerase core includes an exit channel for the RNA which can accommodate a transcript up to 15 nucleotides long. As transcription begins, an RNA–DNA hybrid the size of approximately 9 base-pairs is formed. It is thought that both the RNA–DNA hybrid and the interactions at the exit channel contribute to the stability of the polymerase during the very early steps of transcription.

14.4.2

The General Transcription Factors

14.4.2.1

Initiation Factors

To initiate transcription, RNA polymerase requires general (or basal) transcription factors at the promoter region. Minimally, five transcription factors are required to allow RNAP II to bind the promoter and initiate transcription: TFIID, TFIIE, TFIIF, TFIIE, and TFIIH. TFIID is responsible for recognizing promoters for RNAP II, which is the first step in forming the basal apparatus. TFIID is composed of the TATA binding protein (TBP) which recognizes the TATA box and other subunits called TAFs (TBP-associated factors). If a promoter has a TATA element, TBP binds specifically to DNA, whereas in a TATA-less promoter it may be associated with other proteins that bind to DNA. TBP binds to the minor groove of DNA and surrounds one face of DNA forming a saddle around the double helix bending the DNA by 80° toward the major groove, thus widening the minor groove (Bushnell et al. 2004). Once TBP/TFIID is bound at the promoter, a complex containing RNAP II and the remaining TFs assemble at the site and establish a competent transcription preinitiation complex. First TBP recruits TFIIB, which in turn recruits both RNAP II and TFIIF. TFIIF helps to target RNAP II to the promoter and also destabilizes the nonspecific RNAP II–DNA interactions. At this point, TFIIE is recruited followed by TFIIH. TFIIE modulates TFIIH helicase, ATPase, and kinase activities (Thomas and Chiang 2006). TFIIH contains ten subunits, seven forming the core complex (XPB, XPD, p62, p52, p44, p32, and p8/TTDA) and three forming the cdk-activating complex (CAK, consisting of cdk7, MAT1, and cyclin H), which is linked to the core complex via XPD. TFIIH is ATP-dependent, allowing its helicase activity to unwind the promoter, and the kinase activity to phosphorylate the CTD for promoter clearance (Thomas and Chiang 2006). The helicase activity is associated with the xeroderma pigmentosum complementation group B (XPB) which catalyzes unwinding in the 3¢–5¢ direction and complementation group D (XPD) which catalyzes unwinding in the 5¢–3¢ direction. The ATP/GTP kinase activity is associated with the Cdk7 subunit. Through its helicase activity, TFIIH can torque the DNA and introduce negative superhelical tension in the region of the RNAP II active site (Bushnell et al. 2004). TFIIF is now able to capture the nontemplate strand, permitting the template strand to enter the active site. With the addition of ATP and trinucleotides, RNAP II now catalyzes the first phosphodiester bond to begin transcription initiation.

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267

ATP-Dependent Chromatin Remodeling Complexes

Eukaryotic protein complexes in the cell’s nucleus hydrolyze ATP and perturb or reorganize chromatin by destabilizing or displacing histone-DNA contacts. Such activities act indirectly, removing or altering repressive chromatin structures. This allows for binding of activators and the general transcription apparatus to enhancer and promoter regions. The prototypical member of this family is SWI/SNF, which was first identified by genetic studies in yeast (Gelato and Fischle 2008). Biochemical studies revealed that SWI/SNF proteins are physically associated in a complex. The hallmark subunit of SWI/SNF called Swi2/Snf2 possesses intrinsic DNA-stimulated ATPase activity (Conaway and Conaway 2008). A number of eukaryotic ATP-dependent remodeling complexes, containing subunits homologous to Swi2/Snf2, are found in other organisms, including Drosophila and human. Biochemically, SWI/SNF-type complexes disrupt nucleosomes in vitro and facilitate transcription factor binding in an ATP-dependent manner. Mononucleosome core disruption by the SWI/SNF family of cofactors is catalytic and induces the reversible formation of an altered dimeric form of the mononucleosome. The Swi2/Snf2 subunit of the SWI/SNF complex contains a bromodomain, a 110 amino acid long domain that interacts specifically with acetylated lysine. Purifying and analyzing a SWI/SNF complex without a bromodomain revealed that it is required for SWI/SNF retention on acetylated promoter nucleosomes (Hassan et al. 2002). Bromodomains are also found in a number of protein complexes involved in transcriptional regulation, including the general transcription factor TFIID and the RSC chromatin-remodeling complex. Thus, histone acetylation has the potential to provide high-affinity interaction sites for bromodomain-containing complexes in chromatin. In yeast, the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex, another multifunctional coactivator that regulates transcription, also contains two bromodomains in its Gcn5 and Spt7 subunits and is able to stabilize its own binding to promoter nucleosomes by acetylation of histones (Hassan et al. 2002). This is solely the function of the Gcn5 bromodomain. Additionally, the role of acetylated histones in SWI/SNF promoter suggests a molecular basis for the functional links between SWI/SNF and SAGA, in which the bromodomain of the Swi2/Snf2 subunit might play an important role in anchoring SWI/SNF to promoters. These studies illustrate the central role of bromodomains and histone acetylation in the stable binding of chromatin-modifying complexes to nucleosomes. These interactions are thought to form stable epigenetic marks in chromatin and lead to ordered pathways of chromatin remodeling for transcription. Evidence is accumulating which suggests that SWI/SNF complexes are directed by activators to specific sets of genes. For example, steroid receptors in yeast and in mammalian cells require SWI/SNF complexes for ligand-dependent stimulation of transcription (Aoyagi and Archer 2008). These SWI/SNF complexes physically associate with nuclear receptors. Another example is the erythroid-specific transcription factor, EKLF, which requires a mammalian SWI/SNF complex for full transactivation from a chromatin-assembled b-globin gene template in vitro

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(Bottardi et al. 2006). Additionally, the C/EBPb activator and the c-myc protooncoprotein are shown to bind directly to subunits of mammalian SWI/SNF complexes (Lu¨ scher 2001). Activation domains from various activators linked to heterologous DNA binding domains have also been found to direct the yeast SWI/ SNF complex to promoters in vitro (Cairns 2007). Collectively, these findings suggest that activators may recruit SWI/SNF complexes to enhancer and promoter regions to effect local chromatin remodeling. Nucleosome remodeling complexes are also implicated in inhibition of gene activity by transcriptional repressors, as well as in facilitating DNA replication and repair. This suggests that chromatin remodeling activities are not restricted to aiding transcriptional activators, but assist many types of DNA transactions in the context of chromatin. More work is needed to dissect the relative functional contributions of each nucleosome remodeling complex to transcription of specific genes as well as to other nuclear activities.

14.4.4

Mediator

The precise regulation of gene transcription by RNAP II, controlled by TFs, is vital to gene expression in mammalian cells in response to growth, development, and homeostatic signals. In addition to general TFs, a gigantic (1 MDa) multi-protein complex called Mediator has emerged as a central player in the process by which information is converted into the appropriate response by RNAP II (Kornberg 2005; Tóth-Petróczy et al. 2008). Mediator is recognized as a major conduit of regulatory information. As a multisubunit “adaptor” that bridges RNAP II and its myriad DNA binding regulatory proteins, Mediator transduces both positive and negative signals that turn on and off messenger RNA synthesis. Therefore, Mediator is an essential component of the RNA polymerase II general transcriptional machinery, playing a crucial part in the activation and repression of eukaryotic mRNA synthesis. The yeast Mediator was the first to be defined, being comprised of > 20 distinct subunits, which perform multiple activities in transcription. Also, recent studies have identified the subunit composition and associated activities of mammalian Mediator, revealing that it contains 28–30 subunits organized in a modular fashion, with head, middle, and tail regions (Tóth-Petróczy et al. 2008). When compared, a striking evolutionary conservation of Mediator structure and function from yeast to man is observed. Recent research shows that Mediator subunits are comprised of conserved intrinsically disordered regions (IDRs), which are frequently involved in key biological processes such as cell cycle control, transcriptional and translational regulation, membrane fusion and transport, and signal transduction. The IDRs are thought to contribute to the gene-specific regulatory function of the Mediator via facilitating structural transitions and transmitting transcriptional signals (Tóth-Petróczy et al. 2008). The mechanisms by which mammalian Mediator complexes control mRNA synthesis have not been firmly established. However, evidence argues that Mediator

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activates transcription through direct interactions with DNA-binding transcriptional activators bound at upstream promoter elements and enhancers, with RNAP II and one or more of the general transcription factors bound at the promoter (Conaway et al. 2005). Notably, the transcriptional activation domains (TADs) of different DNA-binding transcription activators are shown to interact with Mediator subunits. For example, mammalian MED1 protein, a subunit of mammalian Mediator, binds to ligand nuclear receptors via its Leu-Xaa-Xaa-Leu-Leu motifs (Conaway et al. 2005). Current research of mammalian Mediator complexes includes identifying other subunits that serve as contact sites for the large collection of DNA-binding transcriptional activators present in cells. Results of biochemical experiments suggest that the presence of mammalian Mediator can increase the efficiency and rate of assembly of the RNAP II preinitiation complex at several steps, including recruitment of TFIID, RNAPII and other transcription initation factors (Kornberg 2005). Mediator is thought to embrace RNAP II and the preinitiation complex, including the GTFs and DNA. This embrace may stabilize the preinitiation complex, promoting its formation or its maintenance for multiple rounds of transcription. Additionally, it may affect the conformation of the complex and its activity in the initiation of transcription. In addition to mammalian Mediator’s role in transcription activation, a portion of Mediator called the Mediator kinase module has an intrinsic potential to negatively regulate transcription (Malik and Roeder 2005). This does not imply that Mediator complexes containing the module would not participate in activation. It could entail conversion of an inactive or repressed transcription complex at the promoter to one that is capable of initiation of transcription. Evidence indicates (1) that the kinase module might interfere with the ability of Mediator complexes to bind RNAP II, either by phosphorylating polymerase and/or Mediator subunits or by steric hindrance; (2) that Mediator-associated kinase might phosphorylate and inactivate one or more of the general initiation factors; and (3) that recruitment of the kinase module to Mediator at promoters might target DNA-binding transcription factors for phosphorylation and ultimately for degradation, providing a mechanism to limit the length of time a given promoter is active (Malik and Roeder 2005; Kornberg 2005).

14.4.5

Elongation Factors

Following promoter escape, RNAP II transitions into the elongation stage of transcription. Research shows that most of the general transcription factors remain at the promoter after the transition from initiation to elongation (Shilatifard et al. 2003). It is not clear whether the Mediator complex remains bound to polymerase during elongation. To understand how RNAP II elongates RNA chains requires understanding the polymerase’s remarkable processivity, how the enzyme grips the DNA template and nascent RNA so tenaciously without dissociating from the DNA template and also understanding why elongating RNAP II is so susceptible to transient pausing and arrest.

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Pausing and arrest result from aberrant backward movement of RNA polymerase on the DNA template and displacement of the 3¢ end of the transcript from the catalytic site. New elongation factors that control the activity of RNAP II on naked DNA and on chromatin templates have been characterized. These include SII, P-TEFb, TFIIF, ELL, elongin, elongator and FACT proteins. There is also direct evidence supporting a role for CTD phosphorylation in the regulation of elongation (Conaway et al. 2000; Shilatifard et al. 2003). Elongation factor SII, the first RNAP II transcription factor to be purified, reactivates arrested polymerase by promoting endonucleolytic cleavage of the nascent transcript realigning the 3¢ end of the RNA in the catalytic site (Shilatifard et al. 2003). SII can also promote nascent transcript cleavage 2 or 3 nucleotides upstream of the transcript’s 3¢ end. General initiation factors TFIIE, TFIIF, and TFIIH also play an important role in early elongation. P-TEFb (positive transcription elongation factor b) is a DRB-sensitive cyclin-dependent protein kinase that is capable of phosphorylating the heptapeptide repeats in the CTD. Unlike TFIIH, which phosphorylates RNAP II at or near initiation, phosphorylation by P-TEFb occurs on polymerase molecules that have moved away from the promoter region and are engaged in elongation (Shilatifard et al. 2003). A number of transcription factors, including TFIIF and members of the ELL and elongin families, stimulate the rate of elongation by RNAP II by suppressing transient pausing by the enzyme at steps of nucleotide addition (Shilatifard et al. 2003). TFIIF is a heterodimer composed of ~30 kDa RAP30 and ~74 kDa RAP74 subunits. TFIIF increases the efficiency of transcription initiation by significantly reducing the frequency at which RNAP II aborts transcription. TFIIF elongation activity could expedite transcription initiation by increasing the rate of synthesis of the first few phosphodiester bonds of nascent transcripts, ensuring that growing transcripts reach a sufficient length to be resistant to abortion (Shilatifard et al. 2003; Thomas and Chiang 2006). ELL functions to stimulate the overall rate of elongation (Shilatifard et al. 2003). The human ELL gene was first identified as a gene that undergoes translocations with the MLL gene in acute myeloid leukemia. ELL, the protein, was initially purified to homogeneity by its ability to stimulate the rate of elongation by RNA polymerase in vitro. The ELL proteins, ELL, ELL2 and ELL3, range in size from ~50 kDa to ~120 kDa and contain a conserved N-terminal elongation activation domain and C-terminal occluding-like domain. Like ELL, elongin was purified to homogeneity based on its ability to stimulate the rate of elongation in vitro (Shilatifard et al. 2003). Elongin is a heterotrimeric protein composed of a transcriptionally active ~100 kDa subunit (elongin A) and two smaller ~18 kDa (elongin B) and ~15 kDa (elongin C) subunits. There is a potential role for elongin as an E3 ubiquitin ligase and RNAP II is a known target for ubiquitination, although the regulatory significance of this modification remains unclear. Recent research shows that elongin A may assemble into an ubiquitin ligase complex in cells and recruit a Cullin/Rbx1 module directly to transcribing RNAP II to target ubiquitination of polymerase or other components of the transcription apparatus.

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Two other proteins, elongator and FACT, appear to promote RNAP II elongation by modifying nucleosomes in ways that facilitate passage of polymerase through chromatin, thus overcoming the negative effects of chromatin of mRNA synthesis (Shilatifard et al. 2003). Elongator was identified through its tight association with hyperphosphorylated RNAP II. The elongator subunit, Elp3, is a HAT. The HAT activity can acetylate both core and nucleosomal histones, and may contribute to the “open” conformation necessary to transcription. Conversely, FACT stimulates RNAP II elongation through nucleosomes by binding them and promoting removal of nucleosomal histones H2A and H2B.

14.5

Conclusion

Synthesis of eukaryotic mRNA by RNAP II is a complex biochemical process that requires a large set of transcription factors. Although much has been learned about gene expression (specifically mRNA synthesis) and its control by local chromatin structure, promoter elements, and interactions of stimulatory and inhibitory protein factors, several issues still need to be resolved. For example, research within the last few years has brought a wealth of information concerning covalent modification of histones and, although it is now clear that chromatin remodeling complexes contribute to the process of transcription, studies addressing their molecular mechanisms are just beginning. In addition to chromatin remodeling, significant progress characterizing the structure and function of the mammalian Mediator complex has been achieved, although, much remains to be learned to gain a better understanding of the Mediator and its role in transcription regulation. In fact, it is expected that an extension of polymerase crystallography to the Mediator complex would one day reveal this regulatory mechanism.

References Aoyagi S, Archer TK (2008) Dynamics of coactivator recruitment and chromatin modifications during nuclear receptor mediated transcription. Mol Cell Endocrinol 208:1–5 Bhaumik SR, Smith E, Shilatifard A (2007) Covalent modifications of histones during development and disease pathogenesis. Nat Struct Mol Biol 14(11):1008–1016 Bottardi S, Ross J, Pierre-Charles N, Blank V, Milot E (2006) Lineage-specific activators affect beta-globin locus chromatin in mulipotent hematopoietic progenitors. EMBO J 25(15):3586–3595 Bushnell DA, Westover KD, Davis RE, Kornberg RD (2004) Structural basis of transcription: an RNA Pol II-TFIIB cocrystal at 4.5 Angstroms. Science 303:983–988 Cairns BR (2007) Chromatin remodeling: insights and intrigue from single-molecule studies. Nat Struct Mol Biol 14(11):989–996 Conaway RC, Conaway JW (2008) The INO80 chromatin remodeling complex in transcription, replication and repair. Trends Biochem Sci 34(2):71–77

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Conaway RC, Sato S, Tomomori-Sato C, Yao T, Conaway JW (2005) The mammalian Mediator complex and its role in transcriptional regulation. Trends Biochem Sci 30(5):250–255 Conaway JW, Shilatifard A, Dvir A, Conaway RC (2000) Control of elongation by Pol II. Trends Biochen Sci 25:375–380 Dvir A (2002) Promoter escape by RNA polymerase II. Biochim Biophys Acta 1577:208–273 Gilmour DS, Ruopeng F (2007) Derailing the locomotive: transcription termination. J Biochem 283(2):661–664 Hahn S (2004) Structure and Mechanism of the RNA polymerase II transcription machinery. Nat Struct Mol Biol 11:394–403 Hassan AH, Prochasson P, Neely KE, Galasinski SC, Chandy M, Carrozza MJ, Workman JL (2002) Function and selectivity of bromodomains in anchoring chromatin-modifying complexes to promoter nucleosomes. Cell 111(3):369–378 Hildmann C, Riester D, Schwienhorst A (2007) Histone deacetylases-an important class of cellular regulators with a variety of functions. Appl Microbiol Biotechnol 75:487–497 Kornberg RD (2005) Mediator and the mechanism of transcriptional activation. Trends Biochem Sci 30:235–239 Kornberg RD (2007) The molecular basis of eukaryotic transcription. Cell Death Differ 14:1989–1997 Kornberg RD, Lorch Y (1999) Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell 98:285–294 Langelier MF, Forget D, Rojas A, Porlier Y, Burton Z (2001) Structural and Functional Interactions of Transcription Factor (TF) IIA with TFIIE and TFIIF in Transcription Initiation by RNA Polymerase II. J Biol Chem 276(42):38652–38657 Lu¨scher B (2001) Function and regulation of the transcription factors of the Myc/Max/Mad network. Gene 277:1–14 Malik S, Roeder RG (2005) Dynamic regulation of pol II transcription by the mammalian Mediator complex. Trends Biochem Sci 30(5):256–263 Phatnani HP, Greenleaf AL (2006) Phosphorylation and functions of the RNA polymerase II CTD. Gene Dev 20:2922–2936 Schneider R, Grosschedl R (2007) Dynamics and interplay of nuclear architecture, genome organization, and gene expression. Gene Dev 21:3027–3043 Shilatifard A, Conaway R, Conaway JW (2003) The Pol II Elongation complex. Annu Rev Biochem 72:693–715 Spangler L, Wang X, Conaway J, Conaway R, Dvir A (2001) TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA. P Natl Acad Sci USA 98(10):5544–5549 Thomas MC, Chiang CM (2006) The general transcription machinery and general cofactors. Crit Rev Biochem Mol 41:105–178 Tóth-Petróczy A, Oldfield CJ, Simon I, Takagi Y, Dunker AK, Uversky VN, Fuxreiter M (2008) Malleable machines in transcription regulation: the mediator complex. PLoS Comput Biol 4(12):1–12 Wang GG, Allis DC, Ping C (2007) Chromatin remodeling and cancer, part II: ATP-dependent chromatin remodeling. Trends Mol Med 13(9):373–380 Yan Q, Moreland RJ, Conaway JW, Conaway RC (1999) Dual roles for Transcription Factor IIF in promoter escape by RNA polymerase II. J Biol Chem 274(50):35668–35675

Chapter 15

Estrogen Signaling Mechanisms Dapeng Zhang and Vance L. Trudeau

15.1

Introduction

The main estrogenic hormone, 17b-estradiol (E2), exerts important regulatory roles in a wide variety of biological processes including reproduction, differentiation, cell proliferation, apoptosis, inflammation, metabolism, homeostasis and brain function (Tsai and O’Malley 1994). The classical mechanism of E2 action is mediated by transcriptional actions of the nuclear estrogen receptors (nERs), ERa and ERb. Upon binding with E2, nERs can be released from inactive complexes containing heat-shock proteins and immunophilins (Ylikomi et al. 1998), homodimerize or heterodimerize (Cowley et al. 1997), and bind to a specific DNA estrogen response element (ERE) which is located in or nearby the promoter regions of target genes. Thereafter, a series of coactivators is recruited to DNA-bound ERs; they commonly induce chromatin remodeling and modulate target gene expression. In addition to direct binding to the ERE, nER can also affect gene expression through protein–protein interaction with other classes of transcriptional factors, such as AP-1 (Gaub et al. 1990) or Sp-1 (Castro-Rivera et al. 2001). In contrast with classical nER action, a new rapid biochemical and physiological effect of E2 has been discovered and specific membrane ERs (mER) are hypothesized to be involved. Upon activation of the mER, various protein kinase cascades, including mitogen activated protein kinase (MAPK), protein kinase A (PKA), protein kinase C (PKC), and phosphatidylinositol 3-OH kinase (PI3K), are activated. Many studies further revealed that either classical nERs or a G protein-coupled receptor (GPCR30) can be the membrane receptor (Norfleet et al. 1999; Pappas et al. 1995; Revankar et al. 2005; Thomas et al. 2005). Moreover, a series of new regulatory pathways has been defined in E2 actions, including mER-initiated signaling to gene expression (Zhang and Trudeau 2006) and the regulatory cascades via third messengers (Carroll et al. 2005; Laganiere et al. 2005). In this chapter,

V.L. Trudeau (*) and D. Zhang Center for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Ontario, Canada, K1N 6N5 e-mail: [email protected]

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_15, © Springer-Verlag Berlin Heidelberg 2010

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we review the basics of E2 action covering both classical nuclear signaling and new membrane actions, as well as present a dynamic regulatory model for E2 action.

15.2

Nuclear Receptor-Mediated Action of E2: The Classical Mechanism

Nuclear actions of E2 are mediated by two main nuclear estrogen receptor subtypes, ERa and ERb, which are members of a superfamily of nuclear receptors that function as ligand- or hormone-dependent transcription factors (McKenna and O’Malley 2002). Two important functional domains in ERa and ERb, the ligand binding domain (LBD) and the DNA binding domain (DBD), are well conserved both at the amino acid sequence and the structural levels. The two major nER subtypes have distinct expressions patterns in tissues (Couse et al. 1997; Shughrue et al. 1997; Shughrue et al. 1998) and ER knockout mice models (aERKO and bERKO) exhibit different phenotypes (Couse and Korach 2001; Hess 2003; Hewitt et al. 2005), indicating distinct biological functions of the receptors. In contrast to the functional deficiencies in the reproductive system in both sexes of aERKO mice, male bERKO mice are fertile while females are subfertile. Maximal transcriptional activity of nER requires recruitment of other transcription factors and multiple coactivators including SRC-1 (steroid receptor coactivitor-1), GRIP-1 (glucocorticoid receptor-interacting protein-1), AIB1 (amplified in breast cancer-1), CBP/p300 (cAMP response element binding protein-binding protein), PGC-1 (peroxisome proliferators-activated receptor gamma coactivator 1a) and p68 RNA helicase (McKenna and O’Malley 2002; Metivier et al. 2003). Coactivator interaction and its availability can greatly influence nER action. In both male and female rodent brain, reduction of SRC-1 and CBP protein with antisense oligonucleotides disrupted nER-mediated induction of progestin receptor gene (Molenda et al. 2002). In male Japanese quail, inhibition of SRC-1 significantly affected the estrogen-dependent male-typical sexual behavior, and is correlated with reduction in the volume of the preoptic medial nucleus and the expression of aromatase and vasotocin proteins (Charlier et al. 2005). Nuclear ER actions can be modulated by numerous cellular signaling pathways. Different kinases, such as PKA (Chen et al. 1999), mitogen activated protein kinase (MAPK) (Tang et al. 2004), and cyclin A-CDK2 (Rogatsky et al. 1999), are induced by extracellular signals (e.g., growth factors) which leads to phosphorylation of several N-terminal serine residues of ERa, for example serines 104, 106, 118, and 167 (Lannigan 2003). These phosphorylation modifications modulate receptor functions, including nER downregulation by the ubiquitin-proteasome pathway (Marsaud et al. 2003), nuclear localization of nER (Lee and Bai 2002), nER dimerization (Chen et al. 1999), and transcriptional activity (Ali et al. 1993; Le Goff et al. 1994). Coregulators of nER are also targeted by these signaling pathways. Mutation of the ERK2-responsive phosphorylation site in GRIP-1 inhibits the ability of this coactivator to enhance ERa transcriptional activity stimulated by

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E2 in human Hela cells (Lopez et al. 2001) and MCF-7 cells (Frigo et al. 2006). Kinase-mediated signaling pathways most likely serve to induce differential activation and recruitment of nuclear receptors and coregulators, which will modulate gene expression in a cell-specific manner (McKenna and O’Malley 2002; Smith and O’Malley 2004; Wu et al. 2005). Importantly, nER can also regulate gene expression without binding directly to DNA. Many promoters of E2 target genes have no consensus ERE, and nER is assumed to act through specific protein–protein interactions with other classes of transcriptional factors to affect gene transcription. Best studied is the Era–Sp1 interaction which induces expression of several genes, including E2F1 (Wang et al. 1999), LDL-R (Li et al. 2001), c-fos (Duan et al. 1998), and cyclin D1 (Castro-Rivera et al. 2001). Interactions between nER and AP-1 proteins (Fos and Jun) increase transcription of ovalbumin (Gaub et al. 1990), IGF-I (Umayahara et al. 1994), collagenase (Webb et al. 1995), and cyclin D1 (Sabbah et al. 1999; Liu et al. 2002), whereas ER-NFkB-C/EBP interaction decreases the expression of interleukin-6 gene (Stein and Yang 1995).

15.3

Membrane Receptor-Mediated Actions of E2

Rapid membrane-initiated actions of E2 have been observed since the late 1960s (Lincoln 1967; Dufy et al. 1976; Pietras and Szego 1977; Nakhla et al. 1994; Watters et al. 1997; Kelly and Levin 2001). E2 exposure can rapidly induce activation of many protein kinases and modulate ion fluxes (i.e., Ca2+, K+) across the plasma membrane. Specific mERs are believed to be involved since such transient events are not prevented by protein synthesis inhibitors. The existence of mERs is also supported by the evidence that treatment with membrane impermeable estrogen, typically E2 conjugated to bovine serum albumin (E2-BSA) in a short time course (seconds to minutes), can mimic the rapid effects of E2 on signal transduction pathways.

15.3.1

Membrane Receptors for E2: Classical Estrogen Receptors or GPCR30?

For a long time, people have tried to verify the nature of the mER, although it still remains controversial. Some experiments favor the hypothesis that the classical nER acting at the level of the plasma membrane may contribute to rapid action of E2. Antibodies directed against different epitopes of nERa have been used to localize it to the region of the plasma membrane (Pappas et al. 1995; Norfleet et al. 1999). Moreover, it has been shown that the translocation of ERa to the plasma membrane is facilitated by two important adaptor proteins, Shc (Src-homology and collagen homology) and IGF-1 (insulin-like growth factor 1 receptor) (Song et al. 2004; Zhang et al. 2004). Using a yeast two-hybrid system, Lu et al. (2004) identified

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another important protein, striatin, which acts as a molecular anchor for ERa in the plasma membrane. A critical role of classical ERs in the rapid action of estrogen was also supported by the data from ERa- and ERb-knockout (ERKO) mice in which the rapid induction of MAPK or PI3K phosphorylations by E2 was lost in either the medial preoptic nucleus (Abraham et al. 2004) or the endothelial cells (Pedram et al. 2006). Moreover, mass spectrometry analysis has verified that ERa is the main protein that locates at plasma membrane and interacts with E2 (Pedram et al. 2006). Other distinct membrane ER (mER) isoforms which are different from the classical ERa and ERb are also likely to exist. Some cell types that do not express either ERa or ERb can still exhibit rapid responses to E2. For example, mouse pancreatic b cells exhibit rapid membrane-mediated non-genomic responses to estrogens and xenoestrogens, yet these cells are immunonegative for membrane-localized classical nERs (Nadal et al. 2000). Filardo et al. (2000) for the first time showed that GPCR30, an orphan receptor member of the GPCR family, may be such a mER. It has been shown that rapid estrogen activation of Erk1/2 phosphorylation is lost in MDA-MB-231 cells where GPCR30 is not expressed; however such action can be recovered by transfection of GPCR30 (Filardo et al. 2000). Thereafter, Thomas et al. (2005) demonstrated that GPCR30 has all the binding and signaling characteristics of a distinct mER in the SKBR3 breast cancer cell line that does not express either ERa or ERb. This is further supported by several studies in which silencing of GPCR30 with either antisense oligodeoxynucleotide (Maggiolini et al. 2004; Sirianni et al. 2008; Vivacqua et al. 2006), siRNAs (Hsieh et al. 2007), or mouse knockouts (Wang et al. 2008) suppresses the E2-induced rapid responses. Moreover, a selective GPCR30 agonist, G-1, has been developed that shows similar potential to E2 in inducing signaling pathways including Ca2+ and PI3K activation, where it has no binding activity to nER (Bologa et al. 2006). It should be noted that there is a disagreement on the cellular location of GPCR30 in the published literature. In contrast with the plasma membrane location reported by Thomas et al. (2005) and Funakoshi et al. (2006), GPCR30 was subsequently found to localize to endoplasmic reticulum membranes of different types of cells (Otto et al. 2008; Revankar et al. 2005). There is also some disagreement regarding the nature of mER. Although a role of GPCR30 in E2 rapid actions has been confirmed, some groups failed to verify it and instead showed that ERa acts as mER (Pedram et al. 2006; Otto et al. 2008). It seems that both classical estrogen receptor and GPCR30 may have a role in membrane-initiated actions of E2 and there may also exist interactions between them in certain cell types, which increases the complexity of estrogen signaling. Importantly, this presents a major challenge in experimental design when determining the contributions of the various ERs to the control of cellular function.

15.3.2

Signaling Pathways Initiated by mER

Several signaling pathways are activated by binding of E2 to membrane ERs, which has been reviewed previously (Zhang and Trudeau 2006). These include

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Ca2+ flux (Improta-Brears et al. 1999), cAMP (Nakhla et al. 1994; Watters and Dorsa 1998), MAPK (Watters et al. 1997; Wade and Dorsa 2003), PKA (Lagrange et al. 1997), PKC (Qiu et al. 2003), PI3K (Simoncini et al. 2000; Alexaki et al. 2004), and Src kinase (Kennedy et al. 2005). Though there are many potential interactions among these pathways, the activation events can be classified into three main signaling pathways: the Ras-Raf-MEK-MAPK, Src-PI3K-Akt-eNOS, and PLC-PKC-cAMP-PKA modules (Fig. 15.1). MAPK activation upon E2 or E2-BSA exposure has been described in breast cancer cells (Migliaccio et al. 1996), endothelial cells (Russell et al. 2000; Klinge et al. 2005), and adipocytes (Dos Santos et al. 2002). MAPK is activated by phosphorylation on specific threonine and tyrosine residues by MAPK kinases (MEKs). MEKs are themselves activated by several kinases such as the Raf proteins, which in turn are regulated by Ras family members. The binding of mER with E2 can induce E E E Membrane

RTK

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+ K PKC

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p p CREB AP1 p Coactivator

p

CyclinD1

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ERE-promoter E p CyclinD1 p nER FoxA1 Coactivator ERE-promoter

Fig. 15.1 An integrative model for E2 action involving both membrane and nuclear estrogen receptors. The figure is adapted from Figure 1 in Zhang and Trudeau (2006). For details refer to main text. Abbreviations: AC, adenylyl cyclase; Akt, protein kinase Akt; Ca2+, calcium; eNOS, endothelial nitric oxide (NO) synthase; CREB, cAMP-response element-binding protein; E, estrogen; G, G protein; GABAR, GABA receptor; GIRK, G protein-gated inwardly rectifying K+ channels; GRB2, growth factor receptor-bound protein 2; MAPK, mitogen activated protein kinase; MEK, mitogen/extracellular signal protein kinase; mER, membrane estrogen receptor (ER or GPCR30); nER, nuclear estrogen receptor; NO, nitric oxide; P, phosphorylation modification; PI3K, phosphatidylinositol 3-OH kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; Src, protein kinase Src; Raf, v-raf-1 murine leukemia viral oncogene; Ras, related RAS viral (r-ras) oncogene homolog; RTK, receptor tyrosine kinase; Shc, Src-homology and collagen homology; SOS, son of sevenless; m, m-opioid receptor

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the activation of receptor tyrosine kinases (RTK) such as EGF and IGF-1 receptors, which transmit signals through protein complexes including adaptor protein Shc, Grb2, and the guanine nucleotide exchange protein Sos, leading to Ras activation (Driggers and Segars 2002; Zhang et al. 2004). MAPK activation can also be modulated by either G protein through a signaling pathway involving non-receptor tyrosine kinase Src and PI3K (Dos Santos et al. 2002), or intracellular calcium which can modulate Ras activation (Improta-Brears et al. 1999; Kupzig et al. 2005). The Src-PI3K-Akt-eNOS module is a pathway in endothelial cells mediating rapid E2-dependent release of nitric oxide (NO). In this process, the signal from mER is transmitted to PI3K through a G-protein and Src complex. In turn, PI3K activates Akt, which stimulates eNOS activation (Segars and Driggers 2002; Haynes et al. 2003). A PLC-PKC-AC-cAMP-PKA module mediates the effect of E2 on the change of K+ fluxes in neurons (Malyala et al. 2005; Gu and Moss 1996). Early evidence showed that E2 can attenuate the ability of m-opioid and GABA receptormediated activation of G protein-gated inwardly rectifying K+ channels (GIRKs) (Kelly et al. 1992). PKA is also involved because E2 action can be mimicked by the PKA activators forskolin and cAMP, whereas PKA antagonist can block this effect (Lagrange et al. 1997). Using a peptide inhibitor that interferes with G-protein receptor signaling and several PKC inhibitors, Qiu et al. (2003) illustrated that the E2 effects are mediated by G protein and PKC. Activation of PKC is likely to be upstream of PKA. Selective PKC inhibition using bisindolymaleimide (BIS) blocked the E2 effects; however forskolin can still mimic the effect of E2 even in the presence of BIS (Qiu et al. 2003). The PLC-PKC-AC-cAMP-PKA pathway is also activated by E2 in intestinal cells and this leads to changes in intracellular Ca2+ (Picotto et al. 1996; Picotto et al. 1999). Note that a change of Ca2+ fluxes can also be induced by PLC through IP3 in the hypothalamus (Malyala et al. 2005). The activation of signaling pathways by E2 is cell type-specific. For example, the effect of E2 on PKC catalytic activity has been observed in the preoptic area (POA) of female rat brain slices, but not in the remaining portion of the hypothalamus posterior to the POA (Ansonoff and Etgen 1998). The activation of the G-protein/Src/PI3K/MAPK pathway by E2 was evident in late, but not early, differentiated rat preadipocytes (Dos Santos et al. 2002). The differential requirement of Src/PI3K or intracellular calcium for MAPK activation is also observed in diverse cell types (Dos Santos et al. 2002; Improta-Brears et al. 1999; Kupzig et al. 2005). It appears that differential expression patterns of some proteins which are involved in signal transduction or the membrane structural organization account for this diversity (Dos Santos et al. 2002). The differential utilization of signal pathways in different cells may contribute to the cell type-specific E2 action.

15.4

mER Signaling to Genomic Transcriptional Regulation

It is well known that activated signaling pathways involving MAPK and PKA can influence the activities of downstream proteins including chromatin remodeling factors, transcription factors (TFs), and also nER and its coactivitors through

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diverse post-translational modifications (e.g., phosphorylation, methylation, or acetylation). The change of states in phosphorylation and other modifications is necessary for the transcriptional activities of these transcription factors, which ultimately leads to changes in transcription of their target genes. Therefore, activation of kinase cascades via the mER is an alternative mechanism for E2 to regulate gene expression.

15.4.1

mER Action can Modulate Gene Expression Through Non-ER Transcription Factors

Many transcription factors have been shown to be regulated by mER-dependent signaling pathways. CREB (cAMP-response element-binding protein) is the most studied target. In a hippocampal cell line (Wade and Dorsa 2003), adipocyte cells (Dos Santos et al. 2002), and colonic carcinoma cells (Hennessy et al. 2005), CREB transcriptional activity can be induced by E2 or E2-BSA through MAPK pathway, independently of PKA pathway. Such activation of CREB induces expression of several genes including c-fos and UCP-2. In contrast, in neuroblastoma cells activation of CREB by mER is mediated through a cAMPPKA pathway, leading to neurotensin gene expression (Watters and Dorsa 1998). This may reflect cell-specific activation of the various signal pathways by mER. Serum response factor (SRF) and ELK1 are also mER-responsive transcription factors, which are involved in regulating c-fos expression (Hennessy et al. 2005; Duan et al. 2001). It should be noted that in human MCF-7 breast cancer cells, SRF can be regulated by either MAPK pathway (Duan et al. 2001) or PI3K pathway (Duan et al. 2002), implying that the different pathways can cooperate to exert actions. The AP-1 protein is another effector of mER action. Both DNA binding activity and transcriptional activity of AP-1 can be increased by E2 through the MAPK pathway (Dos Santos et al. 2002; Bjornstrom and Sjoberg 2004), which further contributes to regulating expression of the cyclin D1 and collagenase genes (Marino et al. 2002; Bjornstrom and Sjoberg 2004). Other targets include several members of the Stat family such as Stat 1, Stat3, and Stat5 (Kennedy et al. 2005; Bjornstrom and Sjoberg 2002). In endothelial cells, activation of both Stat3 and Stat5 by E2 was mediated through signaling pathways involving MAPK, PI3K and Src, and functioned to regulate b-casein expression (Bjornstrom and Sjoberg 2002). Furthermore, a global gene expression group was first identified in human vascular endothelial cells responding to the mER-initiated PI3K signaling pathway (Pedram et al. 2002). E2 exposure for 40 min rapidly up-regulated approximately 250 genes, which was prevented by inhibiting the PI3K pathway with LY294002. With the development of new selective mER agonists, the mER-specific regulated genes have been profiled recently. For example, in MCF-7 breast cancer cells, over 300 genes are shown to be differentially expressed upon treatment with

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estrogen-dendrimer conjugates (EDCs), which only have mER action because of their charge and size (Madak-Erdogan et al. 2008). Additionally, a group of 241 genes in pig arcuate nucleus was identified after treatment with STX, which is a selective mER agonist (Roepke et al. 2008). It should be noted that several typical genes responsible for mER actions, such as c-fos and cyclin D1, were also shown to be regulated by direct interactions between nER and other transcription factors, including Sp1 and AP-1 (Duan et al. 1998; Castro-Rivera et al. 2001; Sabbah et al. 1999; Liu et al. 2002). In future research it will be necessary to consider both contributions of direct interactions of nERs with other transcription factors, as well as mER signaling, in the regulation of gene expression.

15.4.2

Membrane Actions of E2 Signaling to nER

mER-mediated signaling events may also have important modulatory effects on classical nERs and their coactivators (Zhang and Trudeau 2006; Vasudevan and Pfaff 2008). It has been long known that E2 treatment can increase the phosphorylation state of nERs, and mutation of important phosphorylation sites reduces the transcription activity of nERs (Ali et al. 1993; Le Goff P et al. 1994). Also AIB1, one nER coactivator, has been reported to be phosphorylated following E2 expsoure in human HEK293 cells and MCF-7 cells (Wu et al. 2004). Multiple cellular signaling pathways, including p38, JNK, and ERK1/2, are involved. RNAi-mediated knockdown of these kinases diminishes the nER-induced expression of endogenous pS2, which implies a critical contribution of mER signaling in nER actions through AIB1. Furthermore, induction of MAPK and PI3K pathways by mER are both necessary and sufficient for the transcription of non-nER regulated genes, but also for the transcription of nER-regulated genes (Acconcia et al. 2003). Using a two-pulse paradigm of hormone administration, Vasudevan et al. (2001) demonstrated that rapid E2 action can potentiate slower nER-mediated transcriptional processes in neuroblastoma cells. Intracellular signaling cascades were shown to be important in such potentiation; inhibition of PKA, PKC, MAPK or PI3K in the first pulse decreased this effect (Kow and Pfaff 2004). Besides modifying phosphorylation state, mER-initiated signaling may also regulate nER actions through other types of modifications. For example, nER binding to ERE is inhibited by an S-nitrosylation modification induced by NO (Garbán et al. 2005), which is a major product in the mER-initiated signaling pathway of SrcPI3K-Akt-eNOS module. Moreover, the effect of mER action on nER action has been further verified using global gene expression profiling (Madak-Erdogan et al. 2008). There are about 243 genes in MCF-7 breast cancer cells whose expression is commonly induced by both E2 and EDCs. The inductive effect of both E2 and EDCs on target genes was prevented by treatment with inhibitors of MAPK kinase and c-Src.

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E2-Induced Transcriptional Regulatory Cascades via Third Messengers

Besides mER-initiated signaling to nER, recent studies have illustrated another regulatory cascade; that is, early induction of some TFs via mER or nER can modulate subsequent nER actions. Global gene expression analysis by microarray or CHIP-chip combined with functional analysis and genome transcriptional factor binding site analysis has revealed many so-called third messengers (Kovacs 1998) such as cyclin D1 (Yang et al. 2006), E2F members (Bourdeau et al. 2007), FoxA1 (Carroll et al. 2005; Eeckhoute et al. 2006; Laganiere et al. 2005), ANCCA (Zou et al. 2007), c-MYC (Cheng et al. 2006; Musgrove et al. 2008), and NF1C (Eeckhoute et al. 2006) (Fig. 15.2). In this context, the best studied transcription factor is FoxA1, whose expression is induced by nER action. Interestingly, this induction of FoxA1 was found to be necessary for subsequent nER action in human MCF-7 cancer cells (Carroll et al. 2005; Laganiere et al. 2005). The binding sites for FoxA1 are enriched in the nER binding regions of promoters of many genes. Small interfering RNAmediated knockdown of FoxA1 blocks the transcription of other estrogen-regulated genes, namely TFF-1 and vitellogenin-B1. Therefore, early induction of FoxA1 by E2 action defines a specific gene repertoire for later nER action (Carroll et al. 2005; Laganiere et al. 2005). In addition to these third messengers induced by early nER action, those TFs that can be rapidly induced by mER signaling can also be third messengers. For example, the above-mentioned c-fos

Cyclin D1

E2Fs

FoxA1 E2-mER-nER

E2-nER ANCCA

c-MYC

NF1C

Fig. 15.2 E2-induced transcriptional regulatory cascades via third messengers. A series of transcription factors can be induced by early E2 (mER or nER) actions and then further modulate late nER actions in regulating target gene expression

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and cyclin D1 are known coregulators for nER action (Kovacs 1998; Fu et al. 2004; Burd et al. 2005). Therefore, induction of these coregulators by mER action might be another generalized mechanism for early-to-late regulation of E2 action. Utilization of third messengers by early E2 action may also be cell type-specific. Differential sets of third messengers may determine the specific regulated gene repertoire of E2 action. Moreover, there may exist a more complex transcriptional regulatory network in which diverse TFs can coordinate or interfere in a series of cascades induced by E2 action. For example, the fine-tuning of cyclin D1 by E2 action is achieved by induction of FoxA1 to increase expression or induction of NF1C to inhibit expression of cyclin D1 (Eeckhoute et al. 2006).

15.6

An Integrative Model for mER and nER Signaling

Based on the evidence described above, we propose a regulatory model for E2 action in which both membrane-to-nuclear signaling and early induction of third messenger-to-late modulation on nER action are integrated (Fig. 15.1). Considering the controversy over the nature of mER, we used the generic “mER” to represent either GPCR30 or classical ER at the membrane in Fig. 15.1. For cellular location, only one type of membrane is shown to represent either plasma membrane or endoplasmic reticulum membranes. Specifically, E2 initiates membrane actions and nuclear actions through its membrane receptors and nuclear receptors, respectively. In nuclear action, estrogen receptors binding E2 are activated to recruit ERE in the promoter regions, thereby regulating expression of target genes. In contrast, E2 binding to mER rapidly induces the activation of the signaling pathways including the Ras-Raf-MEK-MAPK (Driggers and Segars 2002), Src-PI3K-Akt-eNOS (Segars and Driggers 2002), and PLCPKC-cAMP-PKA modules (Malyala et al. 2005). These signaling pathways can influence the post-translational modifications of a series of transcription factors (e.g., CREB and AP1), which then modulate expression of some genes that do not contain the consensus ERE in the promoter region. At the same time, mERinitiated signaling has a modulatory effect on nER action through targeting nER and its coactivators. Moreover, induced expression of third messengers (e.g., c-fos, cyclin D1, and FoxA1) is another important mechanism linking the earlier and later estrogen actions. Thus, all these regulatory cascades including membrane-to-nuclear signaling and early induction of third messager-to-late modulation on nER action are combined to specify estrogen action in a cell-dependent manner. Acknowledgements The financial support of the University of Ottawa International Scholarship program (to DZ), and the NSERC Discovery and Strategic programs (to VLT) is acknowledged with appreciation.

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Chapter 16

Signal Transduction Pathways Involved in Glucocorticoid Actions Peter J. Barnes

16.1

Introduction

Glucocorticoids (glucocorticosteroids or corticosteroids) are widely used to treat a variety of inflammatory and immune diseases. The most common use of glucocorticoids today is in the treatment of asthma and other allergic diseases, and inhaled glucocorticoids are now established as first-line treatment in adults and children with persistent asthma. Despite intense efforts by the pharmaceutical industry it has proved extraordinarily difficult to find any new treatment that comes close to the therapeutic benefit of glucocorticoids in asthma (Barnes 2004). This chapter focuses on the molecular mechanisms of actions of glucocorticoids that are relevant to the treatment of inflammatory diseases such as asthma. There have been major advances in understanding the molecular mechanisms whereby glucocorticoids suppress inflammation, based on recent developments in understanding the fundamental mechanisms of gene transcription (Rhen and Cidlowski 2005; Barnes 2006b). This has important clinical implications, as it will lead to a better understanding of the inflammatory mechanisms of many diseases and may lead to the development of new antiinflammatory treatments in the future. The new understanding of these molecular mechanisms also helps to explain how glucocorticoids are able to switch off multiple inflammatory pathways, and it also provides insights into why glucocorticoids apparently fail to work in patients with steroid-resistant asthma and in patients with chronic obstructive pulmonary disease (COPD).

P.J. Barnes Section of Airway Disease, National Heart and Lung Institute, Dovehouse St, London, SW3 6LY, UK e-mail: [email protected]

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The Molecular Basis of Inflammation

Understanding the molecular mechanisms involved in chronic inflammation is necessary in order to understand how glucocorticoids so efficiently suppress inflammation in complex inflammatory diseases such as asthma. Patients with asthma and allergic rhinitis have a specific pattern of inflammation in the airways that is characterized by degranulated mast cells, infiltration of eosinophils and increased number of activated T helper 2 (Th2) cells (Barnes 2008b). Suppression of this inflammation by glucocorticoids controls and prevents these symptoms in the vast majority of patients. Multiple mediators are produced in allergic diseases and over 100 known inflammatory mediators that are increased include lipid mediators, inflammatory peptides, chemokines, cytokines and growth factors (Barnes et al. 1998; Barnes 2008a). Inflammation is mediated by the increased expression of multiple inflammatory proteins, including cytokines, chemokines, adhesion molecules, and inflammatory enzymes and receptors. Most of these inflammatory proteins are regulated by increased gene transcription, which is controlled by proinflammatory transcription factors, such as nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) that are activated in asthmatic cells (Barnes 2006c). For example, NF-kB is markedly activated in epithelial cells of asthmatic patients and this transcription factor regulates many of the inflammatory genes that are abnormally expressed in asthma. NF-kB may be activated by rhinovirus infection and allergen exposure, both of which exacerbate asthmatic inflammation.

16.2.1

Chromatin Remodeling

Chromatin consists of DNA and basic proteins called histones which provide the structural backbone of the chromosome. It has long been recognized that histones play a critical role in regulating the expression of genes and determine which genes are transcriptionally active and which ones are suppressed (silenced). The chromatin structure is highly organized as almost 2 m of DNA have to be packed into each cell nucleus. Chromatin is made up of nucleosomes which are particles consisting of 146 base pairs of DNA wound almost twice around an octomer of two molecules each of the core histone proteins H2A, H2B, H3 and H4 (Kouzarides 2007). Expression and repression of genes is associated with remodeling of this chromatin structure by enzymatic modification of the core histone proteins, particularly by acetylation. Each core histone has a long N-terminal tail that is rich in lysine residues, which may become acetylated, thus changing the electrical charge of the core histone. In the resting cell, DNA is wound tightly around core histones, excluding the binding of the enzyme RNA polymerase II, which activates gene transcription and the formation of messenger RNA. This conformation of the chromatin structure is described as closed and is associated with suppression of gene expression. Gene transcription only occurs when the

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chromatin structure is opened up, with unwinding of DNA so that RNA polymerase II and basal transcription complexes can now bind to DNA to initiate transcription.

16.2.2

Histone Acetyltransferases and Coactivators

When proinflammatory transcription factors, such as NF-kB and AP-1, are activated they bind to specific recognition sequences in DNA and subsequently interact with large coactivator molecules, such as cyclic adenosine monophosphate response element binding protein (CREB)-binding protein (CBP). These coactivator molecules act as the molecular switches that control gene transcription and all have intrinsic histone acetyltransferase (HAT) activity (Kouzarides 2007). This results in acetylation of core histones, thereby reducing their charge which allows the chromatin structure to transform from the resting closed conformation to an activated open form. This results in unwinding of DNA, binding of TATA box binding protein (TBP), TBP-associated factors and RNA polymerase II, which then initiates gene transcription. This molecular mechanism is common to all genes, including those involved in differentiation, proliferation and activation of cells. Of course this process is reversible and deacetylation of acetylated histones is associated with gene silencing. This is mediated by histone deacetylases (HDACs) which act as corepressors, together with other corepressor proteins which are subsequently recruited. These fundamental mechanisms have now been applied to understanding the regulation of inflammatory genes in diseases such as asthma and COPD (Barnes et al. 2005; Adcock et al. 2006). In a human epithelial cell line activation of NF-kB, by exposing the cell to inflammatory signals such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a or endotoxin, results in acetylation of specific lysine residues on histone H4 (the other histones do not appear to be so markedly or rapidly acetylated) and this is correlated with increased expression of genes encoding inflammatory proteins, such as granulocyte-macrophage colony stimulating factor (GM-CSF) (Ito et al. 2000).

16.2.3

Histone Deacetylases and Corepressors

The acetylation of histone that is associated with increased expression of inflammatory genes is counteracted by the activity of HDACs, of which 11 that deacetylate histones are now characterized (de Ruijter et al. 2003; Thiagalingam et al. 2003). There is now evidence that the different HDACs target different patterns of acetylation (Peterson 2002). In biopsies from patients with asthma there is an increase in HAT and a reduction in HDAC activity, thereby favoring increased inflammatory gene expression (Ito et al. 2002). With this background it is now

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possible to understand better why glucocorticoids are so effective in suppressing this complex inflammatory process that involves the increased expression of multiple inflammatory proteins. HDACs act as corepressors in consort with other corepressor proteins, such as NCoR and SMRT, forming a corepressor complex that silences gene expression (Lonard et al. 2007).

16.3

Glucocorticoid Receptors

Glucocorticoids diffuse readily across cell membranes and bind to GR in the cytoplasm. Cytoplasmic GR are normally bound to proteins, known as molecular chaperones, such as heat shock protein-90 (hsp90) and FK-binding protein, that protect the receptor and prevent its nuclear localization by covering the sites on the receptor that are needed for transport across the nuclear membrane into the nucleus (Wu et al. 2004). The mechanism of nuclear translocation involves the nuclear import protein importin-a (karyopherin-b) and importin-13 (Goldfarb et al. 2004; Tao et al. 2006). There is a single gene encoding human GR but several variants are now recognized, as a result of transcript alternative splicing, and alternative translation initiation (Lu and Cidlowski 2004). GRa binds glucocorticoids whereas GRb is an alternatively spliced form that binds to DNA but cannot be activated by glucocorticoids. GRb has a very low level of expression compared to GRa. The GRb isoform has been implicated in steroid resistance in asthma, although whether GRb can have any functional significance has been questioned in view of the very low levels of expression compared to GRa (Lewis-Tuffin and Cidlowski 2006). GR may also be modified by phosphorylation and other modifications, which may alter the response to glucocorticoids by affecting ligand binding, translocation to the nucleus, trans-activating efficacy, protein–protein interactions or recruitment of cofactors (Ismaili and Garabedian 2004). For example, there are a number of serines/threonines in the N-terminal domain where glucocorticoid receptors may be phosphorylated by various kinases. Once glucocorticoids have bound to GR, changes in the receptor structure result in dissociation of molecular chaperone proteins, thereby exposing nuclear localization signals. This results in rapid transport of the activated glucocorticoid receptor–glucocorticoid complex into the nucleus, where it binds to DNA at specific sequences in the promoter region of glucocorticoid-responsive genes known as glucocorticoid-response elements (GRE). Two glucocorticoid receptor molecules bind together as a homodimer and bind to GRE, leading to changes in gene transcription. Interaction of GR with GRE classically leads to an increase in gene transcription (trans-activation), but negative GRE sites have also been described where binding of GR leads to gene suppression (cis-repression) (Dostert and Heinzel 2004) (Fig. 16.1). There are few well documented examples of negative GREs, but some are relevant to glucocorticoid side effects, including genes that regulate the hypothalamic–pituitary axis (proopiomelanocortin and

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Fig. 16.1 Glucocorticoids may regulate gene expression in several ways. Glucocorticoids enter the cell to bind to glucocorticoid receptors (GR) in the cytoplasm that translocate to the nucleus. GR homodimers bind to GRE in the promoter region of steroid-sensitive genes, which may encode antiinflammatory proteins. Less commonly, GR homodimers interact with negative GREs to suppress genes, particularly those linked to side effects of glucocorticoids. Nuclear GR also interact with coactivator molecules, such as CREB-binding protein (CBP), which is activated by proinflammatory transcription factors, such as nuclear factor-kB (NF-kB), thus switching off the inflammatory genes that are activated by these transcription factors. Other abbreviations: SLPI: secretory leukoprotease inhibitor; MKP-1: mitogen-activated kinase phosphatase-1; IkB-a: inhibitor of NF-kB; GILZ: glucocorticoid-induced leucine zipper protein; POMC: proopiomelanocortin; CRH: corticotrophin releasing factor

corticotrophin-releasing factor), bone metabolism (osteocalcin) and skin structure (keratins).

16.4

Glucocorticoid Activation of Gene Transcription

Glucocorticoids produce their effect on responsive cells by activating glucocorticoid receptors to directly or indirectly regulate the transcription of target genes. Relatively few genes per cell are directly regulated by glucocorticoids, but many are indirectly regulated through an interaction with other transcription factors and coactivators. Glucocorticoid receptors homodimers bind to GRE sites in the promoter region of glucocorticoid-responsive genes. Interaction of the activated

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glucocorticoid receptor dimer with GRE usually increases transcription. Glucocorticoid receptors may increase transcription by interacting with coactivator molecules, such as CBP, thus activating histone acetylation and gene transcription. For example, relatively high concentrations of glucocorticoids increase the secretion of the antiprotease secretory leukoprotease inhibitor (SLPI) from epithelial cells (Ito et al. 2000). The activation of genes by glucocorticoids is associated with a selective acetylation of lysine residues 5 and 16 on histone H4, resulting in increased gene transcription (Ito et al. 2000) (Fig. 16.2). Activated GR may bind to coactivator molecules, such as CBP, as well as steroid-receptor coactivator-1 (SRC-1) and glucocorticoid receptor interacting protein-1 (GRIP-1 or SRC-2), all of which possess HAT activity (Kurihara et al. 2002).

Fig. 16.2 Glucocorticoids’ activation of antiinflammatory gene expression. Glucocorticoids bind to cytoplasmic GR which translocate to the nucleus where they bind to GRE in the promoter region of steroid-sensitive genes and also directly or indirectly to coactivator molecules such as CREB-binding protein (CBP), which have intrinsic histone acetyltransferase (HAT) activity, causing acetylation of lysines on histone H4, which leads to activation of genes encoding antiinflammatory proteins, such as secretory leukoprotease inhibitor (SLPI), mitogen-activated kinase phosphatase-1 (MKP-1), inhibitor of NF-kB (IkB-a) and glucocorticoid-induced leucine zipper protein (GILZ)

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295

AntiInflammatory Gene Activation

Several of the genes that are switched on by glucocorticoids have antiinflammatory effects, including annexin-1 (lipocortin-1), SLPI, interleukin-10 and inhibitor of NF-kB (IkB-a). However, therapeutic doses of inhaled glucocorticoids have not been shown to increase annexin-1 concentrations in bronchoalveolar lavage fluid (Hall et al. 1999) and an increase in IkB-a has not been shown in most cell types, including epithelial cells (Newton et al. 1998). Glucocorticoids also switch on the synthesis of two proteins that affect inflammatory signal transduction pathways, glucocorticoid-induced leucine zipper protein (GILZ), which inhibits both NF-kB and AP-1 (Mittelstadt and Ashwell 2001) and mitogen-activated protein kinase phosphatase-1 (MKP-1), which inhibits p38 MAP kinase and Jun kinase (Clark et al. 2008). However, it seems unlikely that the widespread antiinflammatory actions of glucocorticoids could be entirely explained by increased transcription of small numbers of antiinflammatory genes, particularly as high concentrations of glucocorticoids are usually required for this effect, whereas in clinical practice glucocorticoids are able to suppress inflammation at low concentrations.

16.4.2

Side Effect Genes

Relatively little is known about the molecular mechanisms of glucocorticoid side effects, such as osteoporosis, growth retardation in children, skin fragility and metabolic effects. These actions of glucocorticoids are related to their endocrine effects. The systemic side effects of glucocorticoids may be due to gene activation. Some insight into this has been provided by mutant glucocorticoid receptors which do not dimerize and therefore cannot bind to GRE to switch on genes. In transgenic mice expressing these mutant glucocorticoid receptors, glucocorticoids show no loss in their antiinflammatory effects and are able to suppress NF-kB-activated genes in the normal way (Reichardt et al. 2001). As indicated above, several of the genes associated with side effects, including the hypothalamo–pituitary–adrenal axis, bone metabolism and skin structure, appear to be regulated by interaction of glucocorticoid receptors with negative GRE sites (Dostert and Heinzel 2004).

16.5

Suppression of Inflammatory Genes

In controlling inflammation, the major effect of glucocorticoids is to inhibit the synthesis of multiple inflammatory proteins through suppression of the genes that encode them (Table 16.1). Although this was originally believed to be through interaction of GR with negative GRE sites, these have been demonstrated on only a few genes, which do not include genes encoding inflammatory proteins (Dostert and Heinzel 2004).

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Table 16.1 Effect of glu cocorticoids on gene transcription Increased transcription (trans-activation) Annexin-1 (lipocortin-1, phospholipase A2 inhibitor) β2-Adrenoceptors Secretory leukoprotease inhibitor Clara cell protein (CC10, phospholipase A2 inhibitor) Interleukin-1 receptor antagonist Interleukin-1R2 (decoy receptor) IkB-a (inhibitor of NF-kB) GILZ (glucocorticoid-induced leucine zipper protein) MKP-1 (mitogen-activated protein kinase phosphatase-1) Interleukin-10 (indirectly) Decreased transcription (trans-repression) Cytokines IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-12, IL-13, IL-16, IL-17, IL-18, TNF-a, GM-CSF, SCF Chemokines IL-8, RANTES, MIP-1a, MCP-1, MCP-3, MCP-4, eotaxins Adhesion molecules ICAM-1, VCAM-1, E-selectin Inflammatory enzymes Inducible nitric oxide synthase (iNOS) Inducible cyclooxygenase (COX-2) Cytoplasmic phospholipase A2 (cPLA2) Inflammatory receptors Tachykinin NK1-receptors, NK2-receptors Bradykinin b2-receptors Peptides IL: interleukin; TNF: tumor necrosis factor; GM-CSF: granulocyte-macrophage colony stimulating factor; SCF: stem cell factor; RANTES: released by normal activated T cells expressed and secreted; MIP: macrophage inflammatory protein; MCP: monocyte chemoattractant protein; ICAM: intercellular adhesion molecule; VCAM: vascular-endothelial cell adhesion molecule

16.5.1

Interaction with Transcription Factors

Activated glucocorticoid receptors have been shown to interact functionally with other activated transcription factors. Most of the inflammatory genes that are activated in asthma do not have GRE sites in their promoter regions, yet are potently repressed by glucocorticoids. There is persuasive evidence that glucocorticoids inhibit the effects of proinflammatory transcription factors, such as AP-1 and NF-kB, that regulate the expression of genes that code for many inflammatory proteins, such as cytokines, inflammatory enzymes, adhesion molecules and inflammatory receptors (Barnes 2006c). Activated GR can interact directly with other activated transcription factors by protein–protein binding, but this may be a particular feature of cells in which these genes are artificially over-expressed, rather

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than a property of normal cells. Treatment of asthmatic patients with high doses of inhaled glucocorticoids that suppress airway inflammation is not associated with any reduction in NF-kB binding to DNA, yet is able to switch off inflammatory genes, such as GM-CSF, that are regulated by NF-kB (Hart et al. 2000). This suggests that glucocorticoids are more likely to be acting downstream of the binding of proinflammatory transcription factors to DNA, and attention has now focused on their effects on chromatin structure and histone acetylation.

16.5.2

Effects on Histone Acetylation

Activated glucocorticoid receptors may bind to CBP or other coactivators directly to inhibit their HAT activity (Ito et al. 2000), thus reversing the unwinding of DNA around core histones and thereby repressing inflammatory genes. More importantly, particularly at low concentrations that are likely to be relevant therapeutically in asthma treatment, activated GR recruit HDAC2 to the activated transcriptional complex, resulting in deacetylation of histones, and thus a decrease in inflammatory gene transcription (Ito et al. 2000) (Fig. 16.3). Using a chromatin immunoprecipitation assay we have demonstrated that glucocorticoids recruit HDAC2 to the acetylated histone H4 associated with the GM-CSF promoter (Ito et al. 2000). Using interference RNA to selectively suppress HDAC2 in an epithelial cell line, we have shown that there is an increase in the expression of GM-CSF and reduced sensitivity to glucocorticoids (Ito et al. 2004a). By contrast, knockdown of HDAC1 and HDAC3 had no such effect on steroid responsiveness. An important issue that is not yet resolved is why glucocorticoids selectively switch off inflammatory genes, while having no effect on genes that regulate proliferation, metabolism and survival. It is likely that glucocorticoid receptors only bind to coactivators that are activated by proinflammatory transcription factors, such as NF-kB and AP-1, although we do not yet understand how this specific recognition occurs.

16.5.3

Other Histone Modifications

It has now become apparent that core histones may be modified not only by acetylation, but also by methylation, phosphorylation and ubiquitination, and that these modifications may also regulate gene transcription (Peterson and Laniel 2004). Methylation of histones, particularly histone H3, by histone methyltransferases usually results in gene suppression. The antiinflammatory effects of glucocorticoids are reduced by a methyltransferase inhibitor, 5-aza-2¢-deoxycytidine, suggesting that this may be an additional mechanism whereby glucocorticoids suppress genes (Kagoshima et al. 2001). Indeed there may be an interaction between acetylation,

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Fig. 16.3 Glucocorticoids’ suppression of activated inflammatory genes. Inflammatory genes are activated by inflammatory stimuli, such as interleukin-1b (IL-1b) or tumor necrosis factor-a (TNF-a), resulting in activation of IKK2 (inhibitor of IkB kinase-2), which activates the transcription factor nuclear factor kB (NF-kB). A dimer of p50 and p65 NF-kB proteins translocates to the nucleus and binds to specific kB recognition sites and also to coactivators, such as CREB-binding protein (CBP), which have intrinsic histone acetyltransferase (HAT) activity. This results in acetylation of core histone H4, resulting in increased expression of genes encoding multiple inflammatory proteins. GR after activation by glucocorticoids translocate to the nucleus and bind to coactivators to inhibit HAT activity directly and recruiting HDAC2, which reverses histone acetylation leading in suppression of these activated inflammatory genes

methylation and phosphorylation of histones, so that the sequence of chromatin modifications (the so-called “histone code”) may give specificity to expression of particular genes (Wang et al. 2004).

16.5.4

GR Acetylation

It has been increasingly recognized that many regulatory proteins, particularly transcription factors and nuclear receptors, are also regulated by acetylation that is controlled by HATs and HDACs (Popov et al. 2007). Acetylation plays a key role in the regulation of androgen and estrogen receptors, and it has now been shown that this is also the case for GR (Ito et al. 2006). GR is acetylated within the nucleus at specific lysine residues close to the hinge region and only binds to its DNA

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Fig. 16.4 Acetylation of GR. Binding of a corticosteroid to GR results in its acetylation by histone acetyltransferases (HAT), such as CREB-binding protein (CBP), and a dimer of acetylated GR then binds to GRE to activate or suppress genes (such as side effect genes). Deacetylation of GR by HDAC2 is necessary for GR to interact with CBP and inhibit nuclear factor-kB (NF-kB) to switch off inflammatory genes

binding site in its acetylated form. However, in order to inhibit NF-kB-activated genes it is necessary to deacetylate the receptor and this is achieved by HDAC2 (Fig. 16.4).

16.5.5

Non-Transcriptional Effects

Although most of the actions of glucocorticoids are mediated by changes in transcription through chromatin remodeling, it is increasingly recognized that they may also affect protein synthesis by reducing the stability of mRNA so that less protein is synthesized. Several inflammatory proteins are regulated posttranscriptionally at the level of mRNA stability (Anderson et al. 2004). This may be an important antiinflammatory mechanism as it allows glucocorticoids to switch off the ongoing production of inflammatory proteins after the inflammatory gene has been activated. The stability of some inflammatory genes is determined by regulation of AU-rich elements (ARE) in the 3¢- untranslated regions of the gene which interact with several ARE binding proteins such as HuR and tristetraprolin that may stabilize mRNA (Dean et al. 2004) by binding to the 3¢ AU-rich untranslated region of

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mRNAs (Smoak and Cidlowski 2006). Some inflammatory genes, such as the genes encoding TNF-a, GM-CSF and cyclooxygenase-2 (COX-2), produce mRNA that is particularly susceptible to the action of ribonucleases that break down mRNA, thus switching off protein synthesis. Glucocorticoids may have inhibitory effects on the proteins that stabilize mRNA, leading to more rapid breakdown and thus a reduction in inflammatory protein expression (Bergmann et al. 2000; Newton et al. 2001).

16.5.6

Effects on Signal Transduction Pathways

Glucocorticoids have complex effects on signal transduction pathways through trans-repression of critical enzymes involved in inflammatory cascades, or through increased transcription of endogenous inhibitors of these pathways (Barnes 2006a). Mitogen-activated protein (MAP) kinases play an important role in inflammatory gene expression through the regulation of proinflammatory transcription factors. There is increasing evidence that glucocorticoids may exert an inhibitory effect on these pathways. Glucocorticoids may inhibit AP-1 and NF-kB via an inhibitory effect on c-Jun N-terminal kinases (JNK) which activate these transcription factors (Barnes 2005). Glucocorticoids reduce the stability of mRNA for some inflammatory genes, such as COX2, through an inhibitory action on another MAP kinase, p38 MAP kinase (Dean et al. 2004). p38 MAP kinase regulates multiple inflammatory genes, including TNF-a, IL-1b, IL-6, GM-CSF and IL-8 which have ARE sites in their 3¢ untranslated regions, by stabilizing their mRNA so that synthesis of the inflammatory protein is increased (Dean et al. 2004). The inhibitory effect of glucocorticoids is mediated via the rapid induction of a potent endogenous inhibitor of p38 MAP kinase, MKP-1, which is one of the genes switched on by glucocorticoids (Clark 2003) (Fig. 16.5). Glucocorticoids not only induce the MKP-1 gene, but also reduce its degradation. MKP-1 inhibits all MAP kinase pathways and therefore inhibits JNK and to a lesser extent extracellularly regulated kinase (ERK), in addition to p38 MAP kinase (Clark et al. 2008). This indicates that glucocorticoids have the capacity to inhibit all MAP kinase pathways, but the selectivity of MKP-1 for different MAP kinases appears to vary from cell to cell (Engelbrecht et al. 2003).

16.6

Glucocorticoid Resistance

Although glucocorticoids are highly effective in the control of asthma and other chronic inflammatory or immune diseases, a small proportion of patients with asthma fail to respond even to high doses of oral glucocorticoids (Adcock and Lane 2003; Leung and Bloom 2003) and patients with COPD are largely unresponsive to

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Fig. 16.5 Inhibition of p38 mitogen-activated protein (MAP) kinase by glucocorticoids. p38 MAP kinase is activated by inflammatory stresses though activation of MAP kinase kinase (MKK)-3 and -6. p38 phosphorylates (P) MAP kinase-activated protein kinase (MAPKAPK)-2, which plays a role in stabilizing messenger RNA (mRNA) encoding several inflammatory proteins, such as tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, IL-6, IL-8, GM-CSF and cyclooxygenase (COX)-2. This mRNA is characterized by AU-rich elements (ARE) in the 3¢ untranslated region, which make the mRNA unstable and rapidly degraded. ARE-binding proteins (AREBP) stabilize these proteins and may be activated (probably indirectly) by MAPKAPK-2. Glucocorticoids induce the expression of MAP kinase phosphatase (MKP)-1, which inhibits p38 and thus prevents the stabilization of multiple inflammatory proteins

glucocorticoids. Resistance to the therapeutic effects of glucocorticoids is also recognized in nonpulmonary inflammatory and immune diseases, including rheumatoid arthritis and inflammatory bowel disease (Barnes and Adcock 2008). Steroid-resistant patients present considerable management problems as there are few alternative antiinflammatory treatments available. The new insights into the mechanisms whereby glucocorticoids suppress chronic inflammation have shed light on the molecular basis for steroid resistance in asthma and COPD.

16.6.1

Glucocorticoid-Resistant Asthma

There may be several molecular mechanisms for resistance to the effects of glucocorticoids and these may differ between patients (Barnes and Adcock 2008). It is

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likely that there is a spectrum of steroid responsiveness, with the very rare resistance at one end, but a relative resistance is seen in patients who require high doses of inhaled and oral steroids (steroid-dependent asthma).

16.6.2

p38 MAP Kinase

Biopsy studies have demonstrated the typical eosinophilic inflammation in the bronchial mucosa in these patients, with increased expression of Th2 cytokines. There is also resistance to the antiinflammatory effects of glucocorticoids in circulating mononuclear cells (Adcock et al. 1995). Certain cytokines (particularly IL-2, IL-4 and IL-13 which show increased expression in bronchial biopsies of patients with steroid-resistant asthma) may induce a reduction in affinity of glucocorticoid receptors in inflammatory cells such as T-lymphocytes, resulting in local resistance to the antiinflammatory actions of glucocorticoids. The combination of IL-2 and IL-4 induces steroid resistance in vitro through activation of p38 MAP kinase, which phosphorylates glucocorticoid receptors and reduces glucocorticoid binding affinity within the nucleus (Irusen et al. 2002). The therapeutic implication is that p38 MAP kinase inhibitors now in clinical development might reverse this form of steroid resistance.

16.6.3

GRb

Another proposed mechanism for steroid resistance in asthma is increased expression of GRb, which may theoretically act as an inhibitor by competing with GRa for binding to GRE sites or preventing GRa from interacting with coactivator molecules (Hamid et al. 1999; Lewis-Tuffin and Cidlowski 2006). However, there is no increased expression of GRb in the mononuclear cells of patients with steroid-dependent asthma which have a reduced responsiveness to glucocorticoids in vitro. Furthermore, GRa greatly predominates over GRb making it unlikely that it could have any functional inhibitory effect (Gagliardo et al. 2000), and GRb protein is undetectable in blood monocytes of asthmatic patients (Torrego et al. 2004). In addition, there is no evidence for induction of GRb in response to IL-2/IL-4 exposure, which induces steroid-resistance in mononuclear cells, convincingly demonstrating that GRb cannot account for steroid resistance in asthma (Torrego et al. 2004).

16.6.4

Interaction with Transcription Factors

Another proposed mechanism is a failure of GR to inhibit the activation of inflammatory genes by transcription factors, such as NF-kB and AP-1. Indeed, there is

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defective inhibition of AP-1 in response to glucocorticoids in mononuclear cells of steroid-resistant patients (Adcock et al. 1995). This may be due to increased activation of AP-1 due to excessive activation of the JNK pathway, which has been demonstrated in the cells of steroid-resistant asthma patients (Sousa et al. 1999).

16.6.5

Defective Histone Acetylation

Mononuclear cells from asthmatic patients who are steroid-dependent or resistant show reduced suppression of cytokine release and a reduction in histone H4 acetylation in the nucleus following treatment with a high concentration of dexamethasone (1 mM) (Matthews et al. 2004). In one group of patients nuclear localisation of GR in response to a high concentration of glucocorticoids is impaired and this accounts for the reduced histone acetylation, since there is a direct correlation between the degree of histone acetylation and the GR nuclear localisation (Matthews et al. 2004). This may be a result of GR nitrosylation leading to reduced dissociation of GR from hsp-90 (Galigniana et al. 1999). However, in another group of patients the defect in acetylation of histone acetylation is found despite normal nuclear localization of GR. This may be a result of GR phosphorylation within the nucleus due to the activation of p38 MAP kinase (Irusen et al. 2002), which may result in a failure to recruit a distinct coactivator(s). This may result in failure of glucocorticoid receptors to trans-activate steroid-responsive genes (Szatmary et al. 2004). In this group of patients specific acetylation of histone H4 lysine-5 by glucocorticoids is defective (Matthews et al. 2004). This presumably means that glucocorticoids are not able to activate certain genes that are critical to the antiinflammatory action of high doses of glucocorticoids, but whether this is a rare genetic defect is not yet known.

16.6.6

Steroid Resistance in COPD

Although inhaled glucocorticoids are highly effective in asthma, they provide relatively little therapeutic benefit in COPD, despite the fact that active airway and lung inflammation is present. This may reflect the fact that the inflammation in COPD is not suppressed by glucocorticoids, with no reduction in inflammatory cells, cytokines or proteases in induced sputum even with oral glucocorticoids. Furthermore, histological analysis of peripheral airways of patients with severe COPD shows an intense inflammatory response, despite treatment with high doses of inhaled glucocorticoids (Hogg et al. 2004). There is increasing evidence for an active steroid resistance mechanism in COPD, as glucocorticoids fail to inhibit cytokines (such as IL-8 and TNF-a) that they normally suppress (Keatings et al. 1997; Culpitt et al. 1999). In vitro studies show that cytokine release from alveolar macrophages is markedly resistant to the antiinflammatory effects of glucocorticoids, compared to cells from normal smokers and these in turn are more resistant

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than alveolar macrophages from nonsmokers (Culpitt et al. 2003). This lack of response to glucocorticoids may be explained, at least in part, by an inhibitory effect of cigarette smoking and oxidative stress on HDAC function, thus interfering with the critical antiinflammatory action of glucocorticoids (Ito et al. 2001). As discussed above, HDAC2 is required for the deacetylation activated nuclear GR in order for GR to inhibit NF-kB activity and therefore the expression of inflammatory genes. The reduced activity of HDAC2 in COPD patients is associated with increased acetylation of GR, which may be a major mechanism accounting for corticosteroid resistance in COPD (Ito et al. 2006; Barnes 2008c) (Fig. 16.6). In addition, the increased acetylated GR may promote gene activation and gene suppression by binding to GR recognition sequences (GRE) in steroid-sensitive genes, such as genes involved in side effects of corticosteroids.

Fig. 16.6 Proposed mechanism of glucocorticoid resistance in COPD, severe asthma and smoking asthma. Stimulation of normal and asthmatic alveolar macrophages activates nuclear factorkB (NF-kB) and other transcription factors to switch on histone acetyltransferase leading to histone acetylation and subsequently to transcription of genes encoding inflammatory proteins, such as tumor necrosis factor-a (TNF-a), interleukin-8 (IL-8) and GM-CSF. Glucocorticoids reverse this by binding to GR and recruiting HDAC2. This reverses the histone acetylation induced by NF-kB and switches off the activated inflammatory genes. In COPD and smoking asthmatic patients cigarette smoke generates oxidative stress (acting through the formation of peroxynitrite) to impair the activity of HDAC2. This amplifies the inflammatory response to NF-kB activation, but also reduces the antiinflammatory effect of glucocorticoids, as HDAC2 is now unable to reverse histone acetylation. A similar mechanism may operate in severe asthma where increased oxidative stress is generated by airway inflammation

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It is likely that oxidative and nitrative stress in COPD specifically impairs HDAC2 (Ito et al. 2004b), resulting in steroid resistance (Barnes et al. 2004). Although this is seen in all stages of COPD it is most marked in the patients with the most severe disease (Ito et al. 2005). Even in patients with COPD who have stopped smoking, the steroid resistance persists and these patients are known to have continuing oxidative stress. Oxidative stress is also increased in patients with severe asthma and during exacerbations, so that a reduction in HDAC may also account for the reduced responsiveness to glucocorticoids in these patients and the relative unresponsiveness of acute exacerbation of asthma to glucocorticoids (Bhavsar et al. 2008).

16.7

Interaction with b2-Adrenergic Receptors

Inhaled b2-agonists and glucocorticoids are frequently used together in the control of asthma and it is now recognized that there are important molecular interactions between these two classes of drug (Barnes 2002; Giembycz et al. 2008). As discussed above, glucocorticoids increase the gene transcription of b2-receptors, resulting in increased expression of cell surface receptors. This has been demonstrated in human lung in vitro (Mak et al. 1995a) and nasal mucosa in vivo after topical application of a glucocorticoid (Baraniuk et al. 1997). In this way glucocorticoids protect against the down-regulation of b2-receptors after long-term administration (Mak et al. 1995b). This may be important for the nonbronchodilator effects of b2-agonists, such as mast cell stabilization. Glucocorticoids may also enhance the coupling of b2-receptors to G proteins, thus enhancing b2-agonist effects and reversing the uncoupling of b2-receptors that may occur in response to inflammatory mediators, such as interleukin-1b through a stimulatory effect on a G-protein coupled receptor kinase (Mak et al. 2002). There is also evidence that b2-agonists may affect GR and thus enhance the antiinflammatory effects of glucocorticoids. b2-Agonists increase the translocation of GR from cytoplasm to the nucleus after activation by glucocorticoids (Roth et al. 2002). This effect has now been demonstrated in sputum macrophages of asthmatic patients after an inhaled glucocorticoid and inhaled long-acting b2-agonist (Usmani et al. 2005). This suggests that b2-agonists and glucocorticoid enhance each other’s beneficial effects in asthma therapy.

16.8

Conclusions

There is now a much better understanding of how glucocorticoids act so effectively in asthma and also why they are relatively ineffective in COPD, based on a better understanding of their molecular mechanisms. Glucocorticoids exert their

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antiinflammatory effects through influencing multiple signal transduction and gene expression pathways. Their most important action is switching off multiple activated inflammatory genes through inhibition of HAT and recruitment of HDAC2 activity to the inflammatory gene transcriptional complex. In addition, glucocorticoids may activate several antiinflammatory genes and increase the degradation of messenger RNA encoding certain inflammatory proteins. This broad array of actions may account for the striking efficacy of glucocorticoids in complex inflammatory diseases such as asthma and the difficulty in finding alternative antiinflammatory drugs. There is now a better understanding of how the responsiveness to glucocorticoids is reduced in severe asthma, asthmatic patients who smoke and in patients with COPD. An important mechanism now emerging is a reduction in HDAC2 activity as a result of oxidative stress. There is a two-way interaction between GR and b2-adrenoceptors since glucocorticoids increase the expression of b2-receptors, whereas b2-agonists increase GR nuclear translocation and may thereby enhance the antiinflammatory effects of glucocorticoids. These new insights into glucocorticoid action may lead to new approaches to treating inflammatory lung diseases and in particular to increasing efficacy of steroids in situations where they are less effective.

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Barnes PJ, Ito K, Adcock IM (2004) A mechanism of corticosteroid resistance in COPD: inactivation of histone deacetylase. Lancet 363:731–733 Barnes PJ, Adcock IM, Ito K (2005) Histone acetylation and deacetylation: importance in inflammatory lung diseases. Eur Respir J 25:552–563 Bergmann M, Barnes PJ, Newton R (2000) Molecular regulation of granulocyte macrophage colony-stimulating factor in human lung epithelial cells by interleukin (IL)-1b, IL-4, and IL-13 involves both transcriptional and post-transcriptional mechanisms. Am J Respir Cell Mol Biol 22:582–589 Bhavsar P, Hew M, Khorasani N, Alfonso T, Barnes PJ, Adcock I, Chung KF (2008) Relative corticosteroid insensitivity of alveolar macrophages in severe asthma compared to non-severe asthma. Thorax 63:784–790 Clark AR (2003) MAP kinase phosphatase 1: a novel mediator of biological effects of glucocorticoids? J Endocrinol 178:5–12 Clark AR, Martins JR, Tchen CR (2008) Role of dual specificity phosphatases in biological responses to glucocorticoids. J Biol Chem 283:25765–25769 Culpitt SV, Nightingale JA, Barnes PJ (1999) Effect of high dose inhaled steroid on cells, cytokines and proteases in induced sputum in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 160:1635–1639 Culpitt SV, Rogers DF, Shah P, de Matos C, Russell RE, Donnelly LE, Barnes PJ (2003) Impaired inhibition by dexamethasone of cytokine release by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 167:24–31 de Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB (2003) Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 370:737–749 Dean JL, Sully G, Clark AR, Saklatvala J (2004) The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation. Cell Signal 16:1113–1121 Dostert A, Heinzel T (2004) Negative glucocorticoid receptor response elements and their role in glucocorticoid action. Curr Pharm Des 10:2807–2816 Engelbrecht Y, de Wet H, Horsch K, Langeveldt CR, Hough FS, Hulley PA (2003) Glucocorticoids induce rapid up-regulation of mitogen-activated protein kinase phosphatase-1 and dephosphorylation of extracellular signal-regulated kinase and impair proliferation in human and mouse osteoblast cell lines. Endocrinology 144:412–422 Gagliardo R, Chanez P, Vignola AM, Bousquet J, Vachier I, Godard P, Bonsignore G, Demoly P, Mathieu M (2000) Glucocorticoid receptor a and b in glucocorticoid dependent asthma. Am J Respir Crit Care Med 162:7–13 Galigniana MD, Piwien-Pilipuk G, Assreuy J (1999) Inhibition of glucocorticoid receptor binding by nitric oxide. Mol Pharmacol 55:317–323 Giembycz MA, Kaur M, Leigh R, Newton R (2008) A Holy Grail of asthma management: toward understanding how long-acting beta(2)-adrenoceptor agonists enhance the clinical efficacy of inhaled corticosteroids. Br J Pharmacol 153:1090–1104 Goldfarb DS, Corbett AH, Mason DA, Harreman MT, Adam SA (2004) Importin alpha: a multipurpose nuclear-transport receptor. Trends Cell Biol 14:505–514 Hall SE, Lim S, Witherden IR, Tetley TD, Barnes PJ, Kamal AM, Smith SF (1999) Lung type II cell and macrophage annexin I release: differential effects of two glucocorticoids. Am J Physiol 276:L114–L121 Hamid QA, Wenzel SE, Hauk PJ, Tsicopoulos A, Wallaert B, Lafitte JJ, Chrousos GP, Szefler SJ, Leung DY (1999) Increased glucocorticoid receptor beta in airway cells of glucocorticoidinsensitive asthma. Am J Respir Crit Care Med 159:1600–1604 Hart L, Lim S, Adcock I, Barnes PJ, Chung KF (2000) Effects of inhaled corticosteroid therapy on expression and DNA-binding activity of nuclear factor-kB in asthma. Am J Respir Crit Care Med 161:224–231 Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, Pare PD (2004) The nature of small-airway obstruction in chronic obstructive pulmonary disease. New Engl J Med 350:2645–2653

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Irusen E, Matthews JG, Takahashi A, Barnes PJ, Chung KF, Adcock IM (2002) p38 Mitogenactivated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma. J Allergy Clin Immunol 109:649–657 Ismaili N, Garabedian MJ (2004) Modulation of glucocorticoid receptor function via phosphorylation. Ann N Y Acad Sci 1024:86–101 Ito K, Barnes PJ, Adcock IM (2000) Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits IL-1b-induced histone H4 acetylation on lysines 8 and 12. Mol Cell Biol 20:6891–6903 Ito K, Lim S, Caramori G, Chung KF, Barnes PJ, Adcock IM (2001) Cigarette smoking reduces histone deacetylase 2 expression, enhances cytokine expression and inhibits glucocorticoid actions in alveolar macrophages. FASEB J 15:1100–1102 Ito K, Caramori G, Lim S, Oates T, Chung KF, Barnes PJ, Adcock IM (2002) Expression and activity of histone deacetylases (HDACs) in human asthmatic airways. Am J Respir Crit Care Med 166:392–396 Ito K, Adcock IM, Barnes PJ (2004a) Knockout of histone deacetylase-2 by RNA interference enhances inflammatory gene expression and reduces glucocorticoid sensitivity in human epithelial cells. Am J Respir Crit Care Med 169:A847 Ito K, Tomita T, Barnes PJ, Adcock IM (2004b) Oxidative stress reduces histone deacetylase (HDAC) 2 activity and enhances IL-8 gene expression: role of tyrosine nitration. Biochem Biophys Res Commun 315:240–245 Ito K, Ito M, Elliott WM, Cosio B, Caramori G, Kon OM, Barczyk A, Hayashi M, Adcock IM, Hogg JC, Barnes PJ (2005) Decreased histone deacetylase activity in chronic obstructive pulmonary disease. New Engl J Med 352:1967–1976 Ito K, Yamamura S, Essilfie-Quaye S, Cosio B, Ito M, Barnes PJ, Adcock IM (2006) Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-kB suppression. J Exp Med 203:7–13 Kagoshima M, Wilcke T, Ito K, Tsaprouni L, Barnes PJ, Punchard N, Adcock IM (2001) Glucocorticoid-mediated transrepression is regulated by histone acetylation and DNA methylation. Eur J Pharmacol 429:327–334 Keatings VM, Jatakanon A, Worsdell YM, Barnes PJ (1997) Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 155:542–548 Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705 Kurihara I, Shibata H, Suzuki T, Ando T, Kobayashi S, Hayashi M, Saito I, Saruta T (2002) Expression and regulation of nuclear receptor coactivators in glucocorticoid action. Mol Cell Endocrinol 189:181–189 Leung DY, Bloom JW (2003) Update on glucocorticoid action and resistance. J Allergy Clin Immunol 111:3–22 Lewis-Tuffin LJ, Cidlowski JA (2006) The physiology of human glucocorticoid receptor beta (hGRbeta) and glucocorticoid resistance. Ann N Y Acad Sci 1069:1–9 Lonard DM, Lanz RB, O’Malley BW (2007) Nuclear receptor coregulators and human disease. Endocr Rev 28:575–587 Lu NZ, Cidlowski JA (2004) The origin and functions of multiple human glucocorticoid receptor isoforms. Ann N Y Acad Sci 1024:102–123 Mak JCW, Nishikawa M, Barnes PJ (1995a) Glucocorticosteroids increase b2-adrenergic receptor transcription in human lung. Am J Physiol 12:L41–L46 Mak JCW, Nishikawa M, Shirasaki H, Miyayasu K, Barnes PJ (1995b) Protective effects of a glucocorticoid on down-regulation of pulmonary b2-adrenergic receptors in vivo. J Clin Invest 96:99–106 Mak JC, Chuang TT, Harris CA, Barnes PJ (2002) Increased expression of G protein-coupled receptor kinases in cystic fibrosis lung. Eur J Pharmacol 436:165–172 Matthews JG, Ito K, Barnes PJ, Adcock IM (2004) Defective glucocorticoid receptor nuclear translocation and altered histone acetylation patterns in glucocorticoid-resistant patients. J Allergy Clin Immunol 113:1100–1108

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Mittelstadt PR, Ashwell JD (2001) Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem 276:29603–29610 Newton R, Hart LA, Stevens DA, Bergmann M, Donnelly LE, Adcock IM, Barnes PJ (1998) Effect of dexamethasone on interleukin-1b-(IL-1b)-induced nuclear factor-kB (NF-kB) and kB-dependent transcription in epithelial cells. Eur J Biochem 254:81–89 Newton R, Staples KJ, Hart L, Barnes PJ, Bergmann MW (2001) GM-CSF expression in pulmonary epithelial cells is regulated negatively by posttranscriptional mechanisms. Biochem Biophys Res Commun 287:249–253 Peterson CL (2002) HDAC’s at work: everyone doing their part. Mol Cell 9:921–922 Peterson CL, Laniel MA (2004) Histones and histone modifications. Curr Biol 14:R546–R551 Popov VM, Wang C, Shirley LA, Rosenberg A, Li S, Nevalainen M, Fu M, Pestell RG (2007) The functional significance of nuclear receptor acetylation. Steroids 72:221–230 Reichardt HM, Tuckermann JP, Gottlicher M, Vujic M, Weih F, Angel P, Herrlich P, Schutz G (2001) Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor. EMBO J 20:7168–7173 Rhen T, Cidlowski JA (2005) Antiinflammatory action of glucocorticoids–new mechanisms for old drugs. New Engl J Med 353:1711–1723 Roth M, Johnson PR, Rudiger JJ, King GG, Ge Q, Burgess JK, Anderson G, Tamm M, Black JL (2002) Interaction between glucocorticoids and b2 agonists on bronchial airway smooth muscle cells through synchronised cellular signalling. Lancet 360:1293–1299 Smoak K, Cidlowski JA (2006) Glucocorticoids regulate tristetraprolin synthesis and posttranscriptionally regulate tumor necrosis factor alpha inflammatory signaling. Mol Cell Biol 26:9126–9135 Sousa AR, Lane SJ, Soh C, Lee TH (1999) In vivo resistance to corticosteroids in bronchial asthma is associated with enhanced phosyphorylation of JUN N-terminal kinase and failure of prednisolone to inhibit JUN N-terminal kinase phosphorylation. J Allergy Clin Immunol 104:565–574 Szatmary Z, Garabedian MJ, Vilcek J (2004) Inhibition of glucocorticoid receptor-mediated transcriptional activation by p38 mitogen-activated protein (MAP) kinase. J Biol Chem 279:43708–43715 Tao T, Lan J, Lukacs GL, Hache RJ, Kaplan F (2006) Importin 13 regulates nuclear import of the glucocorticoid receptor in airway epithelial cells. Am J Respir Cell Mol Biol 35:668–680 Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF (2003) Histone deacetylases: unique players in shaping the epigenetic histone code. Ann N Y Acad Sci 983:84–100 Torrego A, Pujols L, Roca-Ferrer J, Mullol J, Xaubet A, Picado C (2004) Glucocorticoid receptor isoforms alpha and beta in in vitro cytokine-induced glucocorticoid insensitivity. Am J Respir Crit Care Med 170:420–425 Usmani OS, Ito K, Maneechotesuwan K, Ito M, Johnson M, Barnes PJ, Adcock IM (2005) Glucocorticoid receptor nuclear translocation in airway cells following inhaled combination therapy. Am J Respir Crit Care Med 172:704–712 Wang Y, Fischle W, Cheung W, Jacobs S, Khorasanizadeh S, Allis CD (2004) Beyond the double helix: writing and reading the histone code. Novartis Found Symp 259:3–17 Wu B, Li P, Liu Y, Lou Z, Ding Y, Shu C, Ye S, Bartlam M, Shen B, Rao Z (2004) 3D structure of human FK506-binding protein 52: implications for the assembly of the glucocorticoid receptor/Hsp90/immunophilin heterocomplex. Proc Natl Acad Sci USA 101:8348–8353

Part V

Reactive Signaling Molecules

Chapter 17

Cellular Signaling by Reactive Oxygen Species: Biochemical Basis and Physiological Scope Michel B. Toledano, Simon Fourquet, and Benoît D’Autréaux

17.1

Introduction

Reactive oxygen species (ROS) is a collective term assigned to the chemical species that are formed upon incomplete reduction of oxygen and include the superoxide anion (O2−), hydrogen peroxide (H2O2) and the hydroxyl radical (HO•). ROS are thought to mediate the toxicity of oxygen because of their greater chemical reactivity than oxygen. They also operate as intracellular signaling molecules, a function widely documented but still controversial. Skepticism stems from the apparent paradox between the specificity that is required for signaling and the reactive nature of ROS that make them seem indiscriminate and potentially lethal oxidants (D’Autreaux and Toledano 2007; Nathan 2003). However, this paradox fades when considering that signaling is mainly operated by H2O2, an oxidant less reactive than other ROS. The mild reactivity of H2O2 with its target then must be compatible with the nanomolar range of concentrations of the oxidant in the cell, above which it becomes toxic and leads to cell death (Antunes et al. 2001; Winterbourn and Hampton 2008). Lastly, targets should have unique reactivity to outcompete interactions of H2O2 with other targets, in order to account for the specificity required in signaling (Winterbourn and Hampton 2008). In microorganisms ROS signaling has been firmly established based on the existence of signaling pathways controlling ROS intracellular homeostasis. Studies of these pathways have, and still are providing fundamental principles of ROS-based redox regulation that explain how specificity can be achieved in ROS signaling. These pathways generally make use of ROS sensors that “measure” by a redox-based mechaM.B. Toledano (*) and S. Fourquet CEA, IBITECS, SBIGEM, Laboratoire Stress Oxydants et Cancer, CEA-Saclay, bat 142 91191, Gif-sur-Yvette, France e-mail: [email protected] B. D’Autréaux ICSN, Centre National de la Recherche Scientifique, 91191, Gif-sur-Yvette, France

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_17, © Springer-Verlag Berlin Heidelberg 2010

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nism the intracellular concentration of ROS and proportionally set the expression of specific ROS scavengers, thereby maintaining ROS concentration below a toxic threshold. These pathways regulate a physiological response fitted to a ROS signal, in which the ROS signal is the agonist, and the sensor, a specific ROS receptor (D’Autreaux and Toledano 2007). In mammals, potential equivalents of such ROS homeostatic pathways are uncommon. Rather, it seems that ROS signals have been diverted from their primary function of ROS homeostasis to the regulation of other cellular processes. Still, it is often not clear whether the mechanisms identified constitute true examples of physiological ROS signaling or aberrant pathophysiological responses to oxidative stress. Nevertheless, the discovery of NADPH oxidase in non-phagocytic cells, the primary function of which is to deliberately generate ROS (Lambeth 2004), provides a very strong argument in favor of physiological ROS signaling. In this chapter, we discuss the redox mechanisms of ROS homeostatic pathways in microbial models and their potential equivalents in mammals. We then compare these mechanisms with other types of ROS signaling pathways, emphasizing the aspect of their physiological relevance.

17.2 17.2.1

ROS Chemistry Dictates Target Specificity Reactivity Specifies Reaction Kinetics

Signaling is classically achieved through the noncovalent binding of a ligand to its cognate receptor by virtue of complementarities of shapes, whereas ROS operate in signaling by chemical reactions with specific atoms of target proteins leading to their covalent modification (Nathan 2003). It is thus necessary to consider the chemistry of ROS and their chemical reactivity towards biological molecules (see Figs. 17.1 and 17.2). Reactivity specifies the kinetics of a reaction, and conditions ROS signaling by establishing which reaction is the fastest and should therefore prevail (Winterbourn and Hampton 2008). Considering both reaction kinetics and the concentration of ROS and their potential targets helps predict which reactions can occur in the cell.

17.2.2

ROS Biological Targets

ROS have very distinct biological properties resulting from their chemical reactivity, half-life and lipid solubility (see Fig. 17.1). Whereas the HO• has indiscriminate reactivity towards all biological molecules (rate constant ~109–1010 M−1 s−1), O2− and H2O2 each have preferred biological targets. This preference is best exemplified by the use of the SoxR and OxyR redox sensors that enable Escherichia coli (E. coli) to discriminate and regulate distinct responses towards ROS (see below). Such chemical discrimination is a hallmark of the high atomic reactivity of O2− with iron–sulfur (Fe-S) clusters, which constitute the SoxR redox center, and of H2O2 with reactive Cys residues that constitutes the OxyR redox center.

17

Cellular Signaling by Reactive Oxygen Species ROOH

HOCl

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myelo-peroxidase

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- Flavins, quinones - NADPH oxidase

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+

+ HO–

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- [FeS] cluster, Fe release - [FeS] clusteroxidation -[FeS] cluster, Fe release -Radicals (NO , semi quinones) - Metallo-enzymes (Oxidation) -SelenoCys, Cys, Met, oxidation

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- Lipidsperoxidation - DNAoxidation - Aminoacidoxidation

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–1

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0.01 M–1.s–1

H2O

+ 2.33 V

e–

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–1

Fe2+

Protein-Met-S-CH3

Fig. 17.1 ROS biochemical properties. (a) Oxygen is rather unreactive despite being a di-radical; its univalent reduction leads to chemically more reactive species (ROS) that include the superoxide anion (O2–), hydrogen peroxide (H2O2) and the hydroxyl radical (HO•). Due to intrinsic chemical properties, each ROS reacts with “preferred” biological targets. (b) Reactivity of H2O2 towards biological molecules. H2O2 toxicity is essentially the consequence of its reduction to HO• by metalcatalyzed Fenton chemistry (Imlay 2003). H2O2 is a poor oxidant, reacting mildly with [Fe–S] (k2 ~102–103 M−1 s−1) and loosely bound metals (k2 ~103–104 M−1 s−1) and very slowly with GSH and cysteine (k2 ~2–20 M−1 s−1) and methionine (k2 ~10−2 M−1 s−1) residues (Imlay 2003; Winterbourn and Metodiewa 1999). Reactivity towards typical cysteine residues can be significantly increased depending on protein environment (10–106 M−1 s−1). In fact, the main reaction of H2O2 is with selenothiol, heme peroxidases, and other transitional metal centers. As a corollary, H2O2 is relatively stable (cellular half-life ~1 ms, steady-state levels ~10−7 M). Diffusion of H2O2 might be modulated by changes of membrane permeability or upon transport through aquaporins (Bienert et al. 2006). Selective reactivity, stability and good diffusion makes H2O2 fit for signaling. O2– has a very low intracellular concentration (~10−11 M) (Winterbourn 2008; Winterbourn and Metodiewa 1999), which reflects its instability resulting from spontaneous and SOD-catalyzed dismutation and to reaction with [Fe–S] clusters. Due to its anionic charge, O2– is unable to diffuse through membranes and is thus not fit for signaling. O2– is attracted to and oxidizes iron–sulfur ([Fe–S]) clusters with high rate constant ~106 M−1 s−1) and releases iron. Its reaction with thiol, which involves formation of a thiyl radical and regeneration of O2–, is too slow to occur in vivo (~103 M−1 s−1). The highly toxic HO• has high indiscriminate reactivity, which limits its diffusion to sites of production (half-life 10−9 s). Hypochlorous acid (HOCl) reacts nonselectively with Cys residues in the thiol and thiolate anion forms and oxidizes them to sulfenic, disulfides, thiosulfonate and sulfonamides at very high reaction rates (107 M−1 s−1) (Peskin and Winterbourn 2001; Winterbourn and Brennan 1997). HOCl is also particularly reactive with methionine and with other residues

17.2.2.1

Iron–Sulfur Clusters are the Preferred Superoxide Targets

The avidity of O2− for Fe–S clusters was demonstrated very early by the identification of the [4Fe–4S] proteins aconitase and dihydroxy-acid dehydratase as main cellular targets of the E. coli toxicity of hyperbaric oxygen-derived superoxide (Imlay 2003) (Fig. 17.1). Fe–S clusters do not have a high avidity for H2O2 that is neutral; however they can also react with other molecules constituting the redox

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center of regulators that respond to iron, oxygen and reactive nitrogen species (RNS). The molecular mechanisms that generate specificity of Fe–S clusters towards a given signal are not known.

17.2.2.2

Cysteine Has Broad Reactivity Towards Electrophiles

The cysteine residue is amongst the most vulnerable of all amino acids to electrophiles (Winterbourn 2008), and its modification can be a stigma of oxidative, nitrosative or electrophilic stress, or the reflection of a redox-regulated process. The cysteine thiol group contains a sulfur atom that becomes nucleophilic when deprotonated; it can then react with various ROS including OH• (1010 M−1 s−1), HOCl (107 M−1 s−1), O2− (~103 M−1 s−1), NO, and with a multitude of electrophilic xenobiotics.

17.2.2.3

Cysteine Has Poor Reactivity Towards H2O2

In contrast to their broad reactivity towards most electrophiles, the reaction of typical thiols with H2O2 is generally very slow (~2–20 M−1 s−1) (Winterbourn and Metodiewa 1999), which explains why the cysteine residue is not the main target of H2O2 toxicity (Fig. 17.1) (Le Moan et al. 2006). Similarly, the low molecular weight thiol glutathione (GSH) cannot directly react with H2O2 in vivo (Winterbourn and Metodiewa 1999); GSH participates in peroxide scavenging essentially by assisting glutathione peroxidases (GPxs) or by forming S-glutathionylated adducts with protein-SOHs produced by H2O2 oxidation. S-Glutathionylation is a protective mechanism preventing further irreversible protein-SOHs oxidation to the sulfinic and sulfonic acid forms (Shelton et al. 2005), and may have also protein-regulatory functions (Ghezzi 2005). S-Glutathionylation is reported to affect the function of Ras, actin, the HIV protease, and the NF-kB and AP1 transcription factors (Shelton et al. 2005). Contrary to previous belief, GSH has a much lower redox buffering capacity than total cellular protein-thiols (Hansen et al. 2009). This idea is based on the observation that 6–9% of all cellular protein-thiols – including those in membranes – are engaged in disulfide bond formation, but only < 0.1% of these are S-glutathionylated proteins.

17.2.2.4

Cysteine is Best Suited for Signaling H2O2

In spite of a poor H2O2 reactivity, the Cys residue appears ideally suited for H2O2 signaling. This is because the reactivity of the Cys residue can increase to very high values depending on the protein context, which provides the basis for selectivity and specificity (D’Autreaux and Toledano 2007). Reactivity is dictated by the solvent-exposed nature of the residue and its ionization state, the thiolate anion

17

Cellular Signaling by Reactive Oxygen Species

RS•

O2 R’SH

OH•

RSH

H2O2

NHR’R’’

RSNR’R’’

RSOH

317

RSSR’ + O2•–

O2•– H2O2

RSO2H

H2O2

RSO3H

R’SH

RSSR’

H2O2

RS(O)SR’

H2O2

RS(O)2SR’

Fig. 17.2 Products of the oxidation of Cys residues by H2O2, O2− and HO•. Reaction of the cysteine thiol group (–SH) with the two-electron oxidant H2O2 proceeds by sulfur-mediated nucleophilic attack breaking the peroxide O–O bond that releases H2O and produces a sulfenic acid (–SOH). Stability of the highly reactive –SOH is influenced by availability of a proximal –SH with which it condenses to a disulfide, or of H2O2 with which it further reacts to sulfinic (–SO2H) or sulfonic (–SO3H) acids. The –SOH can also be stabilized by a proximal amide to form a sulfenyl-amide (Salmeen et al. 2003; van Montfort et al. 2003). All these oxidation forms, except sulfinic and sulfonic acids, can be reversed to the –SH form (Winyard et al. 2005). Reduction of –SOH proceeds via formation of a disulfide that is then reduced by thiol-disulfide exchange by either of the two disulfide-oxidoreductases thioredoxin (Trx) or glutaredoxin (Grx). The 2-Cys Prxs sulfinic acid (R–SO2H) is an exception to the irreversibility of this form, being reduced by ATP-dependent sulfiredoxin (Biteau et al. 2003) (see Fig. 17.3)

(–S−) being far more nucleophilic than its protonated counterpart (Poole et al. 2004). A thiol must have a pKa < intracellular pH (6.8–7.2) in order to be in the –S− form in vivo (free cysteine pKa = 8.5). Electrostatic interaction with neighboring positively charged residue is predicted to helps stabilize –S−, decreasing thiol pKa. Nevertheless, as pKa decreases, stability of –S− also increases, decreasing availability of the negative charge and thus reactivity (Gilbert 1990). A recent compilation of about fifty proteins of diverse families known to carry a Cys–SOH and thus predicted to be reactive towards H2O2, however, revealed features that contradict these previous dogmas (Salsbury et al. 2008). Solvent exposure of the Cys residue is not the rule. Charged residues are under-represented in the structure near the reactive Cys residues, whereas threonine and other polar residues seem to exert a large influence on the Cys pKa by stabilizing the sulfenate through hydrogen-bonding interactions. Further, in some cases, the pKa is only affected by backbone features. It is not clear however from this study whether the features identified specify reactivity of Cys residues or stability of their sulfenate form. These seeming contradictions evidently call for further elucidation of the structural and biochemical basis of the H2O2 reactivity of Cys residues. The ability to cycle between different stable redox forms is the other attribute of the Cys residue endowing it with redox-regulatory properties (Fig. 17.2). It is thus not surprising that the Cys residue constitutes the catalytic center of the thiolperoxidase class of peroxide scavengers and the main regulatory target of H2O2.

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Except for PerR in bacteria (Mongkolsuk and Helmann 2002) and calciumcalmodulin (Ca++/CaM)-dependent protein kinase II (CaMKII) (Erickson et al. 2008) (see below) that use an iron center and methionine (Met), respectively, all known H2O2-specific regulatory mechanisms involve the oxidation by H2O2 of unique Cys residues. Cysteine is also a major target of NO signaling undergoing reversible S-nitrosylation, a modification of utmost importance in redox signaling that will not be dealt here (for a review see Hess et al. 2005). Cys residue can also serve in redox regulation by coordinating zinc, which provides a redox control of metal binding and metal control of cysteine redox reactivity (Ilbert et al. 2006). As a Lewis acid, zinc lowers the pKa of its coordinating thiol and potentially modifies its reactivity. Reciprocally, cysteine oxidation leads to zinc release, which results in a change of conformation that alters protein function.

17.2.2.5

Selenocysteine and Methionine

The selenocysteine (SeCys) residue, which constitutes the catalytic center of mammalian GPxs (Tosatto et al. 2008), is even more reactive towards H2O2 than reactive Cys residues. SeCys is thus the best suited of all amino acids for operating in H2O2based redox regulation, but no example for such function exists apart from GPx4 (see below). Methionine is another H2O2 target that can be oxidized at its sulfur atom to a methionine sulfoxide; however, the very slow reaction rates are not compatible with an in vivo regulatory function.

17.3

Prokaryotic ROS Homeostatic Pathways

Prokaryotic pathways of ROS signaling constitute precise, fast and highly dynamic adjustment mechanisms of intracellular ROS homeostasis (D’Autreaux and Toledano 2007). In all cases the same protein operates both the sensing and signal transduction functions. Another key feature is feedback regulation that originates from the nature of pathway outputs, which are ROS scavengers that extinguish ROS input signals. As these pathways have been recently reviewed, they will be summarized here.

17.3.1

Sensing Superoxide Through [Fe–S] Clusters

The SoxR transcription factor is an O2−-specific sensor (Hidalgo and Demple 1994) that also has a minor role in the response to RNS (Mukhopadhyay et al. 2004). Specificity for O2− is attested by its target genes that specify O2− catabolism.

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319

Oxidation of the SoxR 2Fe–2S cluster by O2− causes a change of SoxR conformation that alters the structure of the SoxR-bound DNA operator, resulting in gene activation. Reduction of SoxR involves an NADPH-dependent membrane system (Koo et al. 2003), which explains the more potent SoxR activation in strains impaired in NADP+ reduction. SoxR is thus not only linked to O2− levels but also to the NAD(P)H/NA(D)P+ ratio (Liochev and Fridovich 1992).

17.3.2

Sensing Oxygen, Iron and ROS through Fe–S Clusters

The oxygen sensor FNR, which activates anaerobic gene transcription, requires integrity of a [4Fe–4S]2+ cluster (Crack et al. 2007). This cluster is oxidized by oxygen to an unstable [3Fe–4S]+ cluster and releases Fe2+ and O2−. H2O2 and O2− can both disassemble the cluster, but their concentration is much lower than oxygen during the aerobic switch (Crack et al. 2007). The Fe–S cluster-containing IscR senses both the Fe–S cluster biosynthesis status and H2O2 and exemplifies the coordinated regulation of iron and ROS metabolism. When its [2Fe–2S] cluster is intact, IscR represses transcription of genes involved in Fe–S cluster biosynthesis (Schwartz et al. 2001). In mammals, coordination of iron and ROS responses is operated by the RNAbinding iron-response protein-1 (IRP1) (Pantopoulos 2004). IRP1 RNA-binding activity is primarily regulated through the cellular iron status-dependent assembly/ disassembly of its [4Fe–4S] cluster, and also possibly by O2− through cluster disassembly.

17.3.3

Active Cysteines or Iron Centers for Peroxide Sensing

OxyR (Zheng et al. 1998), Fur-like PerR (Lee and Helmann 2006) and OhrR (Fuangthong et al. 2001) are bona fide peroxide sensors, each using a distinct sensing mechanism. OxyR and PerR are functional orthologues; they sense both H2O2 and organic peroxides, and regulate many common target genes that specify peroxide scavenging and iron metabolism (Mongkolsuk and Helmann 2002). OhrR only senses organic peroxides, and regulates the organic peroxide-specific thiol-based peroxidase OhrA (Fuangthong et al. 2001).

17.3.3.1

The H2O2-Response Regulator OxyR

OxyR is the archetype of a peroxide receptor; it is activated by low H2O2 levels just exceeding cellular physiological concentration (20 nM) and below the threshold

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toxic level estimated at >1 mM (Aslund et al. 1999). OxyR reacts with H2O2 through a unique Cys residue (Cys198) that oxidizes to a sulfenic acid, which then condenses with Cys208 to an intra-molecular disulfide (Choi et al. 2001; Zheng et al. 1998). OxyR is deactivated by reduction by Grx1, which contributes, together with AhpC and catalase, to OxyR autoregulation. The reaction rate for sulfenic acid formation is very rapid (~105–107 M−1 s−1 depending on the method), which reflects the high H2O2 reactivity of Cys199 (Jang et al. 2004). Oxidation changes the conformation of dimeric OxyR, which triggers activation of OxyR site-specific binding to DNA promoters (Toledano et al. 1994).

17.3.3.2

OhrR

OhrR is a transcriptional repressor that senses organic peroxides by two distinct thiol-based mechanisms, depending on the organism. In Bacillus subtilis peroxide reacts with a unique BsOhrR Cys that oxidizes to a R-SOH (Fuangthong and Helmann 2002), a form still competent for DNA binding. The conformation changes that inhibit OhrR DNA binding occur after reaction of the R-SOH with a low molecular weight thiol with formation of a mixed disulfide, or with a proximal nitrogen with formation of a sulfenyl-amide (Lee et al. 2007). In Xanthomonas campestris, the nascent OhrR–SOH instead forms a disulfide with a cysteine of another subunit (Panmanee et al. 2006), which also causes the allosteric change inhibiting DNA binding. The intersubunit disulfide-based mechanism is efficient at high doses of oxidants, as it protects the reactive Cys residue from irreversible oxidation (Soonsanga et al. 2008a). Linoleic acid hydroperoxide (LHP) is much more potent than cumene hydroperoxide, causing, even at low doses, irreversible oxidation of BsOhrR to the sulfinic and sulfonic acid forms (Soonsanga et al. 2008b). Thus, depending on the oxidant, OhrR can be either reversibly oxidized or can function as a sacrificial regulator. The OhrR specificity towards organic peroxides relates to a long strip of hydrophobic and aromatic residues in the surroundings of its reactive Cys that may provide a docking site for the hydrophobic tail of organic peroxides (Hong et al. 2005).

17.3.3.3

PerR

The H2O2 sensing mechanism of the PerR repressor is unique, as it uses a regulatory metal-ion site that preferentially binds Fe2+ or Mn2+ (Mongkolsuk and Helmann 2002) and not a reactive Cys. Two histidine residues that contribute to PerR Fe2+ coordination become oxidized to 2-oxo-histidine, presumably by HO•, produced in situ by iron-catalyzed H2O2 reduction (Lee and Helmann 2006; Traore et al. 2009). Oxidation releases iron and causes the loss of PerR DNA binding. Full inactivation only occurs in iron-containing medium (Herbig and Helmann 2001), which indicates that PerR not only senses H2O2 but also its toxic potential, which is a function of iron-catalyzed Fenton chemistry (see Fig. 17.1).

17

Cellular Signaling by Reactive Oxygen Species

17.3.4

321

Cysteine-Zinc Redox Switches

Cysteine coordination of zinc constitutes the redox center of regulators that do not qualify as H2O2 sensors; these are Hsp33 (Ilbert et al. 2006) and RsrA (Kang et al. 1999) in prokaryotes, and possibly KEAP1 in mammals (see below). Hsp33 is a redox-regulated chaperone requiring oxidative unfolding for activation. Hsp33, which is activated by either a combination of H2O2 and heat stress, or by HOCl, substitutes for the ATP-dependent DnaK chaperone that becomes inactivated under these severe stress conditions (Winter et al. 2005); it constitutes an integral HOCl stress response pathway preventing protein aggregation caused by this powerful oxidant (Winter et al. 2008). Hsp33 contains a redoxsensitive C-terminal domain that binds zinc through a four-Cys zinc motif, an N-terminal domain and a flexible linker that serves as folding sensor. Upon oxidation of the motif two-distal Cys residues, zinc is released and the zinc-binding domain unfolds. This unfolding destabilizes the linker region, which is now in a dynamic equilibrium between a folded state that prevents oxidation of the motif proximal Cys residues, and a partially unfolded state that allows this oxidation (Ilbert et al. 2007). Disulfide bond formation within the two proximal Cys residues locks the linker region in the unfolded state (Graf et al. 2004), which unmasks the substrate-binding site and dimerization interface and promotes formation of the high affinity dimer of the active chaperone (Graumann et al. 2001). Hsp33 exemplifies the differential reactivity of H2O2 and HOCl (Winter et al. 2008). As a kinetically slow oxidant, H2O2 cannot compete with the fast Hsp33 linker refolding reaction; for oxidizing the proximal Cys residues, it thus requires elevated temperature that shifts the equilibrium to the partially unfolded state. As a kinetically fast oxidant, HOCl oxidizes Hsp33 even in the absence of unfolding conditions. RsrA is an antisigma factor that negatively regulates the sigma R (sR) transcription factor (Kang et al. 1999). RsrA is activated by diamide, which is consistent with its requirement for diamide tolerance. Binding of zinc to the RsrA Cys-zinc motif is of very high affinity, which suggests that zinc has a structural rather than a redox-regulated function (Zdanowski et al. 2006). Diamide leads to zinc release and to formation of a degenerate disulfide bond causing loss of sR-binding by RsrA (Li et al. 2003). Elucidation of the natural signal mimicked by diamide should help understand the mechanism and function of this cysteine-zinc redox switch.

17.4

Yeast ROS Homeostatic Pathways

A feature shared by the ROS homeostatic pathways of Saccharomyces cerevisiae and Saccharomyces pombe is their use of thiol-based peroxidases as H2O2 receptors and transducer modules (for a detailed review see D’Autreaux and Toledano 2007). Thiol-based peroxidases consist of the peroxiredoxin (Prxs) and GPx-like enzyme

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families that are an important subgroup of non-heme peroxide-scavenging enzymes (Fig. 17.3) (Fourquet et al. 2008; Maiorino et al. 2007; Wood et al. 2003b). The exquisite peroxide reactivity of the Prx catalytic Cys has been exploited in yeast for sensing and signaling H2O2. Within the Prx family, 2-Cys Prxs have the unusual attribute of undergoing reversible substrate-mediated inactivation, which regulates both sensing and scavenging functions (see Fig. 17.3).

17.4.1

The S. cerevisiae H2O2 Stress Response Regulator Yap1

17.4.1.1

The H2O2 Sensor Module Orp1-Yap1

In S. cerevisiae, the bZip transcription factor Yap1 regulates a typical H2O2 regulon and constitutes the functional counterpart of OxyR (Toledano et al. 2004). Yap1 is activated by oxidation by H2O2. Oxidation is indirect, requiring the thiol-based GPx-like enzyme Gpx3 also known as Orp1 (oxidant receptor peroxidase), which has also has a role in scavenging hydroperoxides (Avery et al. 2004). Sensing is initiated by reaction of H2O2 with the Orp1-catalytic cysteine that oxidizes to a

Trx

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SH

S Trx

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H2O2

SH

Atypical 2-cys Prx

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SO2H SH

Prx

Fig. 17.3 2-Cys Peroxiredoxins. Typical 2-Cys Prxs are homodimers that reduce H2O2 with the peroxidatic (catalytic) cysteine that oxidizes to a –SOH. The –SOH then condenses with the resolving cysteine of the other subunit to a disulfide, reduced by thioredoxin. The resolving cysteine is nonreactive due to shielding, but becomes solvent-exposed upon oxidation, facilitating reduction. Homodimers form penta-homodimers that further increase reactivity of the peroxidatic cysteine by a conformational effect (Parsonage et al. 2005). In atypical 2-Cys Prxs as in glutathione peroxidase (GPx)-like enzymes, the catalytic –SOH condenses with the resolving cysteine on the same subunit to an intramolecular disulfide. Prxs carry a fascinating but puzzling redox twist; they undergo H2O2-mediated inactivation, by overoxidation of their catalytic cysteine to a sulfinic acid (R–SO2H). Inactivation is unique to eukaryotic Prxs and is reversed by ATP-dependent reduction of the Prx Cys–SO2H by sulfiredoxin (Biteau et al. 2003; Rabilloud et al. 2002; Woo et al. 2003; Wood et al. 2003a), which suggests that this is an acquired gain of function selected for regulatory purposes (Wood et al. 2003a). Inactivation is due to an additional C-terminal helix, which is absent in inactivation-insensitive Prxs, and slows down the rate of peroxidatic Cys–SOH condensation with the resolving cysteine. This kinetic pause allows further oxidation of the R–SOH by H2O2 (Wood et al. 2003a). Overoxidation only occurs during enzymatic cycling and is proportional to the amount of substrate at nonsaturating conditions. Excess substrate also increases the rate of overoxidation by increasing the likelihood of collision of the Cys–SOH with H2O2 (Yang et al. 2002)

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sulfenic acid (Delaunay et al. 2002; Ma et al. 2007). Reaction of this R-SOH with a specific Yap1 Cys residue leads to formation of an intermolecular disulfide, then converted to a Yap1 intramolecular disulfide (Delaunay et al. 2002). Ybp1 (yeast binding protein 1) is critical for the oxidation of Yap1 by Orp1 (Gulshan et al. 2004; Veal et al. 2003), and might favor the interaction between Orp1 and Yap1. Two long-range disulfides linking N- and C-terminal cysteine-rich domains (n- and c-CRD) were initially thought to characterize oxidized, active Yap1 (Wood et al. 2004). A study using an in vitro reconstituted system containing Orp1, Yap1, the thioredoxin system and NADPH then showed that all six Yap1 Cys residues undergo oxidation, thereby generating four distinct oxidized forms of increased thermodynamic stability (Okazaki et al. 2007). The first of these is an n-CRD intradomain disulfide that occurs about 15 s after exposure to H2O2. Three disulfides linking n- and c-CRD subsequently form that increase stability of the active form, which is essential for an efficient and prolonged response (Mason et al. 2006; Okazaki et al. 2007). Formation of the three interdomain disulfides is presumably catalyzed by Orp1, and occurs consecutively, as inhibiting the first disulfide by mutagenesis prevents formation of the others (Delaunay et al. 2000). The generation and function of the n-CRD intradomain disulfide are not understood.

17.4.1.2

The Peroxiredoxin Tsa1 as a Yap1 H2O2 Receptor

The peroxiredoxin (Prx) Tsa1 was identified in a screen for mutations that prevent Yap1 activation, suggesting that Tsa1 is required for this activation (Ross et al. 2000). Such requirement exists but is only observed in yeast strains that carried an inactive allele of the YBP1 gene (ybp1-1) (Tachibana et al. 2009; Veal et al. 2003). In ybp1-1 strains oxidation of Yap1 by H2O2 is however much weaker than in strains with an active Ybp1; the nature of this oxidation is unknown, but different and less stable than the one generated by Orp1, which explains the lower efficiency of the Yap1 response of ybp1-1 cells.

17.4.1.3

Oxidation Regulates Yap1 Nuclear Accumulation

Yap1 disulfide-bond formation inhibits nuclear export of Yap1 by the export receptor Crm1, promoting nuclear accumulation of the protein and gene activation (Kuge et al. 1998). Disulfide-bond formation produces a conformational change that conceals a c-CRD Crm1-cognate nuclear export signal (NES) within a hydrophobic core formed by the interaction of n- and c-CRD (Wood et al. 2004). Reduction of disulfide bonds, probably by thioredoxin (Toledano et al. 2004), exposes the NES and reactivates Yap1 nuclear export. As Yap1 export is more potent that its import, the protein becomes mainly cytoplasmic (Kuge et al. 1998). Oxidation of Yap1 appears to be important also for transcriptional activation (Gulshan et al. 2005).

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Two S. pombe H2O2-Responsive Pathways

S. pombe has two parallel H2O2-responsive pathways, Pap1 homologous to Yap1 and the mitogen-activated protein kinase (MAPK) Sty1 pathway (for a review see Ikner and Shiozaki 2005).

17.4.2.1

The Tpx1-Pap1 Response Pathway

The Pap1 response is unique in being gradually delayed in the presence of increasing H2O2 levels. This delay lies in the use of the unique S. pombe Prx Tpx1 as the pathway sensor device (Bozonet et al. 2005; Vivancos et al. 2005). At high H2O2 concentration, Tpx1 is inactivated by cysteine-sulfinic acid formation (Fig. 17.3), shutting off signaling to Pap1. Tpx1 sulfinic-acid reduction by Sty1-induced Srx1 eventually reactivates the relay. Both catalytic Tpx1 Cys residues are required for Pap1 oxidation, but the mechanism involved is not known. Pap1 inactivation might be required for a build-up of H2O2 at levels that are sufficient to switch on Sty1dependent stress survival (Vivancos et al. 2005).

17.4.2.2

H2O2 Signal Integration in the Sty1 Pathway

H2O2 signal integration in the Sty1 pathway occurs at three levels: at the upstream histidine kinases Mak2/3 (Buck et al. 2001) through a yet undisclosed mechanism, at the MAPKKK Win1 and Wis4 through recruitment of the glyceraldehyde-3phosphate dehydrogenase isozyme 1 Tdh1 (Morigasaki et al. 2008), and at Sty1 itself through Tpx1 (Veal et al. 2004). Tdh1 is present in a complex with the Wis4, Win1 and the Mcs4 response regulator. In response to H2O2, the Tdh1 catalytic Cys residue transiently oxidizes, which enhances the association of the protein with Mcs4 (Morigasaki et al. 2008). Tdh1 is also required for the interaction between the Mcs4 response regulator and the upstream Mpr1 phosphotransfer protein. By engaging the catalytic CysP Cys residue into an H2O2-inducible disulfide linkage with a Sty1 Cys residue, Tpx1 cancels out an inhibitory effect carried by this residue on Sty1 phosphorylation by Wis1 (Veal et al. 2004).

17.4.3

Yeast Redox-Relays Dissociate Sensing from Regulation

Yeast redox relays couple thiol-peroxidases endowed with high H2O2 reactivity with the Yap1/Pap1 regulators that carry stable disulfides (Okazaki et al. 2007; Toledano et al. 2004). The yeast redox-relay architecture also confers quasi-absolute specificity towards peroxides. In S. cerevisiae, only the Orp1-SOH, which is exclusively generated by reaction with peroxides, can promote Yap1 oxidation. In fact, both Yap1 and Pap1 can respond to other thiol-reactive chemicals by alteration of

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their NES by direct modification of c-CRD cysteine residues (Azevedo et al. 2003; Castillo et al. 2002).

17.5

Redox Sensors in Higher Eukaryotes

As stated above, mammalian equivalents of microbial ROS homeostatic pathways are uncommon. In fact, ROS homeostasis cannot be strictly compared between single cell and multicellular organisms (D’Autreaux and Toledano 2007). Mammalian cells that are protected within their host habitat may not require the dynamic ROS concentration adjustments that are critical for single-celled organisms’ survival. A microarray analysis of the H2O2 response of human cells identified a large genomic response that includes cell cycle, cellular repair and death genes, many of which are targets of p53 but lack a coordinated antioxidant transcriptional response (Desaint et al. 2004). Instead, mammalian cells use long-lasting oxidative stress protective responses or cell death if the insults are overwhelming. Another important attribute distinguishing metazoan from protozoan is the ROS regulation of cellular processes that are unrelated to ROS homeostasis, which might be taken as a mechanistic reason for the lack of instant ROS homeostatic adjustment in mammals.

17.5.1

KEAP1-NRF2 as a ROS Homeostatic Pathway

The KEAP1-NRF2 complex constitutes the closest fit to a ROS receptor that regulates environmental and xenobiotic stress-protective responses (for detailed reviews, see D’Autreaux and Toledano 2007; Dinkova-Kostova et al. 2005; Kobayashi and Yamamoto 2006). NRF2 is a CNC-bZip transcription factor, and BTB-Kelch KEAP1 a zinc-binding protein and the adaptor of a CUL3-Rbx ubiquitin ligase complex that marks NRF2 for proteosomal degradation. The role of KEAP1-NRF2 in oxidative and environmental stress tolerance is attested by the nature of its inducers; these include H2O2, lipid oxidation products, nitric oxide, heavy metals and arsenicals and a multitude of natural and synthetic electrophilic compounds. The pathway’s stress protective role is also reflected by the NRF2 target genes, which comprise xenobiotic-phase II enzymes and antioxidants, and by the phenotypes of Nrf2-null mice, which include susceptibility to acetaminophen, diesel exhaust, butylated hydroxytoluene, hyperoxia, UV irradiation and carcinogens. Chemical inducers activate the pathway by inhibiting KEAP1 by direct modification of reactive cysteine residues by alkylation, oxidation or disulfide-bond formation, depending on the nature of the inducer (Dinkova-Kostova et al. 2005; Kobayashi and Yamamoto 2006). Searches for which of the 25 to 27 KEAP1 Cys are reactive identified residues that were common or different depending on the

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inducer and on the study. Among these, Cys273 and Cys288 are not only reactive, but are crucial for zinc coordination and for the KEAP1 ubiquitylation function (Kobayashi et al. 2006; Levonen et al. 2004; Zhang and Hannink 2003). Whether zinc binding is redox-sensitive is not known. Cys151, which has also been identified in most of these searches, is not involved in zinc binding, but is crucial for derepression of NRF2 by inducers (Zhang and Hannink 2003). Modification of these and possibly other Cys residues, possibly through zinc release, might switch KEAP1 to a conformation that inhibits NRF2 ubiquitylation. How the changes of KEAP1 conformation affect NRF2 ubiquitylation is still unclear.

17.5.2

ROS Homeostatic Pathways in Search of ROS Receptors

Several oxidative stress-protective pathways that respond to ROS have been identified, but the mechanism by which ROS signals are transduced is unknown. These pathways participate in large metabolic programs, and respond to many other physiological cues. A function in oxidative stress protection is assigned to the p53 tumor suppressor (Sablina et al. 2005). The PPARg coactivator-1a (PGC1a), which stimulates mitochondrial biogenesis in response to increased energy demand, also regulates an H2O2-inducible antioxidant transcriptional program (St-Pierre et al. 2006), thus coordinating the control of mitochondrial respiration that generates ROS and an anti-ROS program. The oncogene c-Myc increases ROS tolerance by activating transcription of GSH biosynthesis genes in response to H2O2 (Benassi et al. 2006). Transcription factors of the FOXO family are activated by H2O2 and induce either cell death or a quiescent cell state characterized by improved tolerance to oxidative stress that includes expression of Sod1 (Burgering and Kops 2002). ROS signals can reach FOXO through the stress-activated Jun N-terminal kinase (JNK), which induces FOXO protective functions (Essers et al. 2004), or through the sterile-like kinase MST1, which leads to cell death (Lehtinen et al. 2006). Paradoxically H2O2 regulates FOXO both negatively and positively by acetylation by the CREB binding protein CBP, and deacetylation by the NAD-dependent deacetylase SIRT1, respectively (Brunet et al. 2004; van der Horst et al. 2004), possibly indicating a biphasic regulation.

17.5.3

ROS Signals Regulating Other Cellular Processes

ROS signals that regulate ROS homeostasis-unrelated processes originate from the membrane NADPH oxidases complexes, which are ubiquitously expressed in phagocytic and nonphagocytic cells, and/or the mitochondria. Endogenous ROS are believed to modulate a multitude of so-called redox-sensitive signaling pathways (Finkel 2003; Rhee et al. 2005). We will only consider a few of these

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pathways, highlighting some of those that substantiate the concept of physiological redox regulation.

17.5.3.1

Tyrosine Phosphatases’ Redox Regulation

Protein tyrosine phosphatases (PTPs) (both classical and dual-specificity) provide one of the best examples of a physiological target of endogenous H2O2 produced upon growth factor or cytokines-induced cellular stimulation (see for a detailed Tonks 2005). Note that PTPs are also S-nitrosylation substrates.

Current Model In the current model, engagement of a growth factor receptor tyrosine kinase such as epidermal or platelet-derived growth factor receptor or antigen receptor triggers localized production of H2O2. This leads to oxidation and inactivation of the PTP(s) that function to attenuate the signaling response, thereby promoting tyrosine phosphorylation and enhancing signaling. Oxidation occurs at the PTP low pKa catalytic Cys residue that is converted to a sulfenic acid. Further oxidation to a sulfinic or sulfonic acid is prevented either by conversion of the R-SOH to a cyclic sulfenyl-amide species (Cys-S-N-R) as in PTP1B (Salmeen et al. 2003; van Montfort et al. 2003), or by its condensation to a disulfide with a proximal Cys residue as in PTEN (Kwon et al. 2004), CDC25 (Savitsky and Finkel 2002) and low molecular weight PTPs, or by S-glutathionylation (Rinna et al. 2006). These modifications also ensure reactivation of the enzyme by reversion to the thiolate form on reduction.

Limits to the Current Model Important questions still need to be answered regarding the mechanism of PTP redox regulation and its physiological scope. The regulated production of H2O2 can be easily rationalized in settings that involve NADPH oxidases as the oxidant source (Lambeth 2004). However, in other settings, H2O2 does not emanate from NADPH oxidases (Kamata et al. 2005), which raises questions about its source, possibly the mitochondria, and the regulation of its production. Another important question is the basis of the specificity of H2O2 towards the oxidation of particular PTPs. Specificity might be explained by the colocalization of NADPH oxidases and the H2O2 targets, by compartmentalized production of H2O2 within the cell (Li et al. 2006; Ushio-Fukai 2006; Vilhardt and van Deurs 2004), by local modulation of H2O2 fluxes by peroxide scavengers (Wood et al. 2003a) (see below), or by a primary receptor or other yet unknown catalytic oxidation mechanism specifying particular PTPs. Actually, a catalyzed oxidation mechanism should be considered in view of the intrinsic very low reactivity of PTPs towards H2O2 (rate constant

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~1–160 M−1 s−1), despite the low pKa (4.7–5.4) of their catalytic Cys residues (Winterbourn and Hampton 2008).

17.5.3.2

Thiol-/Selenothiol-Based Peroxidase Regulating H2O2 Signaling

Solid demonstrations of the involvement of Prxs in H2O2 signaling are mainly based on genetic grounds (Fourquet et al. 2008; Rhee et al. 2005). The yeast paradigm of thiol-peroxidases serving as receptors and transducers of ROS signaling (see paragraph 4) is suggested for a function of the plant Arabidopsis thaliana AtGpx3, an enzyme similar to yeast Orp1, which relays a H2O2 signal in the abscisic acid (ABA) pathway of guard cells that controls drought stress tolerance (Miao et al. 2006). In mammals, such a function of Prxs has not been yet described, but is reasonable and should be investigated. On the other hand, GPx4 – an enzyme belonging to the selenothiol-based GPx family, absent in microorganisms – carries a function which resembles that of a thiol oxidase (Flohe et al. 2002). In sperm cells, GPx4 promotes polymerization of the sperm mitochondrial capsule by catalyzing disulfide bond formation within structural proteins and itself. GPx4 also contributes to sperm chromatin condensation by oxidation of protamine that replaces histones during maturation (Conrad et al. 2005). GPx4-catalyzed disulfide bond formation is presumably triggered by its oxidation by peroxide, and by its defective reduction by GSH that occurs during sperm maturation. GPx4 also operates as an oxidative sensor, regulating a newly discovered apoptosis-inducing factor (AIF)-operated cell death pathway specifically triggered by 12/15-lipoxygenase-derived lipid peroxidation products (Seiler et al. 2008). GPx4 might modulate this pathway by scavenging these lipid peroxides. Such a function explains the GPx4 gene-knockout embryonic lethality that is accompanied by massive cell death (Yant et al. 2003) and the essential requirement in mammals of GSH that reduces GPx4. If not as specific thiol oxidases, Prxs may affect H2O2 signaling by regulating the fluxes and concentrations of the H2O2 produced upon growth factor or cytokinesinduced cellular stimulation (Rhee et al. 2005). The mitochondrial enzyme PrxIII affects apoptosis signaling by scavenging mitochondrial H2O2 (Chang et al. 2004). PrxII negatively regulates PDGF-R phosphorylation, possibly by modulating the oxidation of a PDGF-R-cognate PTP (Choi et al. 2005). The reversible inhibition of Prxs by overoxidation and/or phosphorylation might add another layer of control on the signaling fluxes of H2O2 reviewed in Fourquet et al. (2008) and Wood et al. (2003b).

17.5.3.3

Other Redox Cell Signaling Mechanisms

Calcium-calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) is part of an integral H2O2 signaling pathway involving NADPH oxidase-dependent H2O2 production, Met oxidation and its reduction by Met sulfoxide reductases

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(Erickson et al. 2008). Increased Ca2+ resulting from b-adrenergic stimulation activates CaMKII. Angiotensin II also induces CaMKII by oxidation of a pair of Met residues through NADPH-dependent production of H2O2. Through an elegant allosteric mechanism requiring prior priming of the enzyme by Ca2+/CaM, Met oxidation causes persistent enzyme activation that leads to cardiomyocyte apoptosis. Met sulfoxide reductases deactivate the kinase by reduction. This study is a solid demonstration of H2O2 signaling, and the first example of the involvement of Met residues in redox regulation, which again raises the question of the mechanism of this oxidation; it also clarifies links between Ca2+ and ROS signaling and between ROS and cardiac diseases. Note that angiotensin II signaling occurs through redox modulation of a PTP. The PKGIa isoform of the guanosine 3¢,5¢-monophosphate (cGMP)-dependent protein kinase (PKG) has been identified as a redox sensor (Burgoyne et al. 2007). NO is known to mediate vasodilatation by binding to and activating soluble guanylate cyclase production of cGMP, which binds and activates PKG. H2O2 provides an alternate NO- and cGMP-independent mechanism of PKG regulation, triggering time-dependent vasorelaxation through oxidative activation of PKGIa to an intersubunit disulfide that stimulates both kinase activity and affinity for substrates.

17.6

Conclusions

ROS receptors that regulate ROS homeostasis are a hallmark of microbial organisms. They define a distinct category of ROS signaling in which the ROS signal is the exclusive agonist that interacts with high specificity with its cognate receptor (D’Autreaux and Toledano 2007). Years of research in several fields has provided tangible explanations for the specificity of these pathways that is built on the high reactivity of agonists for their receptors imprinted in the unique chemistry of both the particular ROS and its atomic target within regulatory proteins. The ROS receptor function of thiol-peroxidases provides a striking example of how nature has used the high H2O2 reactivity of these H2O2 scavengers to operate H2O2 homeostatic signaling. HOCl activation of Hsp33 provides another unique example of how protein dynamics and kinetics has been exploited to account for the indiscriminate high reactivity of the former into the specific activation of the later. These pathways are not yet known to exist in mammals. Instead, mammalian ROS-specific responses, such as those regulated by FOXO and PGC1a, are part of global differentiation programs that integrate ROS protection. The KEAP1-NRF2 pathway devoted to xenobiotic and oxidant elimination is thus an exception to the rule. Lack of instant ROS homeostatic control might in fact be necessary to accommodate the use of ROS as diffusible signals in multiple signaling pathways. An instant ROS homeostatic control would elimination ROS signals. In this view, inactivation of peroxiredoxins by reversible over-oxidation or phosphorylation might be important for allowing signaling by H2O2, despite the presence of antioxidant defenses (Wood et al. 2003a).

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ROS operating signaling of cellular processes that are unrelated to ROS homeostasis is a hallmark of higher organisms. These ROS signals are generally not primary agonists but are superimposed cosignals. Thiol-based redox-regulatory mechanisms linked to oxidation by H2O2 have also been described for almost all classes of cellular regulatory proteins. However, as already mentioned, whether these redox mechanisms are physiological or the reflection of aberrant pathophysiological responses to oxidative stress needs to be determined for many of them. One major question remaining is the paradox between the apparent ubiquitous effects of ROS signals that simultaneously affect many pathways and the requirement for specificity in signaling. As proposed by Nathan (Nathan 2003), mammalian ROS signaling may involve a less stringent specificity of “another kind,” the ubiquitous nature of which may allow integration of cellular activities by recruiting, timing, and tuning multiple signaling paths in accordance with the cell’s metabolic state. However, the start of answers to this question is given by observations of the regulated and compartmentalized production of H2O2 within cells, and of the modulation of H2O2 fluxes by peroxide scavengers. A second major question lies within kinetics considerations, with regard to the mild reactivity of H2O2 towards its targets and the nanomolar range of concentrations of the oxidant in the cell that would make the occurrence of thiol oxidation impossible. Such discrepancy might be solved by the existence of mechanisms that catalyze thiol oxidation that would also provide high specificity. Future research should help answer whether the solid link that exists between ROS biology and diseases (metabolic, inflammatory, neurodegenerative, cancer, and age-related) is the reflection of oxidative stress or of alterations of ROS physiological functions, and should also aim at learning how to specifically master ROS biology for the cure of these diseases. Acknowledgments This work was supported by funds from ANR. M.B.T. is the recipient of a fund program, “Equipe Labellisée Ligue 2009,” from La Ligue Contre le Cancer (LCC).

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Chapter 18

Soluble Guanylyl Cyclase: The Nitric Oxide Receptor Doris Koesling and Ari Sitaramayya

18.1

Introduction

Glyceryl trinitrate (GTN, nitroglycerine), synthesized by Ascanio Sobrero in 1847 and soon found to be useful as an explosive, was later discovered to be highly effective in treating chest pain (angina pectoris) and in lowering blood pressure (Marsh and Marsh 2000). Over 130 years later, it was realized that nitroglycerine and some other nitrogen-bearing compounds such as nitroprusside, azide and hydroxylamine behave much like NO gas in dilating blood vessels. It is generally accepted now that these nitro-compounds, including GTN, become effective vasodilators upon biotransformation to NO (Katsuki et al. 1977; Ignarro et al. 1981). In the 1980s it became clear that not only do blood vessels dilate in the presence of externally supplied NO, but they also produce a vasodilator substance endogenously in response to neurotransmitters such as acetylcholine and bradykinin (Furchgott and Zawadzki 1980; Cherry et al. 1982). This substance was termed endothelium-derived relaxing factor (EDRF) because vessels denuded of endothelial cells did not dilate in response to acetylcholine. EDRF was eventually identified in the 1980s as NO (Ignarro et al. 1987) and, since then, NO has been found to be synthesized enzymatically in many tissues including brain (Bredt and Snyder 1992; Southam and Garthwaite 1993; Forstermann et al. 1994). The vasodilator action of NO is mediated by cyclic GMP (guanosine 3¢,5¢monophosphate), a nucleotide produced from GTP in a reaction catalyzed by guanylyl (or guanylate) cyclase, and hydrolyzed to GMP, catalyzed by cyclic GMP phosphodiesterase. In addition to vasodilation, cyclic GMP is a mediator or second messenger in many other physiological processes influenced by NO including platelet disaggreA. Sitaramayya (*) Department of Chemistry, Oakland University, Rochester, MI, 48309, USA e-mail: [email protected] D. Koesling Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, 44780 Bochum, Germany

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gation (Radomski et al. 1987), olfaction (Breer and Shepherd 1993), fluid secretion (Bailly 1998), and memory (Boulton et al. 1995). Cyclic GMP influences signal transduction pathways by opening nonspecific cation channels, thus influencing the membrane potential; by regulating cyclic AMP phosphodiesterase, thereby altering the cellular concentration of cyclic AMP; or by activating cyclic GMP-dependent protein kinases, which phosphorylate specific proteins and increase or decrease their biological activity (Lincoln and Cornwell 1993). Factors that substantially alter cellular concentration of cyclic GMP by affecting the activity of either cyclase or phosphodiesterase could lead to pathological conditions such as blindness (Dizhoor 2000), hypertension (Azam et al. 1998), and impotence (Bivalacqua et al. 2000). Cyclic GMP is not the sole mediator of all known effects of NO. For example, the cytotoxic effects of high concentrations of NO produced by activated macrophages are probably due to peroxynitrite (Zhu et al. 1992). Likewise, not all cyclic GMP is produced by NO-activated guanylyl cyclase. Two types of guanylyl cyclases are known: membrane forms activated by peptide hormones or intracellular calcium-binding proteins, and soluble forms (sGCs) activated principally by NO. It should, however, be noted that one isoform of NO-sensitive guanylyl cyclase is also found in brain synaptosomal membranes (Russwurm et al. 2001). Membrane forms of guanylyl cyclase play pivotal roles in vision (Semple-Rowland et al. 1998), fluid secretion in kidneys and intestine (Lucas et al. 2000), and olfaction (Gibson and Garbers 2000). sGCs are involved in light adaptation of retina (Sitaramayya 2002), modulation of neurotransmitter release (Kaehler et al. 1999), vasodilation (Moro et al. 1996), platelet aggregation (Radomski et al. 1987), and sodium excretion in the kidneys (Bailly 1998), among others. In this chapter we will briefly review studies on the structural features of sGC that permit activation by NO, the kinetics and mechanisms of activation and deactivation, and the adaptation of the enzyme to NO-rich or -poor environments.

18.2

Subunit Structure

Soluble guanylyl cyclase is a heterodimer of one a and one b subunit. Four subunits a1, a2, b1, and b2 are known with molecular masses of 78 kDa, 82 kDa, 70 kDa, and 76 kDa, respectively (Koesling et al. 1990; Harteneck et al. 1991; Koesling et al. 1988; Yuen et al. 1990). Expressed independently, subunit a1, a2 or b1 has no catalytic activity nor is it activated by NO (Harteneck et al. 1991; Harteneck et al. 1990). Expressed together with the b1 subunit, either a1 or a2 forms a catalytically competent enzyme that is stimulated several hundred-fold by NO (Russwurm et al. 1998). The b2 subunit, originally discovered in a cDNA library of rat kidney (Yuen et al. 1990) and recombinantly expressed, forms heterodimers with a1 or a2 which are only mildly stimulated by NO, about two orders of magnitude less than a1b1 (Gibb et al. 2003). Unlike a1 and b1, the b2 subunit has been reported to form homodimers mildly sensitive to NO (Koglin et al. 2001). Expression of b2 in cultured vascular smooth muscle cells results in reduced activation of the soluble cyclases by NO leading to the suggestion that b2 competes with b1 for the a subunit

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and forms an NO-insensitive enzyme which may be partially responsible for hypertension in rats (Gupta et al. 1997). However, the in vivo expression of the b2 subunit is uncertain as the subunit has never been detected at the protein level in vivo. Its significance to humans is doubtful since the human b2 gene has a frame shift mutation and is not expressed in normal tissues (Behrends and Vehse 2000). Soluble guanylyl cyclase is a hemoprotein: it contains one molecule of heme (Fe-protoporphyrin IX) per heterodimer (a1b1) (Gerzer et al. 1981). The presence of bound heme is essential for activation of the enzyme by NO. Heme is shown to bind to the N-terminal region of the b subunit (Zhao and Marletta 1997), and stretches of 1–194 amino acids in b1 and 1–217 amino acids in b2 have been identified as the heme binding domains (Karow et al. 2005). Histidine 105 has been identified as the residue ligated to heme in the b1 subunit (Wedel et al. 1994). Deletion of 64 amino acids from the N-terminus of the b1 subunit and 131 amino acids from the N-terminus of the a1 subunit does not prevent formation of a1b1 heterodimer with basal catalytic activity (Wedel et al. 1995). However, the mutant enzyme is not activated by NO, consistent with the N-terminal regions being involved in heme binding and activation by NO. In the C-terminal portion, mutation of aspartic acid residues at the 513 and 529 positions of a1 subunit results in the formation of an enzyme that is catalytically inactive and not activated by NO, indicating that these residues are probably located in the active site of the enzyme (Yuen et al. 1994). The C-terminal regions of soluble guanylyl cyclase subunits have domains of about 250 amino acids that have considerable sequence homology with membrane guanylyl and adenylate cyclases. These domains are thought to be responsible for the catalytic activity of the enzymes (Parma et al. 1991; Liu et al. 1997). In the case of sGC, both a and b subunits have this domain and both are required for catalytic activity. In membrane guanylyl cyclases, which have a single subunit, binding of an activator brings about dimerization of the enzyme thus bringing two homologous domains together (Chinkers and Wilson 1992; Yu et al. 1999). Adenylate cyclase is also a single subunit protein but contains two cyclase homology domains (Parma et al. 1991). The conclusion from these observations is that in all these enzymes, two catalytic domains, either intra- or intermolecular, are essential for a catalytically functional enzyme.

18.3

Activation

Crude preparations of soluble guanylyl cyclase are activated by azide, nitrite, hydroxylamine, nitroglycerine, nitroprusside, and NO. It is now understood that NO is the activator and that the other nitro-compounds produce NO or NO-derivatives when incubated with tissues or tissue homogenates (Ignarro et al. 1981) Purified soluble guanylyl cyclase is activated 300–700-fold by NO (Russwurm et al. 1998; Brandish et al. 1998). The variations between reports on the extent of activation are probably due to partial loss of heme during purification. Preparations that have entirely lost their heme are not activated by NO. Such heme-deficient preparations show higher basal activity which is inhibited by the addition of heme.

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A heme-reconstituted enzyme is once again capable of activation by NO (Ignarro et al. 1982a). When reconstituted with protoporphyrin IX (heme minus iron), hemedeficient enzyme demonstrates as much activity as the enzyme reconstituted with NO-bound heme (Ignarro et al. 1982b). This led to the hypothesis that activation of soluble guanylyl cyclase by NO involves removal or displacement of iron from the protein-bound heme (Ignarro et al. 1984). Spectroscopic measurements from Marletta’s laboratory show that purified, heme-containing sGC has a Soret peak at 431 nm and a broad second peak at 555 nm characteristic of 5-coordinate heme with iron bound to His 105 (proximal His) as the axial ligand (Stone and Marletta 1994). NO binds to the enzyme and forms a 6-coordinate complex with the histidine-bound heme of the cyclase. This shortlived 6-coordinate sGC with an absorption maximum at 420 nm can be observed in studies at 4°C (Zhao et al. 1999). In a subsequent step, the iron–histidine bond is broken leading to the formation of a 5-coordinate nitrosyl-heme complex which marks the activated form of the enzyme with an absorption maximum at 399 nm (Stone and Marletta 1996; Stone et al. 1995; Zhao et al. 1998). The suggestion that the activated enzyme has a 5-coordinate heme is in agreement with earlier observations by Ignarro et al. (1982a) that heme-depleted, NO-insensitive sGC could be fully activated by preformed NO-heme. Rapid spectrometric studies at 4°C on purified sGC show that NO binding as a 6-coordinate complex is fast (Kon > 1.4 x 108 M-1 s-1), but the subsequent conversion to 5-coordinate species is at least three orders of magnitude slower (rate constant of 2.4 x 105 M-1 s-1) and thus rate-limiting in sGC activation (Zhao et al. 1999). Furthermore, formation of 5-coordinate complex is dependent on NO concentration suggesting that NO might bind at a second site in addition to the distal side of heme and influence the kinetics of activation. The need for a second NO-binding site is, however, questioned (Bellamy et al. 2002). With or without a second site, activation of cyclase by NO at 37°C occurs in less than 0.1 s (Zhao et al. 1999). Crystallographic and spectroscopic studies on bacterial (Alcaligenes xylosoxidans) cyt c’, another heme protein that binds NO, indicate that NO can bind both to distal and proximal sides of heme and that the stable 5-coordinate molecule has NO on the proximal side, suggesting that activation of sGC in an NO-dependent fashion with the ligand binding at sites other than the distal side of heme is not farfetched (Lawson et al. 2003). Derbyshire and Marletta (2007) have shown that NO activates by fifteen-fold sGC that is partially activated by n-butyl isocyanide that occupies the distal heme site, further evidence of a second site for NO either on the proximal side of heme or elsewhere (Derbyshire and Marletta 2007). Spectroscopic studies assume that sGC with 5-coordinate heme is the activated enzyme. In studies where varying substoichiometric amounts of NO are mixed with purified sGC and the enzyme is purified and tested for stimulated activity, Russwurm and Koesling (2004) have shown that 5-cordinate enzyme (with an absorption maximum at 399 nm) is not necessarily the activated form (Russwurm and Koesling 2004). When 80% of sGC heme is NO-bound, the enzyme exhibits only 20% of maximum possible activated activity. However, when NO is mixed with sGC in the presence of Mg2+-PPi or Mg2+-cGMP, 80% NO saturation of heme leads to 80% of maximum activity. Excess NO, in the presence of Mg2+-PPi or Mg2+-cGMP, can con-

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Fig. 18.1 Model for activation of sGC by NO. NO binding results in the formation of a 6-coordinate state with absorption maximum at 420 nm. It is rapidly converted into the active 5-cordinate form in the presence of the enzyme’s reaction products. In the absence of the reaction products, the enzyme is converted into an inactive form in a NO-dependent manner. Both inactive and active forms absorb maximally at 399 nm. Reprinted from Russwurm and Koesling (2004) with permission

vert the less active form to the fully active, probably through a 6-coordinate intermediate (Fig. 18.1). These results agree with Zhao et al. (1999) that excess NO may be involved in the conversion between two forms of sGC-NO and that NO may bind at more than one site on sGC, possibly on both distal and proximal sides of heme, and also reveal a critical role for either substrate or product in forming a fully activated 5-coordinate form of sGC. In their absence, a less activated 5-coordinate form is generated which cannot be spectroscopically distinguished from the fully activated form. How the two forms are structurally different is not, however, clear. The above observations are confirmed by Cary et al. (2005) who also note that ATP and GTP oppose each other in facilitating the formation of active 5-coordinate enzyme and therefore suggest that under physiological conditions, where both ATP and GTP are present, excess NO is essential to forming the fully active enzyme (Cary et al. 2005). They argue that low concentrations of NO form a persistent, less active sGC-NO which can be transiently converted to a short-lived, highly active form by bursts of higher concentrations of NO. They suggest that the two forms are involved in “tonic and acute” signaling by NO. Studies in vivo on suspensions of platelets and cerebellar astrocytes do not support the existence of tonic and acute phases of activation by NO (Roy and Garthwaite 2006). Assaying cGMP production as a measure of sGC activity, Roy and Garthwaite find that the activation kinetics of sGC is consistent with a single NO-binding site with an EC50 of 10 nM. Activity attributable to stable tonic activity is undetected at NO concentrations up to 500 nM. Difference in results obtained in vitro and in vivo will be discussed further with regard to sGC desensitization. Carbon monoxide also activates sGC (Brune and Ullrich 1987). However, unlike NO which activates cyclase several hundred-fold, CO activates it only about fourfold (Stone and Marletta 1994) thus casting a doubt on its validity as a

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a

b

c

O C FeII

FeII

N

N NH

O N FeII N

NH

NH

Fig. 18.2 Model for partial and full activation of sGC. The view is parallel to the heme plane. (a) Unliganded enzyme with basal activity is 5-cordinate with Fe bound to proximal histidine. (b) Partially activated, 6-coordinate enzyme is formed when carbon monoxide binds. Fe is still attached to the proximal histidine, but heme is slightly puckered away. (c) NO binding results in a 5-coordinate, fully active enzyme in which the Fe–histidine bond is broken. Reprinted from Stone and Marletta (1995) with permission from the American Chemical Society

physiological stimulator of the enzyme. Even so, studies on activation by CO have been helpful in understanding the mechanism of activation. It is suggested that CO binds heme in sGC but fails to break off the histidine–Fe bond, thus leaving the heme iron in a 6-coordinate complex (Stone and Marletta 1998). If breaking off the histidine–Fe bond is essential for activation of the enzyme, how does CO stimulate it even fourfold? Maybe it is not the breakage of the histidine bond but the geometry of the porphyrin plane that influences activation (Fig. 18.2). In basal sGC the heme might be slightly pulled towards histidine, a condition marked by low (basal) activity. In CO-sGC the heme could be slightly pulled away from histidine resulting in low level activation, whereas in NO-sGC the heme is probably pulled further away causing explosive activation (Stone and Marletta 1995). An interesting turn in the activation saga comes with the discovery of NOindependent activators of sGC (Ko et al. 1994). YC-1, 3-(5¢-hydroxymethyl2¢-furyl)-1-benzylindazole, partially activates sGC in the absence of NO and turns NO and CO, particularly the latter, into a potent activator of sGC (Friebe et al. 1996). In the presence of YC-1, CO stimulates sGC to the same level as NO. YC-1 also potentiates activation at submaximally stimulating concentrations of NO. In a recent study with another activator, BAY 41-2272, also thought hitherto to be an NO-independent activator and potentiator of NO activation of sGC, Roy et al. (2008) show that the presumed NO-independent stimulation is in fact due to environmental NO (Roy et al. 2008). That is probably true of YC-1 also. YC1-binding site(s) have been investigated and shown to be on the a subunit, not the heme-bearing b subunit, suggesting that YC-1 is not an agonist but an allosteric activator (Friebe et al. 1999; Stasch et al. 2001). How do YC-1 and BAY 41-2272

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enhance NO-dependent activity? Studies by Friebe and Koesling (1998) have shown that YC-1 potentiates the activation of cyclase by reducing the dissociation of the ligands, CO and NO (Friebe and Koesling 1998). In addition, Kharitonov and colleagues (1999) observe that YC-1 increases the affinity of CO for cyclase (Kharitonov et al. 1999). It also sufficiently weakens the Fe–histidine bond in the ligand-bound enzyme to fully activate it. Breaking the bond may not be necessary when weakening is sufficient (Fig. 18.2). This explains why CO-sGC remains in a 6-coordinate iron complex and yet has high activity in the presence of YC-1.

18.4

Deactivation and Inactivation

A NO-activated enzyme returns to the basal or unactivated state upon dissociation of NO. To study the kinetics of deactivation, NO dissociated from NO-sGC complex has to be trapped so that it does not activate cyclase again. The most commonly used trap in such studies is oxyhemoglobin which has a high affinity for NO (Kharitonov et al. 1997a). In the absence of a trap, the half-life of NO-sGC in vitro is about 90 min at 37°C (Brandish et al. 1998), and accelerates to about 5 s or less in its presence (Kharitonov et al. 1997b; Margulis and Sitaramayya 2000). Deactivation on a similar time-scale is observed in vivo in studies on cerebellar cells (Bellamy et al. 2000). Cyclase which is deactivated rapidly in the presence of oxyhemoglobin can be fully reactivated with nitric oxide showing that the deactivation is reversible (Margulis and Sitaramayya 2000). These observations reveal that in tissues that have oxyhemoglobin, in other words all vascularized tissues, activated cyclase is deactivated very rapidly, thus keeping it in a NO-sensitive state. Inactivation of the enzyme occurs when NO-sGC is modified in a fashion that would not permit reactivation with NO. The effects of oxidizing agents, including ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) fall into this category (Schrammel et al. 1996). Oxidation and loss of heme inactivates the enzyme which can be reactivated by NO only after supplementing the apoprotein with heme (Schrammel et al. 1996; Burstyn et al. 1995; Dierks and Burstyn 1998). Oxidation of cysteine residues in the protein that play a role in heme binding may also inactivate the enzyme (Friebe et al. 1997).

18.5

Desensitization and Hypersensitization

In the early days of industrial production of nitroglycerine as an explosive, it was observed that workers who came into contact with the chemical suffered headaches and dizziness, but soon became tolerant, apparently due to desensitization to the chemical (Marsh and Marsh 2000). The molecular mechanism of such desensitization remained unknown until recently and still remains incompletely understood.

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It is reasonable to assume that one or more of the downstream elements in NO-regulated pathways become less available or less active when a tissue is exposed for a prolonged period of time to NO, thus leading to desensitization. The question is: which elements and how are they affected? Bellamy and colleagues report that when exposed to 1 mM DEA/NO, an NO donor, sGC is desensitized within seconds in platelets and in cerebellar astrocytes (Bellamy et al. 2000). Cells desensitized for 2 min recover sensitivity slowly, but fully, in 10 min. The low concentration of activator required for desensitization in this study, the rapid onset of desensitization, and slower resensitization are characteristics associated with neurotransmitter receptors and reinforce the idea that the relationship between NO and cyclase is similar to that of a neurotransmitter and its receptor (Jones and Westbrook 1996). In addition, these properties distinguish the observations of Bellamy et al. from other studies in which much higher concentrations of NO and longer periods of exposure were used. It is also noteworthy that desensitization does not occur in lysed cells, suggesting involvement of soluble factors. The molecular mechanism of rapid desensitization remains to be determined. In analogy with hormone and neurotransmitter receptors, phosphorylation of one or both of the subunits of NO-activated cyclase is a potential mechanism (Huganir and Greengard 1990). In fact, Zwiller and colleagues report phosphorylation of sGC in purified preparations as well as in intact PC12 cells (Zwiller et al. 1985; Louis et al. 1993). However, this phosphorylation, catalyzed by protein kinase C (PKC), increases cyclase activity, and it is not clear if NO has any influence on it. It is, therefore, unlikely that PKC-mediated phosphorylation is involved in desensitization of cyclase. Dephosphorylation through a tyrosine phosphatase has been shown to inhibit cyclase activity in PC12 cells (Chen et al. 2001). The kinetics of dephosphorylation is, however, not determined. In studies on bovine chromaffin cells, Ferrero et al. (2000) observed that prior elevation of intracellular cyclic GMP reduced the activation of cyclase by subsequent treatment with the NO donor SNP, and that the effect is brought about by dephosphorylation of the b1 subunit of cyclase by a phosphatase activated by a cyclic GMP-dependent kinase (Ferrero et al. 2000). This could potentially be a mechanism of desensitization of cyclase, but it remains to be demonstrated that the kinetics of dephosphorylation is compatible with the rate of desensitization observed by Bellamy et al. (2000). Cyclic GMP-dependent phosphorylation of soluble cyclase, not dephosphorylation, inhibits the enzyme in smooth muscle cells, though the magnitude of inhibition is modest and the kinetics of inhibition unknown (Murthy 2001). This too is a potential mechanism for desensitization. A recent study on primary aortic smooth muscle cells shows that sGC is nitrosylated on cysteine 243 of the a subunit and cysteine 122 of the b subunit on exposure to nitrosylated cysteine. The modified enzyme became less sensitive to stimulation by NO (Sayed et al. 2007). However, even the untreated sGC activity in this study is only about threefold stimulated by NO, unlike the explosive stimulation reported in other studies, and the cells are treated for long periods of time (15–60 min) to desensitize them. It is not clear whether the enzyme is desensitized or inactivated since resensitization is not demonstrated.

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When a tissue is briefly but completely deprived of NO, sGC can become hypersensitive to NO. For example, in blood vessels, NO is produced in endothelial cells in response to stimulation by acetylcholine and it diffuses to smooth muscle cells and activates sGC and causes relaxation. Denuding the vessels of endothelium or incubating them with inhibitors of NO synthesis for 15 min potentiates the vasoconstrictor effects of phenylephrine, decreases the amount of cyclic GMP, and highly sensitizes the soluble cyclase to NO (Moncada et al. 1991). In NO-deprived vascular rings, it takes 20 times less GTN to activate cyclase. The mechanism of supersensitivity remains unknown. However, since supersensitivity develops within minutes of treatment with NOS inhibitors, it is unlikely to be due to excessive production of soluble cyclase. In fact, cyclase subunits are not produced in greater amounts in the aorta of mutant mice in which endothelial NO synthase is knocked out and yet sGC is supersensitive (Brandes et al. 2000), prompting the authors to hypothesize that continuous exposure of cyclase to NO, as in the wildtype animals, oxidizes its heme and makes the enzyme unresponsive to NO, while deprivation of NO or inhibition of NO synthesis helps restore it to the reduced state and available for activation. In short, the pool of NO-sensitive enzyme is higher in NOS knockout animals and in NOS-inhibited tissues. Another explanation for the observed desensitization and hypersensitization is that the processes are mediated through phosphodiesterase. Desensitization could be due to a change in cGMP degradation through a recently described feedback loop, in which cGMP, by binding to the GAF domains of PDE5, activates the enzyme and causes increased cGMP degradation (Mullershausen et al. 2005; Mullershausen et al. 2006). In the case of NO deficiency, PDE5 has less cGMP bound to it and is less active and, as a consequence, the response to NO is enhanced.

18.6

Summary and Future Directions

sGC is the intracellular receptor for NO. How quickly the enzyme is activated by NO, how long the enzyme remains activated, and what happens to the enzyme when exposed to NO for a prolonged period of time are all important questions that have a bearing on the involvement of NO in various physiological processes. At this time it appears that that the activation is very rapid (less than a tenth of a second) and deactivation is slower (half-time of about 5 s). The rapid desensitization observed in brain astrocytes is reversible, even if slowly, suggesting that defense mechanisms do exist, at least in some cells, against exposure to NO for undesirably long periods. Differences between the observations made in vivo and in vitro need to be resolved, particularly with respect to more than one binding site for NO and more than one NO-bound/activated state of sGC. Long-lived low activation (tonic) of sGC by NO appears improbable physiologically, given the much higher affinity of oxyhemoglobin for NO and its relative abundance over sGC. The mechanism of desensitization remains to be investigated. It is likely that soluble factors will be discovered that participate in desensitization. The observation that the a2b1 form

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of sGC is preferentially localized to synaptosomal membranes is an exciting new development (Russwurm et al. 2001). Future research might reveal that sGC is associated with membranes or anchored to other proteins in non-neural cells also.

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Part VI

Cell Cycle, Cell Death and Cancer

Chapter 19

Distinct Roles of the Pocket Proteins in the Control of Cell Cycle Paraskevi Vogiatzi and Pier Paolo Claudio

19.1

Introduction

Over the past 37 years more than twenty thousand papers have been published on the Rb gene family, and the deluge of new articles in the biomedical sciences is leading to information overload. Hence, there is much interest in clarifying the roles of the Rb family of proteins in the complex cell cycle machinery and beyond, in order to explore therapeutic options especially in cancer patients. The Rb gene family includes three members: pRb/p105, and the related (RBR) proteins p107 (RBL1) and p130 (RBL2). The retinoblastoma tumor suppressor Rb gene is the first and best studied of this family, first identified in the pediatric eye tumor, retinoblastoma (Cavenee et al. 1983; Knudson 1971, 1993; Knudson et al. 1975), and found functionally inactivated at high frequency in human cancer (Lee et al. 1988; Hanahan and Weinberg 2000; Horowitz et al. 1990). Collectively, these proteins are called “pocket proteins” because they have a functional domain, named the pocket that can bind and regulate the activity of several cellular proteins. These proteins are renowned cell cycle regulators, sharing the ability to restrain the G1-S transition through the regulation of E2F-responsive genes (Fig. 19.1). Thus, they affect events such as clonal expansion, terminal cell cycle exit and maintenance of the postmitotic state, as well as the induction of tissue-specific gene expression and apoptosis (Genovese et al. 2006). The alteration of any member of the pRb/E2F1 pathway is a hallmark of most human cancers (Alonso et al. 2006, 2008), resulting in uncontrolled cell cycle progression.

P. Vogiatzi Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA P.P. Claudio (*) Department of Biochemistry and Microbiology Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, USA e-mail: [email protected]

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PcGs

E2F subgroup

?

?

E2F8

E2F7

E2F6

p130

E2F5

pRb

p107

E2F4

Biological function

Development Development Differentiation Response to Differentiation DNA damage

Physiological function

Inhibitors

E2F1

E2F2

Apoptosis

E2F3a

Proliferation

Activators

Fig. 19.1 Schematic representation of the E2F transcription factors subgroups in mammals regulated by pocket proteins, their physiological role (i.e., activation or repression) and biological function. The E2F transcription factors can be subdivided in two categories: the first group consists of E2F1, E2F2, E2F3a, which are strong transcriptional activators. E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8, which represent the second group, are considered as inhibitors. In particular, E2F6 acts as a transcriptional repressor, through a distinct direct binding to the polycomb group of genes (PcGs), pocket protein-independent manner. E2F7 and E2F8 factors are critical for embryonic development (Li et al. 2008) and play an important role in dictating the outcome of DNA damage response (Zalmas et al. 2008). Their exact mechanism mediating repression is still to be clarified. Ongoing work takes place in different laboratories

Analysis of germ-line and conditional knockout mouse strains has provided interesting data regarding the physiological and pathophysiological roles of these three proteins (David-Pfeuty 2006; Macpherson 2008; MacPherson and Dyer 2007; Vidal et al. 2007; Wikenheiser-Brokamp 2006). These studies have revealed that different genetic backgrounds result in different phenotypes, providing the idea that there is not complete functional redundancy between family members. Their functional individuality has been demonstrated for the first time in T98G human glioblastoma cell line which is refractory to pRb/p105 and p107 growth suppressive effects, but sensitive to p130 overexpression (Claudio et al. 1994). Furthermore, while p107–/– and p130–/– mice develop normally (Cobrinik et al. 1996), pRb–/– embryos suffer from a faulty erythroid differentiation and die within 13–15 days of gestation with overt anomalies due to a surfeit of cell division and cell death in the liver, lens, central and peripheral nervous systems (CNS/PNS) (Clarke et al. 1992; Jacks et al. 1992; Lee et al. 1992; Morgenbesser et al. 1994). Numerous roles have been attributed to the Rb gene family, including transcription repression and tumor suppression, and new evidence is surfacing. The emerging picture is that the Rb gene family plays an essential role in regulating genomic stability by protecting cells from double-strand breaks (DSBs) that arise during DNA replication or after exposure to toxic substances and radiation. Their activity is concentrated during the DNA damage checkpoint stages (Genovese et al. 2006; Nemajerova et al. 2008). In this chapter, we will review our current understanding of the many roles of this family in the cell’s physiology and physiopathology.

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Structural Organization

The members of Rb gene family are similar in structural characteristics and all of them contain a region that constitutes the pocket domain, which is composed of two conserved domains separated by a spacer region (reviewed by Tonini et al. 2002; Genovese et al. 2006). p130 and p107 show higher similarity with about 50% amino acid identity, while with pRb/p105 they share only 30–35% of identity. The greatest homology between the three proteins lies in the C-terminal pocket domain. p107 and p130 have a conserved region, missing in pRb, which is involved in binding and controlling the activity of cyclin A/CDK2 and cyclin E/CDK2 (Faha et al. 1992; Hannon et al. 1993; Lees et al. 1992; Li et al. 1993). An additional homologous sequence near the amino-terminus is also absent in pRb. Also, the spacer region in p130 and p107 can bind to cyclinA/Cdk2 and cyclinE/Cdk2 complexes, but not in pRB (Claudio et al. 1996; Zhu et al. 1995a,b). The pocket domain is a highly conserved region that accounts for a particular steric conformation of the proteins responsible for their specific interactions and functional activity (Hannon et al. 1993; Lee et al. 1987; Li et al. 1993; Zhu et al. 1993). Especially important in binding this domain is a small block of highly conserved amino-acid residues, the LXCXE peptide sequence (LXCXE is for leucine-X-cysteine-X-glutamate, “X” denoting any amino-acid residue) (Lee et al. 1998), as demonstrated by lack of induction of DNA synthesis and cellular transformation in mutants for this sequence. The pocket region is however a target for some LXCXE-lacking proteins as well, such as E2F transcriptional family proteins, and a number of oncoproteins from the human small DNA tumor virus family, such as the adenoviral E1A, SV40 Large T Antigen, and E7 from the Human Papillomavirus (Nevins 1992; Qin et al. 1992). The occurrence of a different binding mechanism depending on the partner protein has been exploited to investigate the diversity and timing of interactions needed to trigger growth suppression by pRb, such as the recruitment of histone deacetylases HDAC-1 and -2. In addition to HDACs, pRb also interacts with two other chromatin-remodeling enzymes, BRG1 and BRM (Kadam and Emerson 2003). These proteins are ATPases, central components of the human SWI–SNF nucleosome-remodeling complex, which is involved in global transcriptional activation because of its ability to bind DNA, disrupt nucleosomes, and provide transcription factors with access to nucleosomal DNA (Zhang et al. 2000). The ability of pRb to efficiently recruit histone deacetylases seems to be important for the role of SWI–SNF in pRb growth suppression (Dahiya et al. 2001).

19.3

Rb Pathway and Evolution in Model Organisms

The importance of the pRb pathway is underlined by its evolutionary conservation, which allows molecular studies and phylogenetic comparisons in different model organisms (revised by Claudio et al. 2002; David-Pfeuty 2006).

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The retinoblastoma protein (pRb) family plays a conserved and inhibitory role in cell cycle progression in higher eukaryotes. Homologs of all three human pRb family members have been found in mammals. The pocket domain region (starting at residue 211 in human p130 (Pertile et al. 1995) is highly conserved between human p130 and p107, showing 70% identity over the 43 amino acids, while between p130 and pRb/p105 the identity drops to 50%, over 21 amino acids only (Chen et al. 1996). The corresponding region in mouse p107 binds and represses the transcription factor Sp1 (Datta et al. 1995). As in humans, in mice p130 shows a higher similarity in amino-acid sequence to p107 than to pRb/p105. Domains A and B and the carboxy-terminal region are highly conserved between the human and mouse p107 proteins. Domains A and B exhibited 90.6% and 89.4% similarity respectively, and the carboxy-terminal region showed 91.5% similarity. Interestingly, the mouse p130 protein has a 43-amino-acid deletion in the pocket domain compared with the human and other homologs. The rat p130 is almost 90% similar in amino-acid sequence to human p130 (Yeung et al. 1993). While there are two members of Rb gene family (RBF and RBF2) in the common fruit fly’s genome, Drosophila melanogaster, RBF2 is not an essential gene. Paradoxically in Drosophila, the nucleotide sequence of RBF gene is more similar to human p107 and p130 genes than it is to the Rb gene, but the RBF protein sequence has a higher percentage identity with pRb than with p107 or p130 (Du and Pogoriler 2006; Du et al. 1996). The nematode Caenorhabditis elegans has a protein called LIN-35 that has 20% identity to human p130, 19% to p107, 15% to pRb/p105, and 16% to Drosophila RBF gene product. Rb homologs have also been found in tobacco, Chenopodium rubrum (red goosefoot), and Arabidopsis. Most plant species seem to possess only one Rb-related (RBR) gene, but a recent study has shown that in maize there are two types of distinctly regulated RBR proteins, RBR1 and RBR3, and that RBR3 is the equivalent of the mammalian p107 (Sabelli et al. 2005). Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclin-dependent kinases. RBR3 expression is controlled by RBR1 through the activity of the E2 promoter binding factor (E2F)/dimerization partner (DP) family of transcription factors (Sabelli et al. 2005; Sabelli and Larkins 2006). The unicellular green alga Chlamydomonas reinhardtii presents the mat3 gene, which contains a pocket region with two domains separated by a spacer and also has the sequence LXCXE but it seems to have a different role from that of mammalian Rb-like genes (Umen and Goodenough 2001).

19.4

Interview with Pocket Proteins: the E2F Network

In a direct comparison of the “pocket proteins” it has been shown that they share many, at times overlapping, functions in cell cycle regulation. Nevertheless, in vivo studies have proven that each of these proteins also plays distinctive roles depending on the cell status.

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In differentiated cells, for instance, there is an accumulation of mainly p130– E2F-4 complexes that are promptly dissociated when quiescent cells enter the cell cycle, suggesting that p130–E2F-4 complexes may serve as markers for the G0 phase of the cell cycle (Dimova and Dyson 2005). Regarding p107, while its levels are generally quite low in terminally differentiated cells, they rise when quiescent cells are stimulated to proliferate. Interestingly, moderate levels of pRb/p105 can be found in most cell types in quiescent and cycling cells as well. The pRb family members also differ in their binding to the E2F family of transcription factors, and fluctuations in this binding are seen throughout the cell cycle. It is believed that they act in a sequential manner to prevent S-phase entry. This timed regulation is demonstrated through the complexes formed with the E2F family. In vivo studies demonstrate that p130 binds to E2F-4 and -5 in G0 and p107 is associated with E2F-4, especially in G1, whereas pRb/p105 binds to E2F-4 in S phase (De Luca et al. 1997; Ginsberg et al. 1994; Howard et al. 2000; Sardet et al. 1995). Like pRb/p105, p107 is a potent inhibitor of E2F-mediated transactivation, and the overexpression of p107 can inhibit proliferation in certain cell types, arresting sensitive cells in G1. Several experiments, however, showed that growth inhibition by pRb/p105 and p107 did not occur through the same mechanism. In the cervical carcinoma cell line C33A, p107 was able to block cell proliferation, whereas pRb/p105 could not, even though both proteins were potent inhibitors of E2F-mediated transcription in this cell line (Chen et al. 2002; Zhu et al. 1993). Furthermore, various cell cycle regulators rescued growth arrest by either pRb/p105 or p107 in a differential fashion. pRb in conjunction with p107 plays a central role in regulating epidermal homeostasis. Ruiz et al. (2003) have shown that pRb is indispensable for the onset of keratinocyte differentiation in mice, whereas the concomitant absence of p130 and p107 impairs the terminal differentiation of skin. The same scientific group later adopted a conditional knockout approach based on the Cre/loxP system (Ruiz et al. 2004). The targeted deletion of Rb gene was obtained in stratified epithelia by K14-driven Cre recombinase (K14cre). Using this system, the authors demonstrated that pRb loss in the epidermis causes hyperplasia and hyperkeratosis, along with remarkable defects in proliferation and differentiation. Also, they found that simultaneous pRb and p107 loss leads to very high apoptotic rates in the hair follicles. In pRb-deficient skin, epidermal differentiation is highly perturbed; indeed, ectopic expression of K6, suprabasal expression of K5 and a reduction in the percentage of K10-expressing cells are observed. Nevertheless, all these defects appeared more severe in the coabsence of p107, indicating that p107 can partially compensate for pRb loss in the epidermis. By contrast, in skin lacking only p107 and p130, the authors observed that the initial differentiation step was unaffected, whereas noticeable defects in terminal differentiation existed (Ruiz et al. 2004). These results demonstrate that pRb and p107 functions in epidermis only partially overlap, as they also show different roles in suppressing hyperplasia and in regulating skin differentiation. The involvement of pRb and p130 in regulation of differentiation and apoptosis of neural cells has been widely reported as well. In a recent study it was hypothesized

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that pRb and p130 might significantly contribute to marrow stromal stem cell (MSC) differentiation (Jori et al. 2005). Molecular analysis of neural differentiation markers and immunocytochemistry revealed that both proteins (pRb and p130) were highly expressed and upregulated during the neural differentiation but also that their kinetics was very distinct. Furthermore, they assessed that p130 is mainly involved in determination of generic neural properties, whereas pRb mainly acts triggering cholinergic differentiation.

19.5

Regulation of Transcriptional Activity and Apoptosis by Rb Family Members

All pocket proteins can inhibit E2F-responsive promoters, actively repressing transcription and arresting the growth of cells when they are overexpressed (Claudio et al. 1994; Dimova and Dyson 2005) (Fig. 19.1). The Rb gene family is believed to recruit not only components of different chromatin-modifying complexes with HDAC activities (Brehm et al. 1998; Du and Pogoriler 2006; Luo et al. 1998; Magnaghi-Jaulin et al. 1998; Ross et al. 2001) but also various factors with histone methyl-transferase activities and ATP-dependent chromatin remodeling activities in order to prevent gene transcription. The functions of all three members of the pRb family are regulated by cell cycle-dependent phosphorylation. Hypophosphorylated p130 proteins, for example, will remain bound to E2F-4, preventing the transcription of the genes required for cell cycle progression beyond the G0 phase. This has been confirmed by studies performed in p130-/p107-mouse embryonic fibroblasts (Ren et al. 2002), when a loss of these pocket proteins resulted in the derepression of E2F-4. As observed by Liu et al. (2005), in neurons the p130-E2F4 complex also recruits the chromatin modifiers HDAC1 and Suv39H1 to promote gene silencing and neuron survival. The E2F family of transcription factors has the ability to promote apoptotic death at the time of differentiation. However, pRb, p107 and p130 can block E2Finduced apoptosis, providing a level of protection in the differentiation process (Lipinski and Jacks 1999). During the course of normal cellular proliferation, E2F suppression is obtained instead through the Ras-phosphoinositide 3-kinase-Akt signaling pathway and the p53 pathway (Hallstrom and Nevins 2003). Another key target of pRb in order to overcome apoptosis is mouse double minute two (MDM2). MDM2 is an E3 ring-finger ubiquitin ligase that recognizes the N-terminal activation domain (TAD) of proteins belonging to the p53 family. The role of MDM2 as a key negative regulator of p53 has been well established, and a key mechanism is the degradation of p53. It has been experimentally observed that pRb interacts with the MDM2-p53 complex, leading to the formation of a trimer in which MDM2, albeit still bound to p53, becomes unable to degrade it. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of pRb in regulating the apoptotic function of p53 (Hsieh et al. 1999).

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Rb Family: From Proliferation to Differentiation

Loss of pRb may indirectly prevent differentiation through cell cycle block. Spike et al. (2004) showed that fetal liver erythropoiesis in pRb-null mice cannot reach the final stage of differentiation, demonstrating a clear cell-intrinsic requirement for pRb during stress erythropoiesis to regulate erythroblast expansion and promote end-stage erythroid differentiation, and shed light on how aplastic anemia and myelodysplasia might develop in humans. pRb family members play a pivotal role in controlling several aspects of stem cell biology. pRb has a role in myogenesis as well as in cardiogenesis. These effects are not only related to its role in suppressing E2F-responsive genes (Fig. 19.1) but also to its ability to modulate the activity of tissue-specific transcription factors. Daria et al. (2008) investigated the consequences of loss of pRb on hematopoietic stem and progenitor cell (HSPC) function in vivo. Mice genetically deficient in pRb in all hematopoietic cells showed altered contribution of distinct hematopoietic cell lineages to peripheral blood, bone marrow, and spleen; significantly increased extramedullary hematopoiesis in the spleen; and a twofold increase in the number of hematopoietic progenitor cells in peripheral blood. Upon competitive transplantation, HSPCs from these mice contributed with an at least four- to sixfold less efficiency to hematopoiesis compared with control cells. In vitro findings included impaired cellcycle exit upon stress-induced proliferation. The authors concluded that pRb is critical for hematopoietic stem and progenitor cell function, localization, and differentiation. pRb is also involved in adipogenesis through a strict control of lineage commitment and differentiation of adipocytes as well in determining the switch between brown and white adipocytes. Wang et al. (2008) were able to promote mouse preadipocyte 3T3L1 cell differentiation, using a miRNA that targets p130 and is known to promote cell proliferation in various cancers. Jori et al. (2007) investigated the mechanisms governing commitment and differentiation of the cells of the nervous system. The authors overexpressed pRb and p130, which play an important role during nerve cell maturation, in rat neural stem cells (NSCs) and observed that these proteins not only altered the percentage of differentiating NSCs, but also affected their lineage specification. Since pRb and E2F proteins are transcriptional regulators, identification of their targets is critical to our understanding of their biological functions. Homozygosity for a germ-line Rb gene mutation results in embryonic lethality and evokes developmental defects associated with inappropriate S-phase entry and high levels of apoptosis. Weber et al. (2008) analyzed the gene expression profile of pRb-deficient mouse embryo fibroblasts (MEFs), and identified a novel Rb-regulated gene, RbEST47, that is transcriptionally upregulated in pRb-deficient MEFs. RbEST47 gene is conserved from fruit fly to humans. It is expressed in brain, lung, kidney, and testis, and is located on human chromosome 9q34.3, which frequently exhibits loss of heterozygosity in neoplastic diseases. The authors concluded that pRb plays essential and age-dependent roles during cochlear mechanosensory hairy cell (HC) proliferation and differentiation.

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P. Vogiatzi and P.P. Claudio

Rb Family and Cell Cycle Checkpoints Cell Cycle Checkpoints: Definition

Temple and Raff (1986) introduced their cell division counting hypothesis to explain the timing of oligodendrocyte differentiation. The cell cycle was meant to function as a clock, but if this were the case it would be expected that the stages of the cell cycle must function according to some sort of internal pacemaker, that will determine how long a cell-cycle phase should take. The cell cycle checkpoints are therefore operated by composites of protein kinases and adaptor proteins which all play salient roles in the maintenance of the integrity of the division. Cell cycle checkpoints (G1/S and G2/M DNA damage checkpoints) are control mechanisms that ensure the fidelity of eukaryotic cell division. These checkpoints verify whether the processes at each phase of the cell cycle have been accurately completed before progression into the next phase. Multiple checkpoints have been identified, but some of them are less well understood than others. Cells that progress through these points are committed to enter the S-phase, where DNA synthesis and replication will occur. If a cell is not ready, or external conditions are not appropriate for the S-phase, then the cell may enter G0-phase, a quiescent stage. Lack of growth factors causes some cells to arrest at the restriction point. Mutations in factors contributing to cell cycle arrest at the restriction point are thought to be the main contributors to cancer.

19.7.2

Rb Family Members Involved in Particular Checkpoints Controls

Pardee (1974) has presented for the first time the idea of the “restriction point” or the G1 phase checkpoint of cell cycle of animal cells; in yeast cells it is called START point (David-Pfeuty 2006). Loss of the G1/S checkpoint is recognized as a mandatory step in the development of cancer (Foijer and te Riele 2006). The main components of the G1 restriction point are the E2F family of transcription factors, the “pocket” proteins, pRb (exerts most and perhaps all of its effects in the first two thirds of the G1 phase) (Fig. 19.2) (Classon and Harlow 2002) and its homologs p130 and p107 (Dyson 1998; Foijer and te Riele 2006; Weinberg 1995), and the two cell cycle kinases, cdk4/6–cyclin D and cdk2–cyclin E that regulate pRb family members’ activities. During the G1 phase, the pRb–HDAC repressor complex binds to the E2F–DP1 transcription factors, inhibiting the downstream transcription. Phosphorylation of pRb by cdk4/6 and cdk2 dissociates the pRb–repressor complex, leading to the release of bound E2F from pRb. “Free E2F” is now active and ready to drive the transcription of S-phase genes encoding for proteins that switch the cell status from G1 to S phase and that are required for DNA replication (Tonini et al. 2002) (see also Fig. 19.2). Many different stimuli trigger checkpoint

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p130 pRb p107 G0

G1

S

Fig. 19.2 The pocket proteins are differentially expressed throughout the cell cycle machinery. pRb expression is relatively steady throughout the cell cycle, whereas pRb2/p130 expression is highest in arrested cells and p107 expression peaks during S phase. Reprinted with small modifications by permission from Nature Reviews Cancer (Classon and Harlow 2002), copyright (2002) Macmillan Magazines Ltd

control including DNA damage, contact inhibition, replicative senescence, and growth factor withdrawal. The first three act by inducing the transcription of members of the INK4 or Kip/Cip families of cell cycle kinase inhibitors. Growth factor withdrawal activates GSK3b, which phosphorylates cyclin D, leading to its rapid ubiquitination and proteosomal degradation. Ubiquitination, nuclear export and degradation are mechanisms commonly used to rapidly reduce the concentration of cell cycle control proteins. Myc and pRb pathways predominantly regulate the G1/S transition: they are two parallel, cooperating and interacting cascades that converge on the control of the activity of cyclin E–Cdk2, a crucial G1/S-promoting enzyme that is both essential and rate-limiting for S-phase entry (reviewed by Bartek and Lukas 2001; Sherr and Roberts 1999). Although pRb and the p107–p130 pair regulate different E2F-responsive genes, their roles in the cell cycle control are indeed somewhat redundant. It has been observed that, in vitro, fibroblasts lacking pRb or p107–p130 are still able to arrest in G0/G1, whereas those lacking all three pocket proteins fail to arrest in many different conditions (Dannenberg et al. 2000; Sage et al. 2000). Another major cell cycle checkpoint response occurs at the G2/M transition. Wang et al. (2001) suggested that pRb plays a critical role in determining the cell fate following DNA damage. Indeed, pRb-deficient cells cannot undergo G1, mid-S or G2 arrest following DNA damage, although they can activate the G2 checkpoint, which is reversible. pRb-deficient cells are also hypersensitive to DNA damageinduced apoptosis. The G2/M DNA damage checkpoint prevents the cell from entering mitosis (M phase) if the genome is damaged. The Cdc2–cyclin B kinase is pivotal in regulating this transition. During G2 phase, Cdc2 is maintained in an inactive state by the kinases Wee1 and Mt1. As cells approach M phase, the phosphatase Cdc25 is activated by the polo-kinase Pik1. Cdc25 then activates Cdc2, establishing a feedback loop that efficiently drives the cell into mitosis. DNA damage activates the DNAdependent protein kinase (DNA-PK)/ataxia telangiectasia, and the mutated (ATM)/ Rad3-related (ATR) kinases, initiating two parallel cascades that inactivate Cdc2– cyclin B. The first cascade rapidly inhibits progression into mitosis: the Chk (Cell cycle checkpoint kinases) phosphorylate and inactivate Cdc25, which can no longer

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activate Cdc2. The second cascade is slower, as the phosphorylation of p53 dissociates it from MDM2, activating its DNA binding activity (reviewed by Yang et al. 2003). The genes that are activated by p53 are the effectors of this second cascade. They include the following: -3σ, which binds to the phosphorylated Cdc2–cyclin B kinase complex and exports it from the nucleus; the Growth Arrest and DNA Damage or GADD45 protein, which binds to and dissociates the Cdc2–cyclin B kinase complex; and the p21Cip1, an inhibitor of a subset of the cyclin-dependent kinases including Cdc2 (Wang et al. 2001).

19.8

19.8.1

“Essentiality” of Pocket Proteins in Mammalian Cell Cycle Control and DNA Repair Systems DNA Damage Response and Repair Mechanisms

To ensure the high-fidelity transmission of genetic information, cells have evolved mechanisms to monitor genome integrity. Cells respond to DNA damage by activating a complex DNA-damage-response pathway or “DNA repair” (Khanna and Jackson 2001). This kind of defense is present in all examined organisms including bacteria, yeast, Drosophila, fish, amphibians, rodents and humans (Demogines et al. 2008; Sghaier et al. 2008). Cell survival after DNA damage is dependent on two important biological responses: the activation of cell cycle checkpoints and of DNA repair machinery (Hartwell and Kastan 1994). Human cells are constantly threatened from both exogenous agents, like radiation (ultraviolet or UV 200–300 nm radiation from the sun, X-rays and gamma rays), hydrolysis or thermal disruption, chemicals (especially aromatic compounds), certain plant toxins, chemotherapy, radiotherapy, and endogenous sources such as free radicals produced during metabolic processes. Damage caused by exogenous agents comes in many forms. Some examples are: (1) direct DNA damage from UV-B light that causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers; (2) indirect DNA damage from the free radicals produced by UV-A light; (3) breaks in DNA strands by ionizing radiation created by radioactive decay or in cosmic rays; (4) single strand breaks from elevated temperature that increases the rate of depurination (loss of purine bases from the DNA backbone); and (5) crosslinking of DNA due to industrial chemicals, such as vinyl chloride and hydrogen peroxide, and environmental chemicals, such as polycyclic hydrocarbons found in smoke, soot and tar that create a huge diversity of DNA adducts – ethenobases, oxidized bases, alkylated phosphotriesters. Specifically, there are four main types of damage to DNA due to endogenous cellular processes: (1) oxidation of bases and generation of DNA strand interruptions from reactive oxygen species, (2) alkylation of bases (usually methylation), (3) hydrolysis of bases, such as deamination, depurination and depyrimidination,

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and (4) mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted. UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift. Each human cell has to repair the large numbers of different DNA damages encountered each day: around 50,000 single-strand breaks (SSB), 10 DSBs, 10,000 depurinations, 600 depyrimidations, 2,000 oxidative lesions, 5,000 alkylating lesions and 10 interstrand crosslinking events (reviewed by Grillari et al. 2007). Consequently, the DNA repair process is constantly active as it responds to damage in the DNA structure. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states: (1) an irreversible state of dormancy, known as senescence; (2) cell suicide, also known as apoptosis or programmed cell death; or (3) unregulated cell division, which can lead to the formation of a tumor. The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. To name just a few of the hereditary disorders as pathological effects of poor DNA repair: xeroderma pigmentosum, Cockayne syndrome, trichothiodystrophy, Werner syndrome, Bloom syndrome, ataxia–telangiectasia, Fanconi anemia, hereditary breast cancer, and hereditary colon cancer (Hoskins et al. 2008). Most of the DNA repair deficiency diseases show varying degrees of “accelerated aging” or cancer. Rothmund–Thomson syndrome and xeroderma pigmentosum display symptoms dominated by vulnerability to cancer, whereas progeria and Werner syndrome show mostly features of accelerated aging. Hereditary nonpolyposis colorectal cancer (HNPCC) is very often caused by a defective MSH2 gene leading to defective mismatch repair, but displays no symptoms of accelerated aging. The most harmful form of DNA damage is double strand brakes (DSBs) that can be caused by a multitude of exogenous or endogenous factors. These lesions are potentially very dangerous for genomic integrity if not adequately repaired: while they usually lead to cell death through the generation of lethal chromosomal aberrations or direct induction of apoptosis (Khanna and Jackson 2001; Llorente et al. 2008), an inaccurate repair may result in mutations or genomic rearrangements in a surviving and proliferating cell; the consequent genomic instability may in turn lead to malignant cell transformation. The main difference shown by DNA DSBs compared to many other types of DNA lesions is that the complementary strand cannot be used as a template in the repair process. Mechanisms evolved to deal with DNA DSBs in eukaryotes are complex. The two major pathways used by cells to repair DNA DSBs are homologous recombination (HR) (also known as template-assisted repair) and non-homologous end joining (NHEJ). They contribute differently to the repair according to the stage of the cell cycle: this revolves on the fact that HR requires a homologous template DNA

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strand, whereas NHEJ does not. Therefore, HR is performed in the S and G2 phases, when sister chromatids are available, whereas in G1 and G0 DSB, repair has to be mostly elicited through NHEJ, which is an error-prone process. To attempt the recovery of serious lesions through the most appropriate repair mechanisms, the DNA damage response machinery has to trigger the arrest of normal cell cycle progression. This step plays an essential role in the preservation of genomic stability by limiting the propagation of deleterious mutations to the daughter cells (Bosco et al. 2004). Many studies have collected evidence for an immediate and substantial response of mammalian cells to the introduction of DNA DSBs. This response involves the formation of so-called “gH2AX” foci or H2AX histone rapidly phosphorylated on serine 139 on chromosomal regions that encompass megabase lengths of DNA adjacent to the breaks. In addition, foci of DSB-repair proteins, including Rad50 and Rad51, distinctive components of the HR DSB repair pathway, occur specifically within these domains, colocalizing with phosphorylated H2AXs. Undoubtedly, the appearance of gH2AX is a component of the DNA damage checkpoint response and, similarly, the disappearance of gH2AX would probably indicate that the cells have successfully repaired DSBs (Downs et al. 2000). H2AX phosphorylation and dephosphorylation may represent a specialized cycle of chromatin expansions and contractions, necessary in response to the formation and repair of DSBs – a cycle that most likely influences DNA events in both mitotic repair and meiotic recombination (Hunter et al. 2001). In yeast and mammalian cells, DSBs trigger the formation of defined nuclear structures called irradiation-induced foci (IRIF). IRIF are thought to originate by chromatin modification, such as H2AX phosphorylation, at the site of the DSB, followed by the recruitment of signaling and repair factors. For example, MRN complex, composed of Mre11/Rad50/Nbs1, localizes to DSBs and is critical for the formation of IRIF and the consequent response to DNA damage (Petrini and Stracker 2003). Various data have indicated that DNA damage signals prevent pRb phosphorylation, thereby activating the pRb pathway (Downs et al. 2000). Recent evidence also suggests that pRb regulates the expression of several DNA damage repair factors involved in UV damage repair processes including FEN1, XPC, RPA2-3, RFC4, and PCNA (Bosco et al. 2004).

19.8.2

How Rb Family Genes Drive the Cell Cycle to DNA Repair

It is uncertain whether E2F and pRb directly regulate DNA replication during the normal cell cycle progression or whether their functions are restricted only to certain situations, such as in response to DNA damage. There is now an extensive series of links between the E2F/pRb pathway and DNA replication, recombination and repair, in addition to the Rb gene family’s role as an essential mediator of growth and differentiation signals.

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Several tumor suppressor proteins play key roles in the maintenance of an appropriate DNA damage response. In fact, the G1 checkpoint in particular is disabled through a variety of mechanisms, such as loss of p16INK4A, loss of pRb, overexpression of cyclin D1 or overexpression of cdk4 (Sherr 1996). Like the tumor suppressor protein p53, these components of the pRb pathway, although not essential for the cell cycle per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life. pRb is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. pRb activities in the DNA damage checkpoint response can be divided into: (1) transcriptional repression of E2F-regulated genes; (2) induction of cell cycle arrest; and (3) inhibition of DNA DSB accumulation. pRb provides a measure of G1 exit control to cells through its ability to serve as a part of a transcriptional repressing complex and through its phosphorylation state that is tightly regulated during the cell cycle. It is important to note that cells lacking pRb are deficient for the G1 checkpoint response to DNA damage. Data from experiments studying the cell cycle kinetics of the cellular response to ultraviolet (UV) and ionizing radiation (IR) damage in adult mouse fibroblasts (MAF) indicate that a rapid response to UV and IR damage was compromised in pRb-deficient cells. These results imply that pRb loss is sufficient to bypass the rapid checkpoint response to DNA damage (Bosco and Knudsen 2005). During early G1 phase of the cell cycle, pRb is present in its active, hypophosphorylated form and binds to the E2F transcription factors, thereby repressing their function. The mode of pRb-mediated transcriptional repression appears to be promoter-specific and it is dependent on a variety of proteins that are recruited by pRb to promoters (Markey et al. 2002). pRb assembles transcription repression complexes at promoters that are regulated by the E2F-family of transcription factors. These include promoters of cyclin E, cyclin A, cdc2, DNA polymerase d, proliferating cell nuclear antigen (PCNA), thymidine kinase and dihydrofolate reductase. Research conducted in mammalian cells proposed that pRb inhibits DNA replication in part by disrupting the chromatin association of PCNA (Angus et al. 2004). Once activated/ dephosphorylated, pRb can inhibit G1- and S-phase progression. It has been well documented in literature that pRb is inactivated by phosphorylation at G1/S transition through the action of cyclin-dependent kinases and this phosphorylation of pRb is required for S phase. Following DNA damage, the presence of Rb is required for cell cycle inhibition. DNA damage activates pRb by elevating its dephosphorylated form, potentially intervening through the p53 pathway. This response has been assessed in studies utilizing mouse embryonic fibroblasts (MEF) (Bosco and Knudsen 2005). The activation of the p53/p21Cip1 pathway in DNA damage response has the downstream consequence of accumulating pRb in its hypophosphorylated form and therefore in active conformation. pRb is an essential player in this network owing to its regulation of the expression of a wide range of DNA repair factors. pRb has been localized to sites of DNA replication early in S phase. Knudsen et al. (2000) have reported that pRb is required for an intra-S-phase response to

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DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in pRb+/+ but not in pRb−/− MEFs. Niculescu et al. (1998) described that the tendency of pRb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of pRb-negative cancers with genotoxic agents that activate the p53/p21 pathway. In response to irradiation, cell cycle checkpoints are activated to provide time for DNA repair. DNA damage elicits arrest at both G1 and G2 phases of the cell cycle. pRb is required for radiation-induced G1 arrest and it has also been demonstrated to regulate the duration of G2. G1 arrest is brought about through the activation of the p53/p21Cip1 regulatory pathway, which requires functional pRb. Furthermore, while the activation of p53 and p21 is normal in pRb-deficient cells, these cells do not undergo G1 arrest because they lack pRb. As a conclusion, pRb is necessary for the p53/p21 pathway to exert a negative effect on G1/S transition (reviewed by Genovese et al. 2006). The concept that G2 checkpoint is activated to allow time for repair would predict that the duration of G2 arrest should be proportional to the extent of DNA damage. The initiation of the G2 checkpoint is a pRb-independent event, but pRb, similarly to p53, is required for the maintenance of G2 arrest. pRb becomes activated in G2-arrested cells following a high dose of ionizing radiation through p53 and p21Cip1 intervention, participating in a cell cycle exit at G2. The prolongation of G2 arrest in the pRb+/+ cells correlates with a gradual accumulation of hypophosphorylated pRb, and it is believed that radiation-induced damage initiates signaling pathways that lead to pRb dephosphorylation (reviewed by Genovese et al. 2006). However, the activation of G2 checkpoint is independent from the extent of DNA damage and does not require pRb, whereas the duration of G2 arrest most likely depends on the extent of DNA damage and pRb function. The establishment of G2-arrest also relies on Chk1-dependent inactivation of the cyclin B1–Cdc2 kinase. Studies have determined that pRb plays an essential role in regulating the duration of the G2 checkpoint response in human cells. The loss of this pRb-dependent prolongation of G2 arrest sensitizes cells to IR-induced apoptosis and increases the probability of tumor development or progression. Therefore, pRb clearly plays a role in controlling the biological outcome in DNA damage response. It has been proven that when DNA damage occurs in G2 phase and cell cycle is arrested, several genes normally required for G2 and M phase become downregulated (Chang et al. 2002; Ren et al. 2002). In particular, p130 and p107 are critical regulators in the expression of G2/M required genes (e.g., CDK1, Cyclin B1, PLK1, SMC4L1 for chromatin condensation, STATHMIN for spindle formation, and PRC1 and RB6K for cytokinesis). CDE/CHR elements in the promoter sequences of these genes may have a major role in DNA-damage induced repression by p130 and p107. Loss of function of the pathway involving p21/WAF1, CDKs and pRb family proteins results in a lower cellular ability to initiate and maintain a transient G2/M block (reviewed by Genovese et al. 2006). The study by White et al. (1994) in diploid human fibroblasts showed that the expression of the E7 protein of human papillomavirus (HPV)-inactivated pRb, led

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to a p53-independent alteration in cell cycle control, a widespread cytocidal response, and polyploidy as a mechanism of drug resistance. In another work, the inactivation of pRb and the resulting deregulation of one E2F family member, E2F1, led to DNA DSB accumulation in human normal diploid cells. This DSBs’ accretion occurs independently of ATM, p53, caspases, reactive oxygen species and apoptosis. E2F1-associated DSBs are not observed in a p16INK4A-inactivated cell line that retains functional pRb, unless pRb is depleted (Pickering and Kowalik 2006). These reports suggest that pRb is essential not only in the regulation of the proliferation-promoting factor E2F1 but also in preventing DNA damage accumulation in the presence of E2F1 deregulation. A unique role for p130 and p107 in repressing c-myc (key regulators of cellular growth, apoptosis, differentiation and stem cell self-renewal) expression has also been recently revealed (Chen et al. 2002). p130 and p107 are displaced from the promoters of cell cycle-regulated genes during S-phase, but they remain bound at the promoters of apoptotic regulators. Studies focusing on the overexpression effects of p107 protein in fibroblasts demonstrated a subsequent inhibition of Cdk2 activation and a marked delay in S-phase entry (reviewed by Genovese et al. 2006). The inhibition of Cdk2 activity is correlated with the accumulation of p27Kip1, consequent to a decreased degradation of the protein. Based on experimental data, it has been speculated that p27Kip1, an inhibitor of Cdk2, indirectly triggers pRb hypophosphorylation in late G1. Furthermore, immunoprecipitation assays have confirmed an association between the cell growth suppressor p27Kip1, p130 and cyclin E (Howard et al. 2000). It is therefore likely that p27Kip1 binds to the cyclin E/cdk2 complex causing a decrease in cyclin E/cdk2 kinase activity and, consequently, the hypophosphorylation of pRb and p130 in late G1. Induction of p130 expression results in inhibition of cyclin E-associated kinase activity that induces p27Kip1 levels by preventing its degradation (Howard et al. 2000). Most likely, p130 participates in a positive feedback loop in which p130’s inhibition of cdk2associated kinase activity would generate high p27Kip1 levels that in turn would inhibit other cdk activity, thereby preventing p130 inactivation by other cdks (Howard et al. 2000). Also, p130 and p107 contain a motif similar to the one found in the p21/cdk-inhibitor, which grants the ability to bind and inhibit cdks, suggesting that p107 and p130 may also be responsible for an interaction that affects cdk activity (Woo et al. 1997). p130 uniquely also possesses another distinct kinase inhibitory domain, found within its spacer region, which selectively inhibits cdk2 kinase activity (De Luca et al. 1997; Howard et al. 1998).

19.9

Conclusions and Lingering Questions

Studies on the pocket proteins have been at the heart of many of the landmark discoveries in cancer genetics exploring new aspects of the cell cycle machinery. The prototypic tumor suppressor gene Rb is mutated in a number of human tumors. These data have encouraged more and more researchers to study the

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retinoblastoma pathways, and to explore the possibility of replacing the nonfunctioning Rb gene as a potential clinical treatment for human cancers. p130 and p107 have also been found mutated in different types of tumors (Howard et al. 1998; Lara et al. 2008). Loss of pRb compromises critical aspects of proliferation, so that tumors readily arise from a field of aberrant cellular proliferation. In some tissues, loss of pRb could abrogate differentiation programs, whereas in other tissues pRb loss would compromise genomic stability in a manner promoting tumorigenesis. Studies in vitro and in vivo on nonsmall cell lung cancer (NSCLC) showed that pRb deficiency enhances sensitivity to chemotherapeutic challenge, efficient and sustainable response, which is highly dependent on the specific therapeutic regimen, in addition to the molecular environment (Zagorski et al. 2007). 3T3-immortalized and Ras-transformed mouse adult fibroblasts (MAFs) containing conditional Rb alleles were utilized to investigate the consequence of pRb loss on cellular response to cytoxic agents (U0126 and LY294002) and therapies targeting the MEK and PI3K respectively, which specifically function downstream of Ras signaling. Using these models, Stengel et al. (2008) demonstrated that pRb loss modifies response to a variety of therapeutic agents through abrogation of cell cycle checkpoints, and that pRb status alters sensitivity to both cytotoxic and targeted therapies. It is likely that each tissue will have to be tackled separately to understand the action of pRb in suppressing oncogenesis. Activation of tumor suppressor pathways may represent ideal avenues for intervention, and have already proven successful in preclinical studies with p53 (Vassilev et al. 2004). Efforts to exploit direct pRb loss have largely emanated from the field of oncolytic viruses, such as E1A-mutant adenoviruses, called Delta-24, wherein viruses have been engineered to replicate in pRbdeficient cells (reviewed by Gomez-Manzano et al. 2004). Conventional assessment of cytoxic agents has indicated that pRb loss in specific cell-based assays enhances cytoxicity. However, the extent and importance of this phenomenon in vivo is less clear and needs to be evaluated in appropriate models. In spite of the many questions that have been answered, a long path still lies ahead to fully decipher the actions of the pocket proteins and their regulatory partners. Ongoing studies are determining the functional role of novel targets in pRbmediated replication control (Weber at al. 2008). The data collected by future research will be essential in defining the roles of individual retinoblastoma members in different cellular mechanisms and may clarify the significance of their functional loss. Recent studies have improved our understanding of the molecular mechanisms through which pRb/p105, p130 and p107 interact with other cellular components, as well as their impact on G1/S and G2/M control and cell proliferation. It has been proposed that p130 may also contribute to G1 arrest through its unique spacer region, which grants the ability to suppress cdk2 kinase activity, thereby decreasing the activity of kinases that allow the cell to enter S-phase (De Luca et al. 1997). Furthermore, according to several studies using fibroblasts from knockout mouse embryos, the deficiency of pRb or of more than one family member exhibits a shortened G1 phase of the cell cycle and subsequent lengthening of S-phase (Ruiz

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et al. 2004). The binding of pRb family proteins with the E2F family of transcription factors seems to be pivotal in leading cell cycle progression and DNA replication by determining the expression of cell cycle E2F-dependent genes (Frolov and Dyson 2004). However, there may be other undisclosed important participants. Many studies have been conducted on chromatin structure changes in order to attempt to understand the mechanism of postdamage pRb suppression of DNA replication. The Rb gene family’s interaction with chromatin remodeling factors may uncover other crucial players in the complex signaling network responsible for DNA damage response. Ongoing studies focus on the exact mechanisms by which the pRb family permits cell cycle exit upon the occurrence of DNA damage (Bosco et al. 2007; Bosco and Knudsen 2005; Knudsen et al. 2000). New findings in DNA repair mechanisms have continued to revise the picture of how this gene family may perform in enhancing and controlling DNA damage repair dynamics. While significant milestones have been reached in defining the role of pRb family members in cell cycle and DNA damage repair, more goals still have to be attained to gain an active fruition of this knowledge through the development of novel, efficient therapeutic strategies. Acknowledgments We are grateful to Marco Cassone for critical reading of the manuscript, helpful discussions and encouragement. P.V. acknowledges the postdoctoral fellowship at Jefferson University, PA. This work was partially supported by grants from NIH, WV-INBRE, and MU-CDDC to P.P.C.

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Chapter 20

Activation of the p53 Tumor Suppressor and its Multiple Roles in Cell Cycle and Apoptosis Luciana E. Giono and James J. Manfredi

20.1

The p53 Tumor Suppressor

The p53 tumor suppressor protein is widely conserved from zebrafish to humans. It was the first tumor suppressor to be cloned. However, it was not first cloned as a tumor suppressor; p53 was discovered in 1979 as a cellular protein that interacted with the large T antigen of the SV-40 DNA virus and that accumulated in neoplastic rodent cells. p53 levels were increased in human cancer and the protein was able to cooperate with the ras oncogene to transform cells in culture. It was not until almost 10 years later that the cloned p53 was discovered to be mutated and the supposed oncogene was rightfully recognized as a tumor suppressor (Harris 1996). An overwhelming body of literature has, since 1989, proved that p53 is an essential tumor suppressor required to control cell division and cell death, to ensure genome integrity and, by these means, to prevent tumor formation. This has earned p53 its nickname “the guardian of the genome” (Lane 1992). The p53 protein or pathway is mutated or inactivated in most human cancers (Vogelstein et al. 2000). In half of these tumors, p53 is directly inactivated by somatic mutations in the p53 gene frequently accompanied by loss of heterozygosity. In tumors that retain wild-type p53, it is often indirectly inactivated by alterations in proteins that regulate p53 function such as Mdm2 or p14ARF. Germline mutations of p53 have been associated with Li–Fraumeni syndrome, an autosomal disorder characterized by a familial clustering of early onset tumors (Vousden and Lu 2002). Mutations can also confer gain-of-function properties to p53 that behaves then as an oncogene (Strano et al. 2007). Finally, p53 is disabled by binding of viral

J.J. Manfredi (*) Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY, USA e-mail: [email protected] L.E. Giono Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_20, © Springer-Verlag Berlin Heidelberg 2010

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proteins like papillomavirus E6, adenovirus E1B, SV-40 T-antigen, Epstein–Barr virus EBNA-5 and BZLF-1 (Levine 1997). p53-knockout mice are viable and appear normal, suggesting that p53 is not essential during development. However, although the spectrum of tumors might vary depending on the genetic background, p53-deficient mice are highly susceptible to early and spontaneous development of different types of tumors.

20.1.1

p53 Structure

The 393-amino acid (aa) protein encoded by the p53 gene is organized into several domains that include a DNA binding region and transactivation and regulatory domains (Fig. 20.1a). The central DNA binding core of p53 (aa 102–292) contains the major mutation hot-spots found in tumor-derived mutants. p53 binds as a tetramer to two repeats of the palindromic sequence 5¢-RRRCA/TT/AGYYY-3¢, separated by a 0–13 bp spacer (where R is a purine and Y is a pyrimidine). Oligomerization sequences mapped to residues 324–355 are required for tetramer formation. Two independent acidic activation domains have been identified at the N-terminus of p53, between residues 1–42 and 43–73 (Vousden and Lu 2002). These have been shown to interact with factors of the basal transcription machinery such as TFIID (TBP), TFIIH, TAFII31 and TAFII70 (Levine 1997). The oncogenic protein Mdm2, the main negative regulator of p53, also binds to the N-terminal domain of p53 inhibiting its transcriptional activity (Oliner et al. 1993). The proline-rich domain between amino acids 61 and 94 contains five repeats of the sequence PXXP, a motif that has been shown to play a role in signaling through binding to SH3 domains. A cluster of three nuclear localization signals (aa 315– 386) and a nuclear export signal (aa 340–351) regulate p53 subcellular localization and therefore, p53 function (Shaulsky et al. 1991). Finally, a basic regulatory domain is located at the carboxyl terminus of p53, between residues 363 and 393. This domain has been shown to bind to DNA in a nonspecific manner and to negatively regulate specific DNA binding by the central core. Together with the N-terminal domain, the C-terminus of p53 concentrates most of the many posttranslational modification sites that contribute to regulate p53 function (Appella and Anderson 2001).

20.1.2

The p53 Family

Almost 20 years after the discovery of p53, two homologs, p63 and p73, were identified. Both proteins can bind to p53 consensus sites, transactivate some p53 target genes and regulate cell cycle arrest and apoptosis following DNA damage. However, neither p63 nor p73 appear to be mutated in human cancer and therefore would not be traditional tumor suppressors. Consistently, p63- and p73-knockout

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Fig. 20.1 (a) p53 is a transcription factor organized in several well-defined domains, including an N-terminal transactivation domain, a central DNA binding core and a C-terminal region that contains nuclear localization signals (NLS), a tetramerization domain and a regulatory domain. (b) Through alternative splicing, usage of a second internal promoter, and alternative initiation of translation, multiple isoforms of p53 are expressed in different tissues. These isoforms lack different domains of the protein. (Modified from Murray-Zmijewski et al. 2006) (c) Multiple residues clustered at the N- and C-terminus of p53 are post-translationally modified by phosphorylation (P, circles), acetylation (Ac, lozenges), ubiquitination (Ub, squares), sumoylation (S, triangle), methylation (Me, hexagons) and NEDDylation (Nd, gray circle). Through these modifications, p53 integrates signals from multiple stress-activated pathways that involve numerous kinases and other modifying enzymes

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mice do not have increased cancer susceptibility. On the other hand, mice deficient for both p53 homologs exhibit severe developmental abnormalities (Mills et al. 1999; Yang and McKeon 2000). Through alternative splicing, usage of a second internal promoter, and alternative initiation of translation, multiple isoforms are generated from the p53, p63 and p73 genes. Some of these lack the transactivation domain, behaving in a dominant negative manner (Fig. 20.1b). The ability of these three family members and their multiple isoforms to cooperate and interfere with each other’s functions, the subtle balance between the forms, their timing of expression and tissue specificity are only now beginning to be unraveled (Murray-Zmijewski et al. 2006).

20.2 20.2.1

p53 Regulation p53 Stability

Central to p53 regulation is Mdm2, which controls p53 stability, transcriptional activity and cellular localization. In unstressed cells, p53 is maintained at very low levels due to continuous degradation through a ubiquitin-dependent pathway on nuclear and cytoplasmic 26S proteasomes. Mdm2 is a RING Finger E3 ubiquitin ligase that regulates p53 stability by binding to the N-terminus of p53 and ubiquitinating several lysine residues at the C-terminus of p53 (Moll and Petrenko 2003). Mdm2 can inhibit p53 transcriptional activity by two mechanisms: it associates to the N-terminus of p53 blocking its transactivation domain and mediates NEDDylation of p53 at Lys370, 372 and 373, resulting in impaired transcriptional activity (Xirodimas et al. 2004). The significance of the regulation of p53 by Mdm2 is evidenced by the fact that deletion of Mdm2 results in embryonic lethality due to excessive apoptosis, and that deletion of p53 rescues the Mdm2–/– mice (Moll and Petrenko 2003). Mdm2 is in turn regulated by p14ARF, a tumor suppressor encoded by an alternative reading frame at the p16INK4A/ARF locus. p14ARF binds to the RING finger domain of Mdm2 and sequesters it, blocking the Mdm2-dependent degradation of p53. p14ARF is believed not to be involved in the DNA damage pathway but to respond to oncogenic stimuli and is required for the p53-dependent growth arrest provoked by Ras- or E1A-induced apoptosis (Moll and Petrenko 2003). MdmX (or Mdm4) is a protein structurally related to Mdm2 that was identified due to its ability to interact with p53. Like Mdm2, MdmX blocks p53 transcriptional activity and the embryonic lethal MdmX-deficient mice can be rescued by simultaneous deletion of p53. In contrast, MdmX is essentially a cytoplasmic protein and needs to associate to Mdm2 through its RING domain to translocate to the nucleus. Another significant difference between the two proteins is that MdmX cannot function as an E3 ubiquitin ligase and does not, by itself, promote p53 ubiquitination and degradation. Finally, MdmX is degraded following DNA damage in an Mdm2-dependent manner, a process that has been proposed to ensure rapid and maximal activation of p53 (Marine and Jochemsen 2004).

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More recently, Pirh2 and COP1, two E3 ubiquitin ligases that can promote p53 degradation, were identified. Out of several E2 conjugating enzymes, UbcH5B/C have been found implicated in the degradation of both p53 and Mdm2. HAUSP, a ubiquitin-specific protease, is in charge of deubiquitinating p53, and therefore, stabilizes it. Consistently, partial reduction of endogenous HAUSP levels destabilizes p53 and HAUSP overexpression was shown to induce p53-dependent growth arrest and oppose the inhibitory effect of Mdm2 on apoptosis induced by p53. However, in a system where everything appears to be maintained in a very tight balance, complete ablation of HAUSP lead to stabilization and activation of p53. This was shown to be due to the fact that HAUSP also stimulates Mdm2 stability (Brooks and Gu 2006). p53 was also reported to be degraded in an Mdm2 and ubiquitin-independent manner by the 20S proteasome. This degradation is regulated by NAD(P) H:quinone oxidoreductase 1 (NPQO1), which binds to and protects p53 (Asher and Shaul 2005). Finally, more recent evidence suggest that p53 protein levels are also regulated at the level of translation (Halaby and Yang 2007).

20.2.2

p53 Posttranslational Modification

p53 has been shown to be modified on over 30 residues (Fig. 20.1c). These modifications include phosphorylation, acetylation, sumoylation, NEDDylation, methylation and glycosylation and result from the activation of different pathways. Most modifications are observed in response to genotoxic agents in general, but some are rather specific to certain stimuli; some are present in unstressed cells and a few are removed following cellular stress. The role of posttranslational modifications on p53 stability, cellular localization, DNA binding, transcriptional activity and protein–protein interactions has been under extensive scrutiny – not devoid of significant controversy – during the last two decades, as all of these processes have been shown to be affected (Bode and Dong 2004). p53 posttranslational modifications are mainly clustered in the transactivation domain and the C-terminal regulatory region. At the N-terminus, p53 has been found phosphorylated at Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, Thr55, Thr81, Ser149, Thr150 and Thr155. Phosphorylation at Ser15 has been shown to occur in response to ionizing radiation, DNA-damage inducing agents and UV. Ser15 also becomes phosphorylated in human fibroblasts undergoing replicative or ras-induced senescence. Mutation of p53 at this residue (S15A) resulted in impaired transcriptional activity and reduced interaction with the TFIID factor of the basal transcription machinery and the coactivators p300/CBP. The PI3K-related kinases, ATM and ATR, have been implicated in the phosphorylation of Ser15 in response to ionizing radiation and UV, respectively. ATM has also been shown to mediate phosphorylation of p53 at Ser20 after ionizing radiation (IR) (Olsson et al. 2007). Releasing p53 from the inhibitory effect of Mdm2 represents a key step in the activation of p53. This is highlighted by the fact that mere disruption of the association

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of p53 with Mdm2 with small inhibitors, the nutlins, is sufficient to activate p53 (Thompson et al. 2004). The current view is that p53 is relieved from Mdm2 inhibition by stress-induced posttranslational modification of both p53 and Mdm2. Contradictory results were observed regarding the effects of phosphorylation at Ser15, Thr18 or Ser20 of p53 on Mdm2 binding. The analysis of the role played by each individual residue might be obscured in part by the fact that, in cells, phosphorylation of Thr18 appears to require prior phosphorylation of p53 at Ser15 and Ser20, and phosphorylation of Ser20, in turn, requires Ser15 to be phosphorylated. Three reports where p53 N-terminal phosphorylation sites were mutated, alone, in pairs, or all at once, argue that phosphorylation of p53 is not involved in the regulation of stability or transcriptional activity. This apparent discrepancy might be explained in part by an altered behavior of p53 mutants transfected at nonphysiological levels (Appella and Anderson 2001). In addition to p53, and possibly as a safety mechanism, Mdm2 has also been shown to become posttranslationally modified following DNA damage. These modifications are believed to relieve the inhibitory effect of Mdm2 on p53 and may account for the stability of N-terminal mutants of p53. Phosphorylation of Mdm2 at Ser395, Ser407 and Tyr394 was shown to stabilize p53 (Meek and Knippschild 2003). Following DNA damage, a transient auto-degradation of Mdm2 involving Ser395 and a wortmannin-sensitive pathway appear to be required for p53 activation. Finally, MdmX was reported to be subjected to a similar regulation, becoming phosphorylated and degraded in an Mdm2-dependent manner following DNA damage (Pereg et al. 2005). Taken together, the available data depicts a highly sophisticated and complex mechanism of activation of p53 following DNA damage, mediated by ATM and other PI3K-family members, that includes safety mechanisms targeting p53 inhibitors such as Mdm2 and MdmX, to ensure a properly controlled activation of this essential pathway (Meulmeester et al. 2005). p53 is also phosphorylated at Ser33 and Ser46 by p38 and mutation of those two residues or inhibition of p38 resulted in decreased UV-induced apoptosis. Overexpression of p38 led to increased p53 stabilization. Phosphorylation at Ser46 was shown to selectively regulate transactivation of the apoptotic p53 target gene p53AIP-1, and a S46A mutant p53 was impaired in its apoptotic activity (Oda et al. 2000). Phosphorylation of Ser37 and Thr55 were reported to be important for p53 transcriptional activity and induction of apoptosis, respectively. Phosphorylation of Thr81 by JNK increased p53 protein stability and, in contrast, modification of Ser149, Thr150 and Thr155 by the COP9 signalosome stimulated its proteasomedependent degradation (Bode and Dong 2004). The C-terminus of p53 is subject to an even more complex array of posttranslational modifications, including phosphorylation at Ser315, Ser366, Ser371, Ser376, Ser378, Thr387 and Ser392, acetylation at Lys305, 320, 370, 372, 373, 381 and 382, methylation at Lys320 and 372, sumoylation at Lys386, and as mentioned earlier, NEDDylation at Lys370, 372 and 373, and ubiquitination at Lys320, 370, 372, 373, 381, 382, 386 and probably other lysines (Olsson et al. 2007). Ser315 is phosphorylated by cdk2 and cdc2 in a cell cycle-regulated manner and after UV treatment (Bode and Dong 2004). Modification of this residue was reported

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to cause a conformational change on p53, enhancing its sequence-specific DNA binding and altering the binding-site preference of p53 (Wang and Prives 1995). Ser378 is constitutively phosphorylated, whereas Ser376 is also phosphorylated in unstressed cells but becomes dephosphorylated following IR treatment in an ATMdependent manner (Waterman et al. 1998). Ser376 dephosphorylation would allow the binding of 14-3-3 proteins to p53, an interaction that required Ser378 to be phosphorylated. The ability of p53 mutants to interact with 14-3-3 proteins correlated with their ability to induce cell cycle arrest after IR treatment (Stavridi et al. 2001). Ser378 was also found to be phosphorylated by Chk1/Chk2, together with Ser366 and Thr387. The phosphorylation of all three residues was stimulated following exposure to UV, IR, and camptothecin, although Ser378 was also phosphorylated, to a lesser extent, in untreated cells, consistently with the previous report. Interestingly, substitution of these residues to either Ala or Asp, or downregulation of the modifying enzymes Chk1 and Chk2 by siRNA, revealed a complex regulation of the acetylation of p53 by these phosphorylation events. The data suggested that phosphorylated Ser378 in unstressed cells suppresses acetylation of p53 at Lys373/382 but not Lys320, an effect that would be relieved by DNA damage-induced phosphorylation of Ser366 and Thr387 (Ou et al. 2005). Inhibition of Lys382 acetylation by phosphorylation of Ser378 in vitro has previously been reported (Sakaguchi et al. 1998). Ser392 is phosphorylated in vitro by casein kinase II (CKII) (Hupp et al. 1992), and a Ser392-kinase complex containing CKII and the chromatin elongation factor FACT was purified from UV-treated HeLa cells. Phosphorylation of p53 by CKII or the kinase complex stimulated p53 DNA binding in in vitro assays. (Keller et al. 2001). Phosphorylation at Ser392 was decreased in human fibroblasts undergoing p53-dependent replicative senescence, whereas it was increased in replicating fibroblasts following UV treatment, and unchanged after IR or bleomycin treatment (Webley et al. 2000). In this study, partially overlapping but distinct p53 phosphorylation patterns revealed stress-specific modification. Lys372 was found to be methylated by Set9 in response to doxorubicin, a modification that resulted in increased p53 stability, DNA binding and transcriptional activity and apoptosis (Chuikov et al. 2004). Sumoylation of Lys386 correlated with increased transcriptional activity but did not interfere with ubiquitination of p53 (Rodriguez et al. 1999), and a K386R mutant p53 showed partially impaired apoptotic activity (Muller et al. 2000). p300 and CBP (CREB Binding Protein), two closely related histone acetylases, have been shown to interact with and acetylate the multiple lysine residues (Lys370, Lys372, Lys373, Lys381, Lys382) of the C-terminal domain of p53 (Gu and Roeder 1997). Acetylation of p53 occurs in response to a variety of types of cellular stress, including UV, IR, hypoxia (DFX), oxidative stress (H2O2), and deprivation of ribonucleotides (by treatment with N-phosphonoacetyl-l-aspartate, PALA) (Ito et al. 2001). A model was proposed where phosphorylation of the N-terminal serines of p53 stimulates p300/CBP association and acetylation of the C-terminal lysines (Sakaguchi et al. 1998). Furthermore, substitution of Ser15 by Ala dramatically inhibited acetylation at Lys320 and 382 (Saito et al. 2002). p53 was also shown to be acetylated by P/CAF at Lys320, and this site was modified

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in cells after exposure to UV and IR. Acetylation of both Lys320 and Lys382 stimulated p53 sequence-specific DNA binding in vitro (Sakaguchi et al. 1998). Acetylation of Lys382 enhanced p53 transcriptional activity by increasing the association of p53 at promoter sites with coactivators like CBP and TRAPP. Mutant p53, with substitutions at multiple acetylated lysines, was impaired in transcriptional activity, induction of G1 arrest, and coactivator recruitment (Barlev et al. 2001). However, mutation of seven C-terminal lysines to arginine in a mouse model had no effect on p53 stability and transcriptional activity (Krummel et al. 2005). In addition to the effect on DNA binding and transcriptional activity, acetylation of p53 was reported to regulate its stabilization. p300 and Mdm2 were shown to form a ternary complex with p53, and compete to modify the same residues, acetylating and ubiquitinating, respectively, the C-terminal lysines of p53. Accordingly, Mdm2 inhibited p53 acetylation, and acetylation of p53 inhibited ubiquitination by Mdm2 (Brooks and Gu 2003). MdmX shares with Mdm2 its ability to block acetylation of p53 by p300 in cells (Sabbatini and McCormick 2002). Mdm2 was also shown to interact with P/CAF and to inhibit acetylation of p53 at Lys320 (Jin et al. 2002b). The effect of p300/CBP on p53 stability is however less clear. By acetylating the C-terminal lysines of p53, thus preventing ubiquitination, p300/CBP were thought to promote p53 stabilization in response to DNA damage. Surprisingly, p300 was reported to promote its degradation by catalyzing poly-ubiquitination of p53, following mono-ubiquitination by Mdm2 (Grossman et al. 2003). In addition, p300 was shown to stabilize Mdm2, thereby enhancing the Mdm2/p53 negative regulatory loop (Kawai et al. 2001). More recently, a novel p300-acetylated site was identified, Lys305. This residue was acetylated in cells following UV, IR and H2O2 treatment and was reported to play a role in p53 transcriptional activity (Wang et al. 2003). Finally, p53 deacetylation was found to be regulated by PID, a component of the nucleosome remodeling and histone deacetylation (NuRD) complex. PID interacted with p53 and promoted its deacetylation. Moreover, p53 transcriptional activity, as well as growth arrest and apoptotic functions, were reduced by PID overexpression (Brooks and Gu 2003).

20.2.3

Regulation of p53 DNA Binding Activity

The regulation of the ability of p53 to bind to DNA in a sequence-specific manner has been considered a major way of controlling its activity. The last 30 amino acids of p53 have been implicated as the main regulator of p53 DNA binding activity. The C-terminal domain, rich in basic residues, is capable of binding to DNA in a nonspecific way and this was thought to interfere with specific DNA binding by the core domain of p53 (Anderson et al. 1997). Several observations led to the proposal of a model where, under normal conditions, p53 is in a latent form, incapable of

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DNA binding, and requires activating factors or modifications to become capable of sequence-specific binding (Hupp et al. 1992). Modifications were then thought to either neutralize the positive charges of the C-terminus of p53, thus decreasing its DNA binding, or allosterically activate p53. Since the proposal of that model, the role of p53 posttranslational modifications has been extensively studied and has become a source of controversy. In the past few years, several lines of evidence have argued against the existence of a “latent p53.” Using chromatin immunoprecipitation assay, p53 was found associated to several promoters from p53 target genes, before and after different types of DNA damage. The association correlated with p53 levels, suggesting similar binding capabilities in both situations (Kaeser and Iggo 2002). Rather than having a negative effect, the C-terminal domain of p53 was shown to be required for p53 to linearly diffuse along the DNA to find its target sites (McKinney et al. 2004). p14ARF overexpression or treatment with nutlins showed that phosphorylation of p53 on N-terminal serines is not required for p53 activity (Jackson et al. 2004; Thompson et al. 2004). Of note, several of these reports aimed at disrupting the p53–Mdm2 interaction, and by doing so, succeeded in activating p53 function. These observations highlight the critical role of the inhibitory effect of Mdm2 and the necessity of disrupting this complex by posttranslational modification. They also suggest that the absolutely indispensable role of the modifications, if they are indispensable at all, is to allow p53 to become free of Mdm2 and stabilized. Once this is achieved, the wide array of p53 posttranslational modifications might work as safety mechanisms, but also to fine-tune p53 activities depending on the cellular context and type of stress, and maybe modulate the termination of the response.

20.3

p53 Function

p53 functions as a sequence-specific transcriptional regulator and this activity accounts for most its tumor suppressor properties. Through posttranslational modification and protein–protein interactions, p53 integrates signals from multiple stress-activated pathways and regulates the expression of numerous genes involved in many cellular functions (Fig. 20.2a). This chapter will focus on the two main outcomes of p53 activation: cell cycle arrest and cell death. The factors that determine or influence the p53 decision-making process have been, and still are, a long-standing question. p53 responses are often stimuli- and cell typespecific. Many alternative or complementary models have been proposed in which the decision is driven by the affinity of p53 for its response elements on the different target genes, by specific posttranslational modifications, or by the recruitment of different coactivators (Espinosa 2008; Vousden 2000). Finally, it has also been suggested that the final decision is the result of p53-independent mechanisms (Paris et al. 2008).

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Fig. 20.2 (a) p53 target genes: upon DNA damage, hypoxia, oncogene activation and a variety of cellular stimuli, activated p53 regulates the expression of multiple target genes involved in numerous cellular processes including cell cycle arrest, DNA repair, apoptosis, cell survival, energy metabolism, angiogenesis and invasion, autophagy, microRNAs, as well as autoregulation. (b) Through its distinct domains, p53 interacts with many cellular and viral proteins

20.4

p53 and Cell Cycle Checkpoints

Cell cycle checkpoints are mechanisms that monitor the sequence of cell cycle events and that ensure that the progression into late events depends on the successful completion of early events. Checkpoints will therefore delay the progression into the next phase until the previous step is fully completed, and only then will they relieve the arrest. The ultimate goal of these mechanisms is to guarantee that the two daughter cells inherit a complete and faithful copy of the genome of the original cell. Checkpoints can become activated in the presence of damaged DNA, exogenous stress signals, or defects at virtually every step during the replication of DNA, such as presence of unreplicated DNA, or failure of chromosomes to

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attach to the mitotic spindle. Abrogation of cell cycle checkpoints can result in death for a unicellular organism or uncontrolled proliferation and tumorigenesis in metazoans (Nyberg et al. 2002). The G1/S and G2/M checkpoints are activated in response to the presence of damaged DNA and will prevent entry into S-phase and mitosis, respectively, allowing for the damage to be repaired. The “S-phase checkpoint” involves three distinct independent checkpoint types that become activated by stalled replication forks (replication checkpoint), damaged DNA (intra-S-phase checkpoint) and incomplete DNA duplication (S/M checkpoint) (Bartek et al. 2004). Finally, the spindle assembly checkpoint prevents cells from entering anaphase until all chromosomes are aligned at the equator and attached to the microtubules of the mitotic spindle. Central to its function as a tumor suppressor, p53 participates in each one of the cell cycle checkpoints. p53 has a well-documented role in the G1/S and G2/M checkpoints. Its involvement in the S-phase and mitotic spindle checkpoints, however, is more controversial.

20.4.1

The G1/S Checkpoint

Cell cycle progression is driven by the activity of cyclin/cyclin-dependent kinases (cdks) complexes. G1 cyclin/cdk complexes, cyclin D/cdk4, cyclin E/cdk2 and cyclin A/cdk2 are therefore the main targets upon G1/S checkpoint activation (Sherr 1994). This checkpoint prevents initiation of DNA replication in cells in which damaged DNA has been detected and p53 plays an indisputable role in it. p53 activation or ectopic expression arrests cells at the G1/S transition. This is mainly achieved through induction of p21, a cyclin-dependent kinase inhibitor (CKI) that inhibits cdk2-containing complexes. Consistently, p21 overexpression also induced G1 arrest, even in the absence of p53. Furthermore, absence of p21 following DNA damage prevented proper G1 arrest, both in mouse embryo fibroblasts from p21-deficient mice and in human cancer cells in which p21 was deleted by homologous recombination (el-Deiry 1998). More recently, p53 was shown to associate to the c-myc promoter and repress its expression and this was proposed to be required for the p53-dependent G1 arrest (Ho et al. 2005). The cellular response following G1/S checkpoint activation is dual, including a rapid and transient phase followed by a more delayed and sustained arrest. Upon DNA damage, a transient decrease in cyclin E,A/cdk2 activity is caused by phosphorylation and subsequent degradation of its activating phosphatase, Cdc25A, through an ATM-Chk2 pathway. Cyclin D1 has also been shown to be degraded following genotoxic stress and this was required for initiation of the G1 arrest. In addition, upon cyclin D1 degradation, p21 is released and redistributed to cdk2 complexes, which are then inhibited by p21 (Bartek and Lukas 2001b). Its response being at the transcriptional level and, therefore, requiring more time, p53 is responsible for extending the initial delay caused by degradation of Cdc25A and cyclin D providing the cell with sufficient time to repair the damaged DNA (Nyberg et al. 2002).

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The S-Phase Checkpoint

As mentioned earlier, the role of p53 at the S-phase checkpoints remains controversial. While some argue that none of these checkpoints requires p53 (Bartek et al. 2004), some reports suggest otherwise. p53-null cells treated with inhibitors of DNA replication entered mitosis with incompletely replicated DNA, after an extended S-phase. Moreover, under conditions of nucleotide imbalance that cause DNA damage, p53-null cells progressed into mitosis after completing DNA replication. p53 expressing cells, however, failed to enter mitosis following these treatments, suggesting that p53 is required for proper and complete DNA replication (Taylor et al. 1999). In another study, inactivation of ATR and p53 synergized at promoting premature chromatin condensation, a hallmark of mammalian cells that enter mitosis before completing DNA replication (Nghiem et al. 2002). Upon downregulation of Cdc7, a kinase that triggers firing of replication origins, normal human fibroblasts arrested in S-phase, and p53 and p21 were induced. Subsequent downregulation of p53 lead to resumption of abnormal DNA replication, followed by apoptosis, suggesting that p53 prevented progression through a lethal S-phase under limiting amounts of Cdc7 (Montagnoli et al. 2004). The S-phase checkpoint is a transient event and, unlike the G1 checkpoint, is believed to lack a maintenance mechanism. A prolonged arrest at that stage would limit the availability of sister chromatids, required for non-error-prone DNA repair by homologous recombination, and could allow replication origins to regain competence, possibly leading to rereplication (Bartek and Lukas 2001a). This phenomenon was observed in cells where downregulated cdk2 activated an intra-S checkpoint. Interestingly, the number of cells with a > 4N DNA content increased in the absence of p53, arguing for its involvement in this checkpoint (Zhu et al. 2004). Finally, it has been speculated that activation of p53 during S-phase, at a time when E2F activity is high, could induce apoptosis and be detrimental for the cell (Bartek and Lukas 2001a). Moreover, induction of p21 by p53 during S-phase could interfere with multiple activities including DNA replication and repair. Several studies have provided evidence that during S-phase, p53 might initiate a limited response, regulating the expression of only a subset of its target genes (Gottifredi and Prives 2005).

20.4.3

The G2/M Checkpoint

G2/M progression is driven by the cyclin B/cdc2 complex. Activation of this complex, and therefore entry into mitosis, is triggered by its translocation from the cytoplasm to the nucleus, the removal of inhibitory phosphates (Thr14 and Tyr15), by the Cdc25 phosphatases, and the addition of an activating phosphate (Thr161) by the cdk-activating kinase CAK. The G2 checkpoint, which becomes activated

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following DNA damage and stress, targets primarily the cyclin B/cdc2 complex (Nyberg et al. 2002). p53 contributes to the G2/M checkpoint and, as with the G1 checkpoint, it is involved in the maintenance rather than the initiation of the arrest. Elimination of p53 in HCT116 cells led to a transient G2 arrest upon IR treatment, after which cells escaped and progressed into mitosis, while the p53 wild-type cells exhibited a prolonged arrest (Taylor and Stark 2001). p53 regulates the G2/M transition by altering the expression of several target genes. Following DNA damage, a p53-independent pathway involving ATM, ATR and their substrates Chk1 and Chk2 phosphorylates Cdc25C, the phosphatase that promotes mitosis by dephosphorylating cdc2. Modified Cdc25C is then sequestered in the cytoplasm by 14-3-3 proteins. After these events, the p53-dependent maintenance is achieved through transcriptional repression of Cdc25C and induction of 14-3-3s. Upon DNA damage, this 14-3-3 isoform binds cdc2 and anchors the cyclin B/cdc2 complex in the cytoplasm. 14-3-3s-deficient cells failed to maintain the G2 arrest in response to radiation and ultimately died (Piwnica-Worms 1999). Several lines of evidence indicate that p21 also contributes to the G2 arrest. A p53-dependent reduction of cdc2 activity was observed following IR treatment that could not be accounted for by regulation of its phosphorylation status or cyclin B levels. This suggested that p53 might induce a cdc2 inhibitor. p21 expression was reported to peak in G1, go down during S-phase and accumulate again in G2. In the G2 phase, it was found associated with inactive cyclin A/cdk2 and, to a lesser extent, cyclin B-containing complexes. p21, however, was shown to be a poor inhibitor of cdc2 in vitro, compared to other cdks. Another possible mechanism of cdc2 inhibition was suggested by the observation that p21 prevented cdc2 activation by CAK (Taylor and Stark 2001). Furthermore, binding of p21 to cyclin B/cdc2 was proposed to keep it in an inactive state by retaining it in the nucleus, preventing its activation by Cdc25C and recruitment to the centrosome (Charrier-Savournin et al. 2004). Finally, using p21 mutants, it was not p21 binding to cdks that was shown to be required for a sustained G2 arrest, but its binding to PCNA, the processivity factor for DNA polymerases d and e, required for DNA synthesis and repair. PCNA was found associated to Cdc25C and it was proposed that p21, by interacting with PCNA, prevents the incorporation of Cdc25C into the cyclin B/cdc2 complex and its subsequent activation (Ando et al. 2001). Regardless of the mechanisms involved, p21 overexpression caused both G1 and G2 arrest in a variety of human cancer cells, and p21-null HCT116 cells failed to arrest stably in G2 following IR. These results indicate that p21 contributes to the G2 checkpoint (Taylor and Stark 2001). Another p53 target gene involved in the G2 arrest is GADD45. GADD45 binds to cdc2 and inhibits its binding to cyclin B. Induction of GADD45 caused G2 arrest and increased cytoplasmic cyclin B1 (Jin et al. 2002a), and was abrogated by cyclin B and Cdc25C overexpression (Wang et al. 1999). Interestingly, G2 arrest by GADD45 requires the presence of wild-type p53 and depends on the type of DNA damage.

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The Spindle Assembly Checkpoint vs. a G1 Tetraploid Checkpoint

Unlike the other checkpoints that become activated upon specific signals, in vertebrates, the spindle assembly checkpoint pathway is constitutive. From the beginning of mitosis, this pathway is constitutively activated until the checkpoint requirement (attachment of chromosome kinetochores to the mitotic spindle) is satisfied. Only then does the anaphase-promoting complex APC/C become activated and cells are allowed to enter anaphase. APC regulates the degradation of numerous proteins, a prerequisite for mitotic progression. Under certain circumstances, however, cells that have not completed mitosis can enter an apparent state of interphase, a process known as adaptation or mitotic slippage. At this stage, checkpoint failure results in cells exiting mitosis and entering the following cycle with a 4N DNA content, leading to endoreduplication (Rieder and Maiato 2004). For several years, many independent observations seemed to support the idea that p53 played a role in the mitotic spindle checkpoint. However, the belief that p53 arrested cells in mitosis was due to misinterpretation of flow cytometry data that showed an accumulation of cells with a 4N DNA content. More careful examination showed that these cells had exited mitosis and entered a G1-like state, at which point p53 became activated. New analysis and new data suggest that p53 is involved in the 4N G1 arrest and prevents endoreduplication of tetraploid cells that exited mitosis (Vogel et al. 2004).

20.5

Apoptosis

In certain cell types, upon certain stimuli, instead of inducing growth arrest, p53 activation will trigger apoptosis. As mentioned earlier, the rules that govern the outcome of the p53 response are still not fully understood (Oren 2003). Many p53 target genes are involved mostly in the intrinsic, but also the extrinsic, apoptotic pathways. p53 induces the expression of PUMA and Noxa, two “BH3-only” proteins that belong to the Bcl-2 family. These proteins inhibit the antiapoptotic members of the family (Fridman and Lowe 2003). bax, a “multidomain” proapoptotic Bcl-2 family member was one of the first apoptotic p53 targets to be identified. The role of Bax in p53-dependent apoptosis is cell type-specific, as bax-null HCT116 cells have an impaired p53-induced cell death, but thymocytes from a bax knockout mouse have an intact response. p53 also upregulates Peg3/Pw1, which triggers Bax translocation to the mitochondria. p53AIP1, induced upon p53 activation, appears to affect mitochondrial membrane potential and contribute to cell death. The induction of p53AIP1is thought to require phosphorylation of p53 at Ser46 and this involves another p53 target gene, p53DINP1. Apaf1, another direct transcriptional target of p53, is a component of the apoptosome. Upon cytochrome c release from the mitochondria, this complex leads to caspase 9 activation, which in turn activates

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the execution of caspase 3 (Bargonetti and Manfredi 2002). Finally, p53 represses survivin, a member of the inhibitor of apoptosis (IAP) family, that act downstream of mitochondrial cytochrome c release, blocking caspase activation. p53 regulates components of the extrinsic pathway including the death receptors Fas/CD95, DR4 and DR5/Killer, and the Fas ligand TNFRSF6. However, the contribution of this pathway to the p53 apoptotic response is still poorly understood (Fridman and Lowe 2003). Interfering with components of cellular prosurvival pathways constitutes another mechanism by which p53 contributes to apoptosis. p53 can block the IGF-1 survival signaling by repressing the IGF-1 receptor (IGF-1R) and by inducing IGF-BP3 (Sionov and Haupt 1999). p53 transactivates PTEN, a lipid phosphatase that negatively regulates the PI3 kinase/Akt survival pathway. Other p53 target genes involved in apoptosis include proteins related to the production of reactive oxygen species (FDXR and PIG3), and other proteins such as PERP and PIDD (p53-induced protein with a death domain) (Fridman and Lowe 2003). Finally, although most of the studies on p53 function have focused on its transcriptional activity, p53 has been shown to play a transcriptional-independent role in the mitochondrial pathway of apoptosis. Some studies reported localization of p53 to the mitochondria. Studies of a polymorphism at codon 72 of p53 (a proline to arginine change within the proline-rich domain) represented a key support to this model. The Arg72 p53 variant was much more efficient at inducing apoptosis than the Pro72. This difference correlated with a greater ability of the Arg72 p53 to localize to the mitochondria, whereas its transcriptional activity on apoptotic genes was similar to that of Pro72 p53 (Murphy 2006). Subsequent studies showed that p53 could induce cytochrome c release from isolated mitochondria, by a mechanism that required the presence of Bax. p53 was then shown to physically interact with antiapoptotic Bcl-XL and Bcl-2. This interaction is believed to release the proapoptotic Bcl-2 family members Bax and Bak from the inhibition by Bcl-XL and Bcl-2. Interestingly, the site of interaction between p53 and Bcl-XL was mapped to the DNA binding domain of p53, a region that is frequently targeted by mutations in human cancer. The relative contribution of the transcriptional and nontranscriptional activities of p53 to its apoptotic response is still being examined (Schuler and Green 2005).

20.6

Other Targets of p53

p53 is a pleiotropic factor affecting many other cellular functions. Several p53 target genes play a role in the regulation of p53 itself through an auto-regulatory feedback loop: mdm2, Pirh2, COP1 and cyclin G (Brooks and Gu 2006). p53 regulates the expression of numerous genes involved in DNA repair: p21, PCNA, GADD45, p53R2 and the p48 xeroderma pigmentosum protein, and physically interacts with proteins that participate in this process: TFIIH (XPB, XPD), RPA, among others (Fig. 20.2b) (Stewart and Pietenpol 2001). Several p53 target genes

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are involved in the inhibition of tumor angiogenesis and invasion, another important aspect of p53 tumor suppression. p53 directly regulates the expression of thrombospondin-1 (TSP-1), brain-specific antiangiogenesis inhibitor 1 (BAI1), the ephrin receptor EPHA2, MMP2 and Maspin, as well as different components of the extracellular matrix. p53 was also shown to inhibit the transcription of VEGF, bFGF and COX2 by various indirect mechanisms (Teodoro et al. 2007). More recently, p53 has been found to regulate glucose metabolism and oxidative stress by inducing TIGAR, Sestrins, ALDH4, GPX1 and SCO2 and repressing PGM and NQO1 (Bensaad and Vousden 2007). DRAM, another p53 target, is a lysosomal membrane protein involved in autophagy (Crighton et al. 2007). Finally, five recent studies independently identified the microRNAs miR-34a, b and c as targets of p53. Simulation of miR-34 activation was able to recapitulate many p53 effects, in a context-dependent manner. miR-34 induction was shown to downregulate hundreds of genes, many of which were cell cycle regulators such as cdk4, cdk6, cyclin E2 and E2F3. In several cell lines, bcl-2 mRNA was also targeted by miR-34a. The identification of the miR-34s as p53 targets doubly expands the repertoire of p53regulated genes by including miRNAs, but also, by incorporating their numerous targets to the p53 network (He et al. 2007).

20.7

Targeting the p53 Pathway in Cancer Therapy

Intensive research has focused on p53 as a target for cancer therapy. From a “p53 point of view,” tumors can be divided into those expressing a mutated p53, with most mutations affecting its DNA binding domain, and those retaining wild-type p53. Concerning the first group of tumors, the possibility of re-establishing p53 activity has been investigated, namely by the use of small molecular compounds that aim at restoring the DNA binding activity of mutant p53 (Selivanova et al. 1998). Retroviruses and adenoviruses encoding wild-type p53 have also been developed, perhaps the most promising being a defective adenovirus that can only replicate in p53-deficient cells (Bischoff et al. 1996). In the second type of tumors, wild-type p53 expression is maintained, but its activity is impaired in most cases by overexpression of Mdm2 or loss of p14ARF (Vousden and Lu 2002). In this situation, the research has focused mainly on Mdm2 and ways to disrupt its negative influence on p53. Many different approaches were explored, including p53-derived peptides that contain its Mdm2-binding region and would bind to Mdm2 preventing its interaction with p53, and p14ARF-derived peptides that would bind to p53 blocking Mdm2 association (Bottger et al. 1997; Midgley et al. 2000). More recently, a screening of small molecular compounds that disrupt p53–Mdm2 interaction led to the identification of the nutlins. These molecules bind to a hydrophobic pocket present in Mdm2 that contacts side chains from p53 amino acids and can stabilize and activate p53 (Vassilev et al. 2004). In addition, another report provided evidence that the nutlins might even prove to be useful in the treatment of tumors containing mutant p53. In this situation, by

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activating p53 activity and inducing cell cycle arrest in the nontumor cells, the nutlins could protect these cells from the side effects of chemotherapeutic drugs (Carvajal et al. 2005). Whether p53 activity is restored by reactivating mutant p53 or by blocking the inhibition by Mdm2, two major points need to be addressed for an optimal cancer therapy. One aspect is whether the reactivated p53 needs to be posttranslationally modified in order to be active, which would determine if these therapies require concomitant chemotherapy. The other aspect concerns the possibility of directing p53 response and deals with the differential gene-specific effects of p53. Since cancer cells appear to be frequently more resistant to apoptosis compared to normal cells, the possibility of being able to selectively induce p53-dependent apoptosis over cell cycle arrest would be especially appealing.

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Rodriguez MS, Desterro JM, Lain S, Midgley CA, Lane DP, Hay RT (1999) SUMO-1 modification activates the transcriptional response of p53. Embo J 18(22):6455–6461 Sabbatini P, McCormick F (2002) MDMX inhibits the p300/CBP-mediated acetylation of p53. DNA Cell Biol 21(7):519–525 Saito S, Goodarzi AA, Higashimoto Y, Noda Y, Lees-Miller SP, Appella E, Anderson CW (2002) ATM mediates phosphorylation at multiple p53 sites, including Ser(46), in response to ionizing radiation. J Biol Chem 277(15):12491–12494 Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M, Vassilev A, Anderson CW, Appella E (1998) DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev 12(18):2831–2841 Schuler M, Green DR (2005) Transcription, apoptosis and p53: catch-22. Trends Genet 21(3):182–187 Selivanova G, Kawasaki T, Ryabchenko L, Wiman KG (1998) Reactivation of mutant p53: a new strategy for cancer therapy. Semin Cancer Biol 8(5):369–378 Shaulsky G, Goldfinger N, Tosky MS, Levine AJ, Rotter V (1991) Nuclear localization is essential for the activity of p53 protein. Oncogene 6(11):2055–2065 Sherr CJ (1994) G1 phase progression: cycling on cue. Cell 79(4):551–555 Sionov RV, Haupt Y (1999) The cellular response to p53: the decision between life and death. Oncogene 18(45):6145–6157 Stavridi ES, Chehab NH, Malikzay A, Halazonetis TD (2001) Substitutions that compromise the ionizing radiation-induced association of p53 with 14–3-3 proteins also compromise the ability of p53 to induce cell cycle arrest. Cancer Res 61(19):7030–7033 Stewart ZA, Pietenpol JA (2001) p53 Signaling and cell cycle checkpoints. Chem Res Toxicol 14(3):243–263 Strano S, Dell’Orso S, Di Agostino S, Fontemaggi G, Sacchi A, Blandino G (2007) Mutant p53: an oncogenic transcription factor. Oncogene 26(15):2212–2219 Taylor WR, Stark GR (2001) Regulation of the G2/M transition by p53. Oncogene 20(15):1803–1815 Taylor WR, Agarwal ML, Agarwal A, Stacey DW, Stark GR (1999) p53 inhibits entry into mitosis when DNA synthesis is blocked. Oncogene 18(2):283–295 Teodoro JG, Evans SK, Green MR (2007) Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome. J Mol Med (Berlin, Germany) 85(11):1175–1186 Thompson T, Tovar C, Yang H, Carvajal D, Vu BT, Xu Q, Wahl GM, Heimbrook DC, Vassilev LT (2004) Phosphorylation of p53 on key serines is dispensable for transcriptional activation and apoptosis. J Biol Chem 279(51):53015–53022 Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303(5659):844–848 Vogel C, Kienitz A, Hofmann I, Muller R, Bastians H (2004) Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy. Oncogene 23(41):6845–6853 Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408(6810):307–310 Vousden KH (2000) p53: death star. Cell 103(5):691–694 Vousden KH, Lu X (2002) Live or let die: the cell’s response to p53. Nat Rev Cancer 2(8):594–604 Wang Y, Prives C (1995) Increased and altered DNA binding of human p53 by S and G2/M but not G1 cyclin-dependent kinases. Nature 376(6535):88–91 Wang XW, Zhan Q, Coursen JD, Khan MA, Kontny HU, Yu L, Hollander MC, O’Connor PM, Fornace AJ Jr, Harris CC (1999) GADD45 induction of a G2/M cell cycle checkpoint. Proc Natl Acad Sci USA 96(7):3706–3711 Wang YH, Tsay YG, Tan BC, Lo WY, Lee SC (2003) Identification and characterization of a novel p300-mediated p53 acetylation site, lysine 305. J Biol Chem 278(28):25568–25576 Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD (1998) ATM-dependent activation of p53 involves dephosphorylation and association with 14–3-3 proteins. Nat Genet 19(2):175–178

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Chapter 21

Aging and Cancer: Caretakers and Gatekeepers Diana van Heemst

Abstract The accumulation of macromolecular damage which affects the functioning of cells and tissues is thought to underlie both cancer and aging. Caretaker mechanisms that prevent or slow the accumulation of damage are pivotal for maintenance of genome stability and will provide protection against cancer as well as against aging. However, due to evolved limitations in the efficacy of the mechanisms for maintenance and repair, damage will accumulate. Damaged cells may be eliminated from the proliferative pool by either of two gatekeeper mechanisms, induction of apoptosis or induction of cellular senescence. While these mechanisms will provide protection against cancer by inhibiting the proliferation of damaged and potentially (pre)malignant cells, the induction of apoptosis and/or senescence can also exert proaging effects, depending on the magnitude of the responses and the regeneration capacity of the damaged tissue. In elderly individuals, apoptosis and senescence may lead to exhaustion and reduced functionality of stem cell compartments, resulting in impaired tissue regeneration. Moreover, with age, senescent cells may occur in sufficiently high numbers to functionally alter tissue microenvironment, thus increasing the risk of several age-related pathologies, including late life cancer.

21.1 21.1.1

Introduction Aging and Associated Diseases Explained by Evolutionary Biology

The process of aging can best be defined as an increasing risk of death with advancing age. Mechanistically, aging is caused by the continuous accumulation of molecular damage over the lifetime of an individual, a process that may start already in utero.

D. van Heemst Leiden University Medical Center, Department of Gerontology and Geriatrics, PO Box 9600, 2300 RC, Leiden, The Netherlands e-mail: [email protected]

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_21, © Springer-Verlag Berlin Heidelberg 2010

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Numerous agents can lead to persistent damage, including reactive oxygen species, which are produced as byproducts of cellular metabolism. Persistent damage will interfere with the functionality of the different types of vital macromolecules, including DNA, proteins, and lipids, which may disrupt the integrity of cells and tissues and will thus affect the whole organism. To deal with the many different types of macromolecular damage, a multitude of systems for maintenance and repair has evolved to counteract the continuing accumulation of damage. But why do we age? Why has the multitude of systems for maintenance and repair not become sophisticated enough not only to put a brake on the process of accumulation of damage, but also to actually prevent the accumulation of damage from occurring? A glimpse of this possibility is offered by examples of means to push the brake harder. Dramatic differences exist in lifespan between species, being only a few weeks in the roundworm Caenorabditis elegans, a few years in mice, but many decades in humans. But dramatic differences in lifespan can also be observed within species. Most notably, queen honey bees have a 100-fold longer lifespan than nonqueen honey bees (Corona et al. 2005). Over the last decade, a multitude of single gene mutations have been shown to be able to increase C. elegans’ lifespan several-fold (Kenyon 2005). These examples all illustrate that genes and pathways do exist that can slow the rate of aging. But why have these not been positively selected for? Why does a roundworm live approximately two weeks, under standard laboratory conditions, and not several months? Why is it so rare for humans to become over 100 years old? The answer to this question comes from different theories based on evolutionary biology. In their natural habitat, most individuals of a given population will have little chance to survive above a certain age, which is determined by the chance to die from extrinsic causes, including infectious agents, temperature changes, fluctuating food supplies, predators, and risk of accidents. Because, in a natural habitat, the proportion of the population that survives will decrease with advancing age due to death from extrinsic causes, the selection pressure on traits that would confer a late life survival advantage, or against traits that would confer a late life survival disadvantage, will also diminish with age. As stated by Medawar, late life survival occurs in a selection shadow (Medawar 1952). Based on the fact that the selection pressure on late life survival diminishes with age, the mutation accumulation theory states that mutations that confer a late life survival disadvantage will be subjected to very little negative selection and may therefore accumulate (Medawar 1952). The theory of antagonistic pleiotropy hypothesizes that traits that confer a late life survival disadvantage may actually be positively selected for if these same traits will confer an advantage for higher fertility or increased survival at young ages (Williams 1957). Building further on these ideas, the disposable soma theory postulates that, for optimal exploitation of limited metabolic resources, investments in maintenance and repair will need to be balanced with investments in growth and reproduction (Kirkwood and Holliday 1979). Optimal fitness will require a different balance between these investments when extrinsic mortality is high, requiring investments in fast development and timely reproduction, and less in maintenance and repair, than under conditions of low extrinsic mortality, when

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development and reproduction will be slower and the investment in maintenance and repair will be higher. In line with the disposable soma theory, long-lived organisms, as well as the cells derived from these organisms, are often more stress-resistant (Kapahi et al. 1999; Murakami et al. 2003). However, while it will be selectively advantageous as an energy-saving strategy to reduce investments in the maintenance and repair of somatic cells to accelerate development and reproduction, the consequences of these limited investments in maintenance and repair will become dramatically apparent when extrinsic mortality risks suddenly diminish, such as is the case for animals upon domestication, and for humans in modern society.

21.2 21.2.1

History Caretakers and Gatekeepers

In contrast to simple model organisms, such as the roundworm C. elegans and the fruit fly Drosophila melanogaster, whose tissues mainly consist of postmitotic cells, organisms with renewable tissues, including humans, can use stem cells to regenerate tissues by replacing dead, worn-out or damaged cells with new ones. However, while tissue regeneration greatly extended the possibilities for maintenance and repair, it also brought with it a new danger: cancer. During the neoplastic process, a normal cell is transformed into a cancer cell (Hanahan and Weinberg 2000). Tumor suppressor genes encode for proteins that inhibit cell transformation and whose inactivation is advantageous for tumor cell growth and survival. Tumor suppressor functions can be separated into two major categories: gatekeepers and caretakers (Kinzler and Vogelstein 1997). Gatekeepers inhibit tumor growth or promote tumor death. Inactivation of gatekeeper genes directly contributes to cancer formation and progression. In contrast to gatekeepers, caretakers do not directly regulate proliferation, but are required for the maintenance of genome integrity. Inactivation of a caretaker gene will lead to accelerated neoplastic cell transformation by increasing genetic instability, thus enhancing the chances of tumor initiation by inactivation of a gatekeeper gene. Recently, using genomic data mining to compare the features of human caretakers and gatekeepers and their orthologs across species of different biological complexity, a significant phylogenetic rise was observed in gatekeeper ortholog frequency, but not in caretaker ortholog frequency (Zhao and Epstein 2008). These findings suggest that increases in biological complexity may depend more upon gatekeeper than caretaker gene number, supporting the idea that, as multicellularity evolved, stopping the proliferation of potentially tumorigenic cells became increasingly important. Cell growth and differentiation are tightly regulated by complicated interactions between growth-promoting and growth-inhibiting signals. Regulation of proliferation is strongly correlated with regulation of the cell cycle. In mammalian cells, binding of a growth factor to its membrane receptor induces a cascade of intracellular

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signals resulting in cellular growth and cell cycle progression, followed by cell division. For example, upon binding of the insulin-like growth factor-1 (IGF-1) ligand to its receptor (IGF-1R), receptor activation initiates a phosphorylation cascade. The downstream targets of the phosphorylation cascade, which acts through phosphatidylinositol 3-kinase (PI3K) and the protein kinase AKT, include the mammalian target of rapamycin (mTOR), resulting in cellular growth, and the class O forkhead box transcription factors (FOXOs), resulting in cell cycle progression (Kenyon 2005). In response to a wide variety of damage or stress signals, any of a series of cell cycle checkpoints can be activated to transiently halt the cell cycle to allow time for repair and prevent cells with unrepaired lesions from starting DNA replication (the G1/S checkpoint), from progressing with replication (the intra S checkpoint) or from going into mitosis (the G2/M checkpoint) (Zhou and Elledge 2000). In case of persistent damage, cells with mutagenic lesions can be eliminated from the proliferating pool by either of two mechanisms, induction of permanent cell cycle arrest (cellular senescence) or induction of cell death (apoptosis). Tumor suppressor genes play an important role in the cellular responses to damage, with caretakers primarily playing a role in transient cell cycle arrest and DNA repair, and gatekeepers primarily playing a role in the induction of apoptosis and/or cellular senescence. Which type of cellular response to damage will occur will depend on many different factors, including cell type, stage of the cell cycle, location of the damage in the genome and the type and intensity of the stress or damage. For example, with respect to cell type, damaged fibroblasts and epithelial cells have a stronger tendency to undergo senescence, while damaged lymphocytes have a stronger tendency to undergo apoptosis. With respect to type of lesion, two fundamentally different types of lesions exist: mutagenic lesions, for example the oxidative lesion 8-oxodG, which may cause small point mutations if not properly repaired, and cytotoxic lesions, for example DNA double-stranded breaks (DSBs), which may lead to large-scale genomic rearrangements if not properly repaired. Because DSBs will hamper proper genome duplication, as well as an even partitioning of chromatids between daughter cells, DSBs are much more cytotoxic and their persistence is more likely to induce apoptosis or senescence than are small oxidative lesions, such as 8-oxodG, which are mainly mutagenic, as their persistence puts cells at a higher risk of acquiring mutations (Hoeijmakers 2007).

21.3 21.3.1

Current Status Caretakers

Cancer is predominantly a disease of the elderly (Campisi 2000). Most tumors arise in the last quarter of life, with their frequency increasing exponentially with time. It is thought that the increase in cancer incidence with age is due, in part, to the accumulation of somatic DNA mutations over an individual’s lifetime.

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When occurring in oncogenes and tumor suppressor genes, these mutations will put cells at a higher risk of neoplastic transformation (Vogelstein and Kinzler 2004). A variety of mechanisms can result in the activation of oncogenes or inactivation of tumor suppressor genes, including intragenic mutations, chromosomal deletions, and loss of expression. The rate of mutation accumulation with age is estimated to be relatively slow, being 1 x 10−4 point mutations per gene per (mouse liver) cell, even in old age (Dolle et al. 1997). However, large-scale genomic rearrangements will occur roughly as often as point mutations, and these can potentially simultaneously alter the expression of many genes. Recently, it was shown that the cell-to-cell variability in gene expression increases with age, leading to genomic drift and tissue mosaics, illustrating that genome instability increases with aging (Bahar et al. 2006). Caretaker mechanisms play a pivotal role in the maintenance of genome stability. Mutations can arise spontaneously, due to intrinsic flaws in the fidelity of processes related to primary DNA metabolism, such as DNA replication. However, chemical damage to DNA may also become fixed into mutations. Damage to DNA can be caused by spontaneous hydrolysis and by diverse chemical agents, including reactive oxygen species (ROS), which are produced as byproducts of cellular metabolism. DNA damage may also arise from different environmental exposures, including exposure to UV irradiation during sunbathing. Mechanisms that increase the efficiency of energy consumption, which will lower the generation of ROS, will reduce the rate of accumulation of damage. Moreover, to avoid any of the many types of oxidative damage to vital macromolecules, different antioxidant defense systems have evolved, which include enzymatic as well as nonenzymatic ROS scavengers (Hoeijmakers 2001b). Evidence is accumulating that the evolutionarily conserved life-prolonging effects of calorie restriction, as well as those of mutations down-regulating the insulin/IGF-1 pathway, can be attributed, at least in part, to a lower generation of ROS-induced damage (Kenyon 2005). However, despite the numerous strategies to avoid damage, lesions of many different kinds will arise in DNA, and an intricate network of several DNA repair pathways exists to deal with the different sorts of lesions (Hoeijmakers 2001b). Most DNA repair pathways involve detection of the lesion, followed by the nucleolytic resection of the (region containing the) damaged DNA, whereafter DNA polymerization will replace the dissected DNA with new DNA, and ligation will restore the integrity of the phosphodiester backbone. In the case where a lesion occurs on only one of the two strands of the DNA double helix, the complementary DNA strand will be used as a template for repair, such as in the case of base excision repair (BER), nucleotide excision repair (NER) or mismatch repair (MMR). Single-stranded lesions are preferentially repaired by BER in the case of small lesions, such as abasic sites, and by NER in the case where lesions are more bulky and lead to a distortion of the DNA double helix, such as UV-induced 4,6 photoproducts, whereas MMR corrects mispaired bases. In the case of a DNA double-stranded break (DSB), the processed ends can either be religated by nonhomologous endjoining (NHEJ), which can be very precise in the case of blunt, 5¢ phosphorylated ends (van Heemst et al. 2004), but may lead to loss of a few nucleotides around the damaged site in the case of

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staggered ends (van Gent et al. 2001). Alternatively, the resected ends may invade another homologous sequence elsewhere in the genome, preferentially the identical sister chromatid (van Heemst and Heyting 2000), to repair the double-stranded damage by homologous recombination (van Gent et al. 2001). However, besides giving rise to mutations, persistent DNA damage may also physically obstruct essential processes, including DNA replication and transcription. Transcriptioncoupled repair (TCR) specifically removes cytotoxic lesions that interfere with transcription (Ljungman and Lane 2004). The biological significance of DNA repair mechanisms is underscored by their conservation throughout evolution and by the phenotypes of patients with inherited defects in DNA repair genes. Several human cancer-prone syndromes exist that are associated with inborn defects in different DNA repair pathways. Such syndromes include xeroderma pigmentosum (XP) which is associated with defects in NER (de Boer and Hoeijmakers 2000), hereditary nonpolyposis colorectal cancer (HNPCC) which is associated with defects in MMR (Leach et al. 1993), and Nijmegen breakage syndrome (NBS) and ataxia–telangiectasia (AT) which are associated with defects in the complex responses to and repair of DSBs (Savitsky et al. 1995; Varon et al. 1998). The cancer-prone phenotypes of patients with these syndromes collectively support the hypothesis that if DNA repair fails, genetic instability will increase and may put cells at a higher risk of neoplastic transformation. Besides predisposition to cancer, another phenotype that can be associated with defects in caretaker genes is premature aging. Human patients with defects in NER can present with strikingly different clinical phenotypes that have been precisely reproduced in various mutant mouse models. These phenotypes range from an over 1000-fold increased risk of developing UV-induced skin cancer in xeroderma pigmentosum (XP) to severe retardation in growth and development, together with segmented accelerated aging phenotypes in the cases of Cockayne syndrome (CS) and trichothiodystrophy (TTD) (Hoeijmakers 2001a). XP patients and corresponding mouse models show defects in global genome NER (GG-NER), causing a random accumulation of mutagenic lesions (Dolle et al. 2006), which may explain for their cancer-prone phenotype (Fig. 21.1). In contrast, specific defects in the TCR of cytotoxic lesions in actively transcribed genes may lead to the excessive induction of apoptosis and/or senescence (Fig. 21.1), which may explain why mutagenic lesions do not accumulate in the CS and TTD syndromes (Dolle et al. 2006). It has been speculated that excessive induction of apoptosis and/or senescence in response to persistent cytotoxic lesions may explain the absence of cancer as well as the presence of the premature age-related pathologies observed in CS and TTD patients and corresponding mouse models (de Boer et al. 2002; van der Pluijm et al. 2007).

21.3.2

The Gatekeeper p53

One of the factors that is key in determining the cell fate decision between transient cell cycle arrest, permanent cell cycle arrest, and apoptosis is the 53 kDa

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various stresses e.g ROS, UV exposure

repair, e.g. GG-NER

repair, e.g. TCR

mutagenic lesions

cytotoxic lesions

genome instability

apoptosis and/or cellular senescence

cancer

premature age-related pathologies

Fig. 21.1 Phenotypes associated with persistence of mutagenic and cytotoxic lesions. Various stresses (e.g., ROS and UV irradiation) can inflict damage to DNA and an intricate network of several DNA repair pathways exists to deal with the different sorts of lesions, including global genome nucleotide excision repair (GG-NER) and transcription coupled repair (TCR). GG-NER preferentially repairs mutagenic lesions, the persistence of which will put cells at a higher risk of acquiring mutations, leading to genome instability and a primarily cancer-prone phenotype. TCR repairs cytotoxic lesions, the persistence of which will interfere with transcription, which will put cells at a higher risk of being eliminated from the proliferative pool through induction of apoptosis or cellular senescence, and which is associated with the occurrence of (premature) age-related pathologies

transcription factor p53 (Levine 1997). The human gene encoding p53 (TP53) is the best described gatekeeper tumor suppressor gene and is mutated in about half of all human cancers (Levine 1997). Over the past decades, various different p53 mutant mouse models have been created that have helped disentangle the role of p53 in cancer and aging (Attardi and Donehower 2005). Deletion of the mouse gene encoding p53 (p53) shortens lifespan by dramatically increasing cancer incidence (Donehower et al. 1992). Although over-expression of different forms of p53 in mice does decrease cancer risk, it does not automatically increase lifespan. In contrast, in both p53+/m (Tyner et al. 2002) and p44Tg mice (Maier et al. 2004), which contain transgenes encoding for a shorter, truncated form of p53, increased tumor suppression occurs at the expense of shorter lifespan. However, when a transgene is introduced that encodes for full-length p53, such as in the case of super-p53 mice (Garcia-Cao et al. 2002), lifespan is not shortened, but normal. Moreover, in super-Arf/p53 mice, in which the extra gene dose of p53 is accompanied by an extra dose of the gene encoding for the Alternative Reading Frame protein ARF, lifespan is actually increased (Matheu et al. 2007). The differences

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in lifespan between these different p53 mouse models may be explained by differences in the regulation of the activity of p53. In contrast to expression of the shorter forms of p53 in the p53+/m and p44Tg mice, expression of the extra copy of p53 in the super-p53 mice occurs via normal regulatory sequences present on the introduced transgene. Moreover, the different mouse models contain a different ratio of p53 isoforms. The N-terminal transactivator region, which is lacking in the truncated isoforms, is required for the interaction of p53 with the ubiquitin ligase Mouse Double Minute 2 homolog (MDM2), providing a means for MDM2 to inactivate p53 in the absence of stress signals. The constitutively activated short isoforms expressed in the p53+/m and p44Tg mice are thought to deregulate p53 activity. But why would lifespan be shortened in p53+/m and p44Tg mice, but not in super-p53, and be prolonged in super-Arf/p53 mice? It has been suggested that in mice carrying an extra copy of both p53 and Arf, p53 activity is modulated such that p53 will preferentially promote transient cell cycle arrest and DNA repair in response to low, chronic stress (Matheu et al. 2007; Vousden and Lane 2007; Serrano and Blasco 2007). In contrast, in p53+/m and p44Tg mice, p53 activity might be modulated such that p53 will preferentially promote induction of apoptosis and/or senescence in response to low, chronic stress. In p53+/m and p44Tg mice, p53 transcriptional activity might be modulated in a promoter-specific fashion, leading to a preferential activation of targets implicated in cell cycle arrest (Ungewitter and Scrable 2008). Full-length p53 has been described to suppress IGF-1 signaling at different levels, most notably by blocking IGF-1R transcription (Werner et al. 1996). However, in contrast to over-expression of full-length p53, over-expression of the p44 isoform lacks this property, resulting instead in (hyper)activation of IGF-1 signaling, as evidenced by the constitutively high levels of phosphorylated AKT, FOXO and mTOR (Maier et al. 2004; Scrable et al. 2008). It has been speculated that sustained IGF-1R activation by p44 will lead to sustained activation of the extracellular signal-regulated kinase (ERK) subgroup of mitogen-activated protein kinase (MAPK) cascades, which can override proliferative AKT signaling and induce cell cycle arrest via upregulation of p21 (Roovers and Assoian 2000). The differences in size between full-length p53 and truncated (iso)forms may interfere with patterns of posttranscriptional modifications upon induction of stress, which could explain the differential loss of IGF-1R transrepression and gain of p21 transactivation in p53+/m and p44Tg mice (Ungewitter and Scrable 2008). Taken together, the mutant NER and p53 mouse models strongly support the notion that both the accumulation of cytotoxic lesions, as well as changes in the activity of p53, may cause accelerated aging due to excessive induction of apoptosis and/or cellular senescence. Is it likely that these same mechanisms also play a role in the pathologies and degenerative diseases commonly observed in humans at advanced ages? As discussed before, DNA damage does accumulate with age, and will include, besides mutagenic lesions, cytotoxic lesions that will interfere with primary DNA metabolism, making cells more prone to trigger gatekeeper tumor suppression mechanisms (Hoeijmakers 2007). Although evidence is still largely circumstantial, there are indications that p53 activity may also become deregulated with age. The p53 tumor

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suppressor protein has long been considered the only protein expressed by the TP53 gene. Recently, several groups have identified and described the existence of up to ten isoforms of human p53 and have demonstrated that these isoforms play a role in the modulation of p53 activity (Bourdon 2007). The different p53 isoforms have the potential to act either synergistically or antagonistically, depending on their structure and mechanism of production. Interestingly, a truncated form of p53 was recently found to accumulate with age in human peripheral blood mononuclear cells (PBMCs), suggesting a possible means for a change in p53 activity with age in humans (Lanni et al. 2008). Moreover, the activity of the protein deacetylase sirtuin-1 (SIRT1), which may mediate many of the life-prolonging effects induced by calorie restriction through deacetylation of various target proteins including FOXO and p53, also declines with age in mammalian tissues (Sasaki et al. 2006), suggesting another possible means by which p53 activity may change with age. These emerging data raise the possibility that cells from elderly individuals may respond differently to damage than cells from younger subjects, but leave open the question as to how the differential responses of cells to damage and stress may be associated with age-related pathologies.

21.4 21.4.1

Applications Apoptosis

In the case of persistent damage, cells with mutagenic lesions can be eliminated from the proliferating pool by either of two mechanisms, induction of permanent cell cycle arrest (cellular senescence) or induction of apoptosis. Apoptosis involves the process of programmed cell death, through the orderly, caspasemediated proteolytic destruction of cellular contents, leading to cellular (and nuclear) fragmentation. At the final stage of apoptosis, the resulting fragments (apoptotic bodies) are phagocytosed by neighboring macrophages and granulocytes. In mammalian cells, apoptosis can be induced by intrinsic or extrinsic pathways (Fig. 21.2) that converge at the point of mitochondrial cytochrome c release, which induces mitochondrial membrane permeability changes as well as further activation of the caspase cascade (Green and Reed 1998). In the intrinsic pathway, cytochrome c release is triggered by a surplus of proapoptotic over antiapoptotic proteins. The proapoptotic proteins, most notably the Bcl2-associated x protein (Bax) (Chipuk et al. 2004), are produced in response to stress and damage signals coming from inside the cell, in a response that often (although not necessarily) involves p53 activation (Fridman and Lowe 2003). In the extrinsic pathway, cytochrome c release is triggered by cell death receptor mediated activation of the caspase cascade and subsequent cleavage of the BH3 interacting domain death agonist (Bid), in response to stress and damage signals coming from outside the cell, including the Fas ligand and TNF-a (Green and Reed 1998).

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extrinsic pathway

intrinsic pathway

e.g Fas, TNF-α death receptors

e.g DNA damage, ROS

caspases 8, 10

p53

tBid

Bax,Bad/Bcl-2,Bcl-xl

cytochrome c release

caspase 9

caspases 3,6,8

apoptosis Fig. 21.2 Extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway of apoptosis is activated by death receptors. Ligands, e.g., Fas or TNF-a, bind to their cognate receptors, leading to the formation of a death-inducing signaling complex, with caspase activation, production of tBid (from Bid cleavage) and subsequent mitochondrial cytochrome c release. The intrinsic pathway of apoptosis can be initiated by various intracellular stressors (e.g., DNA damage, ROS) that can activate the p53 cascade, but also other stress cascades. Activation of stress cascades leads to a surplus of proapoptotic proteins (e.g., Bax, Bad) over antiapoptotic proteins (e.g., Bcl-xL, Bcl2), causing release of cytochrome c from the mitochondrial membrane. Both pathways activate the same effector caspases that execute the final common pathway of apoptosis

In most human and rodent tissues, age-related gene expression changes have been observed that are consistent with an enhanced expression of proapoptotic genes and/or a decreased expression of antiapoptotic genes (Horton et al. 1998; Joaquin and Gollapudi 2001; Kinkel et al. 2004; Liu et al. 1998; Savory et al. 1999). These gene expression changes may occur in response to the age-related increase in damage load as well as the functional decline in repair. However, when cells were compared that derived from various tissues from young and old mice exposed to high doses of ionizing irradiation (IR), an age-related decline was observed in the apoptotic response mediated by the pathway consisting of the protein encoded by the ataxia–telangiectasia mutated gene (ATM), p53 and p21 (Feng et al. 2007). A similar reduction in mean apoptotic response with age has also been observed for human cells (Camplejohn et al. 2003). The induction of apoptosis in (pre)malignant cells will prevent these cells from proliferating and spreading, thus constituting an important tumor suppression

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mechanism. The effects of apoptosis on tissue homeostasis can however be twofold. First, elimination of division-competent stem cells through apoptosis can disrupt the delicate balance between cell loss and cell renewal, leading to an exhaustion of regenerative capacity. The diverse models of accelerated aging mice discussed above demonstrate that exaggerated cell loss may indeed contribute to premature aging phenotypes. For example, the accelerated aging p53 mouse models display an increased tissue atrophy (Tyner et al. 2002) and a reduced capacity for repair and regeneration (Dumble et al. 2007; Medrano et al. 2007). In these models, it is not always clear whether the disturbed balance between cell loss and cell renewal is caused by apoptosis or by cellular senescence or by a combination of these two processes. However, based on genome-wide expression analyses, gene expression changes have been observed in TTD mice that are consistent with an enhanced (cytotoxic stress-induced) rate of apoptosis (Park et al. 2008). Likewise, higher rates of apoptosis have been observed for hematopoietic stem cells derived from the premature aging p53+/m mice (Dumble et al. 2007). In line with these results, a decrease in the proliferative capacity of stem cells caused by apoptosis has been tentatively linked to diverse human age-related pathologies, including sarcopenia (Phillips and Leeuwenburgh 2005), myocardiopathy (Higami and Shimokawa 2000), Alzheimer’s disease (Higami and Shimokawa 2000), as well as the general decline in immune function observed upon aging (Ginaldi et al. 2000). However, the converse scenario can also be envisioned. If stem cells with mutations persist due to less efficient apoptosis, these mutations might impair stem cell functionality (Van Zant and Liang 2003). Likewise, if in a target tissue, or elsewhere in the organism, malfunctioning cells persist with mutations due to less efficient apoptosis, these might also interfere with stem cell functionality. Such malfunctioning cells might either inhibit or over-stimulate stem cell proliferation, both of which could lead to a premature exhaustion of regenerative capacity, or these cells might interfere with the homing of circulating stem cells or the activation of resident stem cells. All of these effects could be mediated by changes in the local microenvironment as well as by changes in the circulatory system to which the stem cells and target tissues are exposed. In this second scenario, the persistence of cells with mutations due to less efficient apoptosis could actually have a negative impact on the maintenance of tissue homeostasis. In elegant experiments, in which the circulatory systems of old and young mice were connected, circulating substances produced by old mice were found to negatively influence the regenerative capacity of stem cells from young mice, while exposure to blood of young mice positively influenced the regenerative capacity of stem cells from old mice (Conboy et al. 2005).

21.4.2

Cellular Senescence

Cellular senescence is a state of irreversible cell cycle arrest. Cellular senescence was originally described by Hayflick as the cell culture phenotype of humans fibroblasts that undergo replicative exhaustion after approximately 50 cumulative

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population doublings (Hayflick and Moorhead 1961). Over the last decade, it has become increasingly clear that cellular senescence can be triggered in many different cell types by a variety of stresses, including dysfunctional telomeres, DNA damage, chromatin perturbations, and hypermitogenic signaling (Campisi and di Fagagna 2007). Cellular senescence can be induced by the p16/RB and ARF/p53 pathways (Fig. 21.3). The p16/RB and ARF/p53 tumor suppressor pathways interact, but can also act in parallel; their relative contributions are dependent on diverse factors, including species, cell type and severity and type of stress. P16 is a Cyclin Dependent Kinase (CDK) inhibitor that can block G1/S progression via its ability to bind to and inhibit the cyclin D dependent kinases CDK4 and CDK6, thus preventing phosphorylation of the retinoblastoma family of tumor suppressors (RB, p107 and p130) and allowing these to silence proliferationassociated genes via promotion of a repressive heterochromatin environment at E2F target gene promoters (Narita et al. 2003). P53 is normally inactive, partly as a result of its binding to and rapid degradation by the ubiquitin ligase Mouse Double Minute 2 homolog (MDM2; HDM2 in humans). The Alternative Reading Frame protein ARF (p19Arf in mice and p14Arf in humans) binds to and inhibits

various stresses e.g dysfunctional telomeres, DNA damage, hypermitogenic signaling, chromatin perturbations

p16

ARF

CDK4/6, cyclin D

MDM2

RB

p53

E2F

p21

cellular senescence Fig. 21.3 P16/RB and ARF/p53 pathways of cellular senescence. Senescence can be induced through various sorts of stress which engage the p16/RB or the ARF/p53 pathways. P16 binds to cyclin D-dependent kinases CDK4 and CDK6, thus inhibiting CDK4/6-mediated phosphorylation (and inactivation) of RB. Hypophosphorylated RB inhibits the E2F family of transcription factors and thus prevents G1/S phase progression. P53 is negatively regulated by the ubiquitin protein ligase MDM2, and MDM2 is negatively regulated by ARF. P53 activates the p21 member of the CIP-KIP family of cyclin-dependent kinase inhibitors. Both pathways lead to induction of cell cycle arrest and cellular senescence

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MDM2, thereby stabilizing p53 (Weber et al. 1999). Subsequently, p53 transactivation will lead to inhibition of proliferation via pathways including the CDK inhibitor p21 (Marx 1993). Senescence is a potent tumor suppression mechanism, which can limit the proliferative potential of premalignant cells (Campisi and d’Adda di Fagagna 2007) as well as malignant cells (Xue et al. 2007). Moreover, the senescence program may also provide protection against other diseases. Recently, cellular senescence was shown to limit the fibrogenic response to acute tissue damage (Krizhanovsky et al. 2008). In a mouse model for liver fibrosis, damage activates hepatic stellate cells to proliferate and produce the network of extracellular matrix that is the hallmark of the fibrotic scar. Induction of senescence in activated stellate cells was shown not only to put a halt to their proliferation but also to the deposition of extracellular matrix. Gene expression profiling revealed that senescent cells showed a reduced expression of extracellular matrix components, as well as an enhanced expression of genes implicated in the degradation of extracellular matrix. Moreover, senescent cells adopt a secretory phenotype, with enhanced expression of immune modulators, such as proinflammatory cytokines and receptors that enhance immune surveillance by potentiating natural killer cell function. In line with the gene expression data, natural killer cells were shown, both in vitro and in vivo, to preferentially kill senescence activated stellate cells, thereby facilitating the resolution of fibrosis (Krizhanovsky et al. 2008). Likewise, induction of senescence in murine liver carcinoma cells was shown to enhance tumor regression, which was associated with an upregulation of inflammatory cytokines and clearance of senescent cells by the innate immune system (Xue et al. 2007). Upon induction of cellular senescence, a cell undergoes many morphological changes, such as an enlarged flattened cell morphology, as well as several functional changes, such as the accumulation of senescence-associated b-galactosidase activity (Campisi and d’Adda di Fagagna 2007). Although most of the changes induced upon senescence are beneficial, especially in the short run, in the long run some of these changes can also have a negative impact on tissue function and integrity, especially if senescent cells accumulate in sufficiently high numbers. In line with this hypothesis, it has been found that the number of senescent cells does indeed accumulate with age and that senescent cells are present at sites of age-related pathologies (Krishnamurthy et al. 2004; Paradis et al. 2001; Vasile et al. 2001). Both p16 and ARF are effectors of senescence in cultured cells (Kim and Sharpless 2006), and their levels increase with age in many rodent and human tissues (Krishnamurthy et al. 2004; Zindy et al. 1997). Interestingly, it was recently shown that, in contrast to p16, ARF has anti-aging effects in premature aging (BudR1) mice (Baker et al. 2008). Loss of p16 delayed the in vivo accumulation of senescent cells and (premature) aging, while loss of ARF accelerated the in vivo accumulation of senescent cells and (premature) aging in this mouse strain, providing evidence for a causal relation between in vivo induction of senescence and (premature) aging. In mice, an age-dependent rise is observed in p16-positive stem cells in stem cell compartments of the brain, pancreas and hematopoietic system, and it has been suggested that one way in which senescence may contribute to aging is by decreasing the pool of proliferation-competent stem cells (Kim and Sharpless 2006).

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However, besides cell cycle arrest, other changes associated with senescence may contribute to aging and age-related diseases. Senescence can be induced by hypermitogenic signaling, which blocks cell cycle progression, but not cell growth (Chen et al. 2005). Hypermitogenic stimulation in combination with cell cycle arrest may actually be responsible for the enlarged morphology often displayed by senescent cells (Blagosklonny 2006). The hyperactivation of mitogenic signaling pathways (such as AKT/mTOR) may also cause senescent cells to become growth factor-resistant (through feedback inhibition), and may cause these to secrete growth factors in a futile attempt to overcome the cell cycle blockage (Blagosklonny 2006). The growth factors secreted by senescent cells may stimulate premalignant cells to form tumors. Senescent cells were shown to induce preneoplastic characteristics in neighboring cells or even distal cells within tissues (Krtolica and Campisi 2002). In vivo, a precancerous cell may thus be pushed into full-blown malignancy by a senescent, mitogen-secreting neighbor. It is not known why senescent cells accumulate with age. The accumulation of senescent cells in aged tissues may involve different mechanisms, including enhanced induction through the accumulation of damage or altered p53 activity, as well as functional changes induced by the senescence program, such as resistance to clearance by apoptosis (Marcotte et al. 2004; Seluanov et al. 2001). Senescent cells can be cleared by the immune system, and the accumulation of senescent cells may also be due, in part, to an age-associated decline in immune function (Woodland and Blackman 2006). Possibly, different states of senescence may exist that may or may not be induced by specific stressors. Besides the detection of senescence-associated b-galactosidase activity (Dimri et al. 1995) and the protein marker p16 (Krishnamurthy et al. 2004), new markers have recently emerged, and their rigorous testing may allow to discriminate between potential different states of senescence. Such new senescence markers include the protein markers differentiated embryo-chondrocyte expressed-1 (DEC1), p15 (a CDK inhibitor) and decoy death receptor-2 (DCR2), as well as the cytological markers senescence-associated heterochromatin foci (SAHF) and senescence-associated DNA damage foci (SDFs) (Campisi and d’Adda di Fagagna 2007). If different states of senescence indeed exist and are proven to be biologically relevant, it is an intriguing possibility that these may differ in their propensity to accumulate, for example by differential clearance by apoptosis or the immune system. Such different states of senescence may also differ in their biological relevance for aging or specific age-related diseases, for example late life cancer, as some states might be associated with the secretion of specific growth factors, whereas others are not.

21.5

Future Directions

Although available evidence is scarce, some subtle hints suggest that the relations that have been observed in model organisms between induction of gatekeeper tumor suppression mechanisms and age-related phenotypes and diseases may also exist in

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humans. A first hint comes from the observation that long-term survivors of chemotherapy and radiotherapy do show evidence of premature age-related phenotypes (Beeharry and Broccoli 2005). Secondly, centenarians seem to display a lower than expected incidence of cancer, as well as a decline in metastatic rate and a decreased mortality due to cancer (Caruso et al. 2004), and although the underlying mechanisms have not yet been revealed, it is noteworthy that centenarians posses a well-maintained natural killer cell activity (Bonafe et al. 2002). Thirdly, in human subjects, a relation was observed between Pro/Pro carriership of a common codon 72 polymorphism in the human TP53 gene (Weston et al. 1994) and proportionally increased late life cancer mortality (van Heemst et al. 2005). Subsequently, compared to fibroblasts derived from 90-year-old Arg/Arg subjects, fibroblasts derived from 90-year-old Pro/ Pro subjects were found to give rise to a higher dose-dependent increase in the percentages of cells positive for senescence-associated b-galactosidase activity at three days after ionizing irradiation (IR), as well as in the percentages of cells displaying signs of genomic instability, including those containing micronuclei (den Reijer et al. 2008). These results raise the possibility that a relation might also exist in humans between a higher propensity to induction of cellular senescence and/or a higher accumulation of persistent senescent cells and late life cancer risk. However, clearly more research is required to address this important issue as well as other issues, of which some are briefly mentioned below. The different models discussed in this chapter indicate that a relation might exist between the induction of apoptosis and/or cellular senescence and the occurrence of age-related diseases, with net effects depending on the magnitude of the responses and the regenerative capacity of the damaged tissue. However, most evidence for a direct relation between the induction of apoptosis and/or senescence and the occurrence of age-related diseases remains circumstantial, and many important questions remain to be answered. How do cells decide whether to opt for transient cell cycle arrest with stimulation of DNA repair activities, for cellular senescence or for apotosis, and which are the major signals, signal transduction pathways or components implicated in this decision? What exactly is the relative contribution of apoptosis and senescence to the proaging effects observed in different models, tissues and cells, and how do these relate to the many age-related pathologies commonly observed in humans at advanced age?

21.6

Conclusion

It has been argued that some of the processes or pathways implicated in tumor suppression may have negative late life effects on the occurrence of age-related diseases. Caretaker tumor suppression mechanisms that prevent mutations from occurring or persisting through more efficient global genome maintenance and repair are thought be at the root of the slowing of the aging process and enhancement of longevity as well as providing protection against cancer. However, it has been argued that the gatekeeper tumor suppression mechanisms, apoptosis and

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cellular senescence, that occur in response to persistent damage may actually have negative late life effects on the occurrence of age-related diseases. There is indeed evidence that apoptosis and cellular senescence may have negative late life effects related to the exhaustion of division-competent stem cells. Moreover, while clearly beneficial for tumor suppression (and thus for lifespan) in the short run, induction of cellular senescence may have additional negative late life consequences on the development of age-related diseases, which are likely related to the hypermitogenic and secretory phenotype of senescent cells, and which may include, paradoxically, the development of late life cancer.

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Chapter 22

Signal Transduction in Embryonic Stem Cells and the Rise of iPS Cells Solene Jamet, Ruairi Friel, and P. Joseph Mee

22.1

Mouse ES Cell Signal Transduction

When mouse ES cells were initially isolated they were grown in an empirical combination of a bed of feeder cells, with conditioned media mixed with fetal calf serum. In this complex mixture there are clearly potentially many different signaling events from the various cytokines and growth factors, either in serum or produced from the feeders, that may be necessary to give rise to self-renewal (Smith, 2001). Later cytokine leukemia inhibitory factor (LIF) was found to substitute for the conditioned media and was considered a preeminent signal for self-renewal (Smith, 2001; Burdon et al. 1999; Chambers and Smith 2004). The LIF receptor consists of the LIF-specific receptor subunit (LIFR) and the common signal transducer gp130. Following binding of LIF to its receptor, this complex activates associated Janusassociated (JAK) tyrosine kinases that phosphorylate the receptor chains. The transcription factor Src homology 2 (SH2) domain, containing signal transducer and activator of transcription 3 (STAT3), becomes phosphorylated by the JAKs and this in turn promotes dimerisation of STAT3 (Burdon et al. 2002). Translocation of the STAT3 dimers to the nucleus now occurs, where they bind DNA thus activating genes important in ES self-renewal. Interference in recruitment and activation of STAT3 on engagement of the LIFR has been shown to block self-renewal. Thus mutation of the LIFR such that it can no longer activate STAT3, or by using a constructed STAT3 molecule that can be activated by the drug tamoxifen without the need for receptor stimulation, demonstrates a key role for this pathway. Curiously, though, STAT3 is not unique to ES cells and if STAT3 is the sole key mechanism to P. Joseph Mee (*) Stem Cell Sciences, Meditrina Building 260, Babraham Research Campus, Cambridge, CB22 3AT, United Kingdom e-mail: [email protected] S. Jamet New World Laboratories, 500 Cartier Blvd. West, Laval, Quebec, H7V 5B7, Canada R. Friel Department of Technology Transfer, National University of Ireland, University Road, Galway, Ireland

A. Sitaramayya (ed.), Signal Transduction: Pathways, Mechanisms and Diseases, DOI 10.1007/978-3-642-02112-1_22, © Springer-Verlag Berlin Heidelberg 2010

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control self-renewal why does STAT3 expression in other cell types, such as T-cells, not induce pluripotency? Binding of LIF to gp130 also stimulates the Ras/mitogen-activated protein kinase (MAPK) pathway. Here the protein tyrosine phosphatase SHP-2 is recruited to the receptor where it associates with Gab 1. This promotes the activation of Ras which initiates a cascade of transphosphorylations involving Raf kinase and MAPK kinase (MEK) resulting in the activation of Extracellular signal-receptor kinases (ERKs). ERKs can phosphorylate cytoplasmic proteins and can also be translocated to the nucleus where they can modulate the activities of transcriptional regulators. In ES cells, activation of this pathway on stimulation of the LIFR is a prodifferentiation signal rather than contributing to self-renewal. Thus the addition of MEK inhibitors to growth medium can promote self-renewal by preventing the activation of this pathway leading to enhanced STAT3 signaling. A model for the efficiency of ES cell self-renewal is due to the balance of conflicting signals of STAT3 activation contributing to self-renewal and ERKs signaling pushing the cells to differentiate, both emanating from the LIFR on its activation. A major difficulty in understanding the true context of the signaling pathways involved in ES cell self-renewal is the fact that mouse ES cells are predominately grown in medium containing fetal calf serum. Under these culture conditions serum is an essential component, as without serum ES cells differentiate even in the presence of LIF. More recently the bone morphogenic proteins (BMP) have been shown to substitute for serum in these fully defined systems (Chambers and Smith 2004). BMP is a known antineural factor in embryos. As ES cells grown in these particular serumfree conditions develop spontaneously into neural phenotypes it was thought that an antineurogenic factor may play a role in maintaining the self-renewal phenotype. When ES cells are cultured in the presence of BMP4 or growth differentiation factor 6 (GDF6) serum-free media in conjunction with LIF, they maintain pluripotency. BMPs initiate signaling from the cell surface by interacting with heterodimers of type I and type II serine–threonine receptors. Following binding, cytoplasmic proteins called Smads are activated by phosphorylation, form heterodimers with Smad 4 and are translocated to the nucleus where they inhibit or activate target genes. BMP4 has no direct effect on STAT3 signaling, nor does it inhibit the Extracellular signal-receptor kinases (ERKs) pathway, suggesting that it acts in a parallel pathway to LIF/ STAT3. In undifferentiated ES cells, transcripts for types I and II serine–threonine, BMP4 and GDF6 are found, suggesting the possibility of autocrine signaling. Following BMP4 signaling, Smad 1 is rapidly phosphorylated and it induces the expression of Id (inhibitor of differentiation) genes. Id genes encode negative bHLH (basic helix–loop–helix) factors and these proteins bind to ubiquitous HLH factors, the E proteins. Thus, the Id proteins may act in an antidifferentiation manner by sequestering E proteins which normally partner proneural bHLH factors, such as Mash-1 which is expressed in ES cells. The Id proteins were shown to be the factors directly involved in self-renewal, as expression of Id1, Id2 or Id3 in the absence of BMP caused the cells to grow as well as when cultured with LIF and BMP, and it was also seen that serum induces Id gene expression in ES cells (Friel et al. 2005). (Fig. 22.1) The observation that ES cell self-renewal is best studied in highly defined and serum-free cultures has been further studied and surprisingly it has been shown

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Signal Transduction in Embryonic Stem Cells and the Rise of iPS Cells BMP

LIF MAPK pathway JAK

Typell Ser-Thr Receptor Typel Ser-Thr Receptor

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P

ERK Pro-differentiation SMAD

P STAT3 P STAT3

mesoderm endoderm

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neuroectotoderm

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Fig. 22.1 Schematic diagram of the main signaling pathways involved in ES cell self-renewal. LIF activates both the pro-self-renewal JAK-Stat pathway and the prodifferentiation MAPK pathway. BMP binding to its receptor activates the MAPK pathway and induces expression of Id genes which block neurogenesis. Together LIF and BMP can drive ES cell self-renewal in a serum-free medium. LIF alone is insufficient to drive self-renewal in a serum-free medium as it blocks differentiation of mesoderm and endoderm but only weakly blocks differentiation into neuroectoderm. BMP signaling blocks neurogenesis; however its ability to induce differentiation into other cell types is blocked by LIF/Stat3 signaling. This interplay and balance between the two pathways results in self-renewal

that extrinsic stimuli are in fact dispensable for the derivation, propagation and pluripotency of ES cells. Here self-renewal is enabled by the elimination of differentiation-inducing signaling from MAPK via the use of small-molecule inhibitors SU5402 and PD184352 to inhibit fibroblast growth factor (FGF) receptor tyrosine kinases and the ERK cascade, respectively. This, coupled with the use of the selective glycogen synthase kinase 3 (GSK3) b inhibitor, CHIR99021, is sufficient to allow ES cell self-renewal in the absence of any cytokine signaling. The CHIR99021 acts to enhance cell survival by the relief of GSK3-mediated negative regulation of biosynthetic pathways, thus consolidating biosynthetic capacity and suppressing residual differentiation. Complete bypass of self-renewal signals emanating from the LIFR was confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate program for self-replication that does not require extrinsic instruction through receptor signaling. These studies delineate the minimal requirements for self-renewal and provide a defined platform for further dissection of the nature of pluripotency (Fig. 22.2).

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Self-renewal II Chemical inhibition (SU 5402/PD184352)

FGF FGF BMP

pERK pERK

Growth and and Growth Viability Viability

Commitment

Growth and and Growth Viability Viability

Commitment

GSK3 LIF Chemical inhibition (Chiron 99021)

Fig. 22.2 Models of mouse ES cell self-renewal. ES cells can be maintained in a self-renewing state in defined serum-free culture conditions when LIF and BMP act together to inhibit commitment to differentiate and maintain growth (Self-renewal I). This can be simulated using chemical inhibitors that block ERK signaling whilst maintaining growth and survival by the chemical inhibition of GSK-3 (Self-renewal II)

22.2

Human ES Cell Signal Transduction

In 1998 the first human ES cell line was derived from human blastocysts. These stem cells have the ability to self-renew and to differentiate into one or more differentiated cell types. However, there is some controversy about whether these cells represent the same population type as mouse ES cells, particularly because of the ethical impossibility of making chimeras from human ES cells. Human and mouse ES cells differ in many respects. Morphologically, murine ES cells form tight, rounded clumps, while human ES cells form flatter, looser colonies. Human ES cells grow slower and are more difficult to passage than their mouse counterparts. Although both species express many common markers of pluripotency including transcription factors and common markers of pluripotency (e.g., Oct4, FoxD3, and Nanog and alkaline phosphatase), they differ in other marker expression such as stage-specific embryonic antigens (SSEA, surface antigens originally used to characterize murine embryonic development). Thus while human ES cells express SSEA-3 and SSEA-4 when undifferentiated, they only express SSEA-1 as they differentiate: an expression pattern for these molecules that is essentially the reverse of that seen in mouse cells. The most striking difference relating to self-renewal in the two species is the fact that LIF itself cannot sustain human ES cell self-renewal. Human ES cells are instead cultured in media containing activin and/or FGF2 which act as prodifferentiation signals in mouse ES cells. To understand this difference, a chemically

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defined medium (CDM) containing both activin A and FGF2 which maintains human ES cells in an undifferentiated form has been used to culture cells from rodent embryos. This media, which is sufficient for long-term maintenance of human ES cells, had the ability to culture epiblast cells from the late epiblast layer of postimplantation mouse and rat embryos. These so called “EpiSCs” are temporally defined ES cells that have unique properties similar to human ES cells rather than standard mouse ES cells. This observation could reflect a similar late epiblast origin of human ES cells (Bendall et al. 2008; Lovell-Badge 2007).

22.3

Mouse iPS Cells

Embryonic stem cell research has the prospect of future cellular therapeutic applications (Murry and Keller 2008). There are however elements of ethical controversy when it comes to the use of human fertilized embryos in the isolation of human ES cells. In addition the use of human ES cells in clinical applications could be hampered by immune rejection. A way of overcoming these obstacles came from Kyoto University in 2006 (Liu 2008; Yamanaka 2008; Pei 2008). Over previous years expression profiling of mouse ES cells has led to the identification of a number of genes that appeared to play key roles in ES cells; however no single factor was found to have the ability to induce terminally differentiated cells to become pluripotent. Takahashi and Yamanaka developed a systematic approach in which mouse fibroblasts were assayed for their ability to be reprogrammed via monitoring of the expression of a b-geo cassette introduced into the Fbx15 gene. This essentially acts as a reporter to identify those cells that were induced to resemble stem cells (induced pluripotent stem cells; iPS). Their strategy focused on a list of 24 candidates genes that they systematically introduced by retroviral transduction. The Fbx15 gene had been identified as a novel target of Oct3/4 (another key gene previously identified as being expressed in ES cells). Unlike Oct3/4, however, Fbx15 expression had been found to be dispensable for mouse ES cells’ self-renewal. This meant that somatic cells containing the Fbx15-b-geo cassette were sensitive to G418 drug selection as the Fbx15 gene is inactivated, but when the cells are reprogrammed to resemble stem cells the Fbx15 gene would be reactivated and these reprogrammed cells would become drug-resistant, whilst expression from the b-geo gene cassette is easily identified by b-galactosidase staining. Together this provided a powerful reporter assay to monitor the transduction of the candidate factors. By sequentially reducing the number of factors used to infect the somatic cells required to get drug-resistant cell colonies after transduction, they were able to narrow down the list to 4 genes (Oct3/4, Sox2, Klf4 and c-Myc) that together were necessary to create these iPS cells (Yamanaka 2007). Analysis of these first generation iPS cells showed a similar morphology and proliferation rate compared to standard mouse ES cells. Moreover their pluripotency was confirmed both in vitro by differentiation, and in vivo via teratoma formation. They differed however from true ES cells in their gene expression profiles and their DNA methylation

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patterns and were unable to give rise to viable chimeras when these cells were injected into mouse embryos. (Fig. 22.3) Second generation mouse iPS cells were generated by the same group and two other research groups, from Harvard, Massachusetts Institute of Technology (MIT), and the University of Los Angeles. Instead of using Fbx15 expression to select the iPS, they used Oct4 and Nanog (another key candidate gene in ES cells) expression by inserting a drug-selectable cassette into either the Oct4 or the Nanog genes. In these cases the induction of the same four factors into the mouse fibroblasts gave rise to the selection of iPS cells that now had similar characteristics to mouse ES cells (by morphology, alkaline phosphatase expression and SSEA-1 expression) and importantly they had gene expression profiles and DNA methylation patterns similar to those seen in true mouse ES cells. Moreover they were able to form live chimeras as a stringent test of their pluripotent nature. Tumor formation, however, occurred in mice created from retroviral transduced c-Myc. c-Myc is a known proto-oncogene thought to contribute to the formation of iPS cells through causing epigenetic changes necessary for pluripotency. A desire to remove the requirement for the use of a proto-oncogene was aided in studies where mouse iPS were generated by the induction of Oct3/4, Sox2 and Flk4, without c-Myc, which gave rise to viable chimera without tumor formation (albeit less efficiently). Somatic cells have also been transduced with the less tumorigenic n-Myc substituted for c-Myc. Here the use of a drug-selectable cassette to isolate the iPS was also not required. The use of small molecules to target specific transduction signaling pathways in a similar manner to those studied in mouse ES cell self-renewal may be a further way to improve cell reprogramming. One recent example described the targeting of the Wnt pathway in the absence of c-Myc transduction

Similar characteristics to embryonic stem (ES) cells

Somatic cells carrying a drug selectable pluripotency marker 1

Mix population of cells that have integrated the exogenous genes or not

- ES cell marker genes - in vitro and in vivo pluripotency - normal karyotype

2

- telomerase activity - chimaera formation Induced pluripotent stem (iPS) cells

Fig. 22.3 Generation of induced pluripotent stem (iPS) cells. Somatic cells are transduced with retroviruses carrying the four factors. The somatic cells are engineered to carry a selectable marker associated with a pluripotency marker. Drug selection is used to select for the reprogrammed cells and classical ES cell expansion protocols are applied to isolate the ES cell-like colonies

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using soluble Wnt3a to improved cell reprogramming in mouse somatic cells transduced with only Oct3/4, Sox2 and Klf-4. Moreover there is desire to find an alternative use to retroviruses and the associated concerns of integration sites. This includes the use of nonintegrating adenoviral vectors. The field of iPS cell research is rapidly expanding and it will be interesting if small molecules could be used to specifically target the transduction signaling pathways targeted by the four factors.

22.4

Human iPS Cells

The work done to generate mouse iPS cells paved a way to see if the same strategy could be applied to human somatic stem cells (Yamanaka 2008; Pei 2008; Nishikawa et al. 2008). Formation of iPS from human adult fibroblasts was first described by Yamanaka’s group (Nishikawa et al. 2008). After optimizing the protocol of retroviral transduction to yield better transduction efficiency, they transduced the four factors, Oct3/4, Sox2, Klf4 and c-Myc, into human adult dermal fibroblasts (HDF). Twenty-five days later, typical tight and packed human ES celllike colonies were observed amongst many other granulated colonies. The average number of human ES cell-like colonies observed was about 10% of the granulated cell colonies. They found that human iPS cells had expression patterns similar to human ES cells (SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, alkaline phosphatase and Oct3/4, Sox2, Nanog genes) as well as similar telomerase activity. Promoters of the ES cell-specific genes were shown to be active in human iPS cells and the retroviruses were silenced as predicted. In vitro differentiation potency was assayed by embryoid body (EB) formation and expression of markers of the three germ layers was observed. In vivo differentiation potency was confirmed by teratoma formation in immunodeficient mice. Although global gene expression patterns were similar to human ES cells, they were not however identical, and only a small fraction of the HDF had been reprogrammed, underlying the need for further optimized methods to increase the reprogramming efficiency. At the same time, Thomson’s team published their work on the generation of human iPS cells by lentiviral transduction of four other factors, Oct3/4, Sox2, Nanog and LIN28, into human fetal fibroblasts (Nishikawa et al. 2008). LIN28, a marker of undifferentiated human ES cells, was actually found to be not necessary for initiating the reprogramming or maintaining the expansion of the reprogrammed iPS cells. After 12 days, clones of iPS cells became visible with a typical human ES cell morphology, normal karyotype, and a telomerase activity comparable to that in human ES cells. These iPS expressed the human ES cell markers SSEA-3, SSEA-4, TRA-1– 60 and TRA-1–81. Microarray analysis showed a similar gene expression pattern to human ES cells as well as a similar demethylation pattern. In vitro and in vivo pluripotency was confirmed by EB formation and teratoma formation. Similar results were obtained by lentiviral transduction of the same four factors into human

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foreskin fibroblast. Once again the iPS were similar to human ES cells but not identical. In April 2008, human iPS similar to those obtained by Yamanaka and Thomson were generated from human foreskin fibroblasts but by lentiviral transduction of a combination of six genes described previously: Oct3/4, Sox2, Nanog, LIN28, c-Myc and Klf-4 (Nishikawa et al. 2008). Using this combination, human iPS colonies were visible after seven days, and the efficiency of cell reprogramming was 10-fold higher than seen with the four factors. This new generation of iPS exhibited the human ES cell markers (SSEA-3, SSEA-4, TRA-1–60, TRA-1–81 and AP), a typical human ES cell morphology, and differentiated into the three germ layers in vitro. The addition of c-Myc and Klf-4 to Thomson’s four factors thus improved the efficiency of cell reprogramming, possibly by preventing apoptosis or regulating the cell cycle. To allow clinical application of iPS cells a number of issues must be addressed. In particular the delivery of the genes into the cells by retroviruses or lentiviruses allows the integration of these vectors into the host DNA, which has oncogenic potential and therefore recently a move has been made to use nonintegrating adenoviral vectors. Moreover, although c-Myc may be removed from the reprogramming cocktail, there is a compromise in the efficiency of reprogramming which needs to be addressed. To fully harness the power of iPS cells will ultimately rely on having a fundamental understanding of the mechanisms of cell reprogramming. It has been hypothesized that Oct3/4 and Sox2 acting together drive reactivation of the endogenous genes associated with pluripotency, while Flk4 and c-Myc facilitate the access of these transcription factors to their targets on the DNA by inducing modification of the chromatin structure. In fact it will be interesting if small molecules could be used effectively to substitute for gene transduction by targeting signaling pathways. iPS cell research continues apace and much progress is anticipated in terms of understanding of the fundamental processes involved in the creation of iPS cells at a molecular level, better and more efficient reprogramming protocols and improved safety considerations.

22.5

Further Prospects

Further probing into mouse and human stem cell biology with a particular emphasis on the molecular basis of both their pluripotent nature, as well as the molecular events that occur on the differentiation of these cells, is likely to be a major focus of research in this area for some time to come. The ultimate goal of the utilization of these pluripotent stem cells as a source of therapeutic material depends on having an exquisite control over their biology which can only be done through careful analysis of their molecular signaling events (Blum and Benvenisty 2008). Safety also remains a key goal in harnessing these cells as means to facilitate cellular therapeutics, and to this end there will clearly be a shift in emphasis away from

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embryo-derived pluripotent stem cells to those induced from patients’ own tissues (Yu and Thomson 2008).

References Bendall SC, Stewart MH, Bhatia M (2008) Human embryonic stem cells: lessons from stem cell niches in vivo. Regen Med 3:365–376 Blum B, Benvenisty N (2008) The tumorigenicity of human embryonic stem cells. Adv Cancer Res 100:133–158 Burdon T, Chambers I, Stracey C, Niwa H, Smith A (1999) Signaling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells. Cells Tissues Organs 165:131–143 Burdon T, Smith A, Savatier P, Smith AG (2002) Signaling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol 12:432–438 Chambers I, Smith A (2004) Self-renewal of teratocarcinoma and embryonic stem cells Oncogene 23:7150–7160 Friel R, van der Sar S, Mee PJ (2005) Embryonic stem cells: understanding their history, cell biology and signaling. Adv Drug Deliv Rev 57:1894–1903 Liu SV (2008) iPS cells: a more critical review. Stem Cells Dev 17:391–397 Lovell-Badge R (2007) Many ways to pluripotency. Nat Biotech 25:1114–1116 Murry CE, Keller G (2008) Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132:661–680 Nishikawa S, Goldstein RA, Nierras CR (2008) The promise of human induced pluripotent stem cells for research and therapy. Nat Rev Mol Cell Biol 9:725–729 Pei D (2008) The magic continues for the iPS strategy. Cell Res 18:221–223 Smith AG (2001) Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 17:435–462 Yamanaka S (2007) Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1:39–49 Yamanaka S (2008) Pluripotency and nuclear reprogramming. Philos Trans R Soc Lond B Biol Sci 363:2079–2087 Yu J, Thomson JA (2008) Pluripotent stem cell lines. Genes Dev 22:1987–1997

Index

A Abnormal DNA replication, 286 Abnormal tumor vessels, 177 Accelerated aging, 363 Acetylation, 209, 381 histone H4, 71 p53, 381 Acetylcholinesterase (AChE), 28 Action at a distance, 8 Activating Gs, 84 Activation of GPCRs, 48 of phospholipase D, 49 ADAM family metalloproteinases, 120, 129, 138 Adaptor protein complex-2 (AP-2), 87 Adaptor proteins, 123 Adenoviral E1A, 355 Adenoviruses, 390 Adhesion, 3 β2-Adrenergic receptor kinase, 10 β2-Adrenoceptors, 33 A−F classification system, 3 Age-related macular degeneration (AMD), 178 Agonist, 11 β2-Agonists, 305 AKAP79/150 complexes, 189 AKAP-Lbc, 187 AKAP Yotiao, 187 AKT, 126 Allergic rhinitis, 290 Allosteric, 14 Allosteric two-state model, 17 Alzheimer’s β-amyloid precursor protein (APP), 226 Alzheimer's disease, 112 α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), 184 Amphipathic helix, 184

Angiogenesis, 159, 169 Angiopoietin-1 (Ang-1)/Tie2 signaling, 174 Annexin-1, 294 Anorexigenic effects, 143 Anorexigenic peptides, 143 Antagonists, 11, 44 Anti-aging effects, 409 Antidepressants, 69, 72 Antidepressant treatment, 74 Anti-nociceptive tolerance, 33 Apoptosis, 202, 380, 388, 405 Apoptotic machinery, 211 Arachidonic acid, 240 ARF, 409 Arf6, 51 Arf family GTPases, 44 Arf GTPases, 48 Arrestin, 66 Arrestin 2, 36 Arrestin gene family, 67 β-Arrestins, 67, 28, 224 Asthma, 290 Ataxia telangiectasia (AT), 402 ATPases, 355 AU-rich elements (ARE), 300 Axonal compartment, 27 5-Aza-2'-deoxycytidine, 297

B Base excision repair (BER), 401 BAY 41-2272, 342 Bcl-2 family members, 124, 128 Biased agonism, 14 Binding assays, 17 Biochemical theories of pathogenesis, 73 Biological role of AKAPs, 185 Bleomycin treatment, 381 Blood-brain barrier (BBB), 143

427

428 Bone morphogenic proteins (BMP), 418 Brain-specific antiangiogenesis inhibitor 1 (BAI1), 390 Breast cancer, 133, 139 Breast cancer cells activation, migration, 91 Bromodomain, 267

C Caenorhabditis elegans, 356 Calcium homeostasis, 241 Calcium-release-activated calcium (CRAC), 238 cAMP, 278 cAMP flux, 185 cAMP response element (CRE), 14 cAMP signal transduction cascade, 190 Camptothecin, 381 cAMP transduction cascades, 211 Cancer, 83, 176, 363, 400 Cancer therapy, 390 Cannabinoid receptor (CB1R), 27 Capillary electrophoresis, 17 5’-Capping, 258 Carbon monoxide, 341 Carboxy-terminus (CTD), 261 Cardiogenesis, 259 Cardiomyocytes, 186 Caretakers, 399 Ca2+-selective calcium current (ICRAC), 238 Cav-1, 240 Caveolae, 28 Caveolar LRDs, 240 Caveolin, 240 Caveolin-1 (Cav-1), 240 Caveolin scaffolding domain (CSD), 244 Cavin, 240 Cdc25C overexpression, 387 cdc2 inhibition, 387 Cell cycle arrest, 400, 405 checkpoints, 360, 384 death, 400 fate, 107 proliferation, 86 Cellular dielectric spectroscopy, 17 Cellular senescence, 407 Central leptin resistance, 145 Central nervous system (CNS), 70 insulin signaling, diabetes and localized therapies, 112 Cervical ganglion neurons (SCGs), 189 c-fos immunoreactivity, 71

Index Charcot-Marie-Tooth (CMT) subtype 2A, 200 Chemokines, 290 Chenopodium rubrum, 356 Cholesterol, 240 Chromatin, 258, 290 Chromatin immunoprecipitation assay, 383 Chromatin remodeling complexes, 267–268 Chromatin-remodeling enzymes, 355 Chromatosome, 259 Chronic hypercholinergic mice, 34 Chronic hyperdopaminergic mice, 34 Chronic insulin treatment, 192 Chronic obstructive pulmonary disease (COPD), 291 c-Jun NH2-terminal kinase (JNK), 220 Class III Arf proteins, 51 Clathrin, 28 Clathrin adaptors, 95 Cloning novel AKAPs, 184 cMyc, 422 Coactivators, 280 Cognitive impairment, 112 Concomitant chemotherapy, 391 Conformational coupling hypothesis, 238 Constitutive chronic receptor, 28 Constitutive endocytosis, 27 Constitutively activating mutations (CAMs), 10, 12 Constitutively active Gi2, 86 Conventional insulin signaling, 102–103 diabetes and therapies, 107 5-Coordinate complex, 340 COP1, 379 COP9 signalosome, 380 Core Class II Promoter, 260 Corepressors, 291 C-reactive protein (CRP), 148 CREB binding protein (CBP), 381 Cross-talk, 129 networks, 282 Culture conditions, 418 CXCR4 receptor, 92 Cyclic adenosine monophosphate response element binding protein (CREB), 282 Cyclic GMP, 338 Cyclic-nucleotide-gated (CNG), 190 Cyclin B/cdc2 complex, 386 Cyclin/cyclin-dependent kinases (cdks) complexes, 385 Cycloheximide, 32 Cysteine, 318 Cysteine-rich 61 protein (Cyr61), 48 Cytokine-independent survival kinase (CISK), 92

Index Cytokines, 290 Cytoskeletal rearrangement, 47

D Damage caused by exogenous agents, 362 Damage to DNA, endogenous cellular processes, 362 Delta-like 4/Notch signaling, 169 Delta opioid receptor (DOR), 33 Deregulation of ErbB receptors, 132, 134 Described light sensitivity, 111 Desensitization, 343 Deubiquitinating enzymes (DUBs), 92 Dexamethasone, 303 Dexamethasone suppression test (DST), 73 Diabetic retinopathy, 179 1,2 Diacylglycerol (DAG), 8 Diet-induced obesity (DIO), 144 Differential engagement, 14 Differentiation, 359 Diffusible factor hypothesis, 238 Dissociation of NO, 243 DNA-damage-response pathway, 362 DNA double stranded breaks (DSBs), 400 DNA repair process, 363 Dnm1p in yeast, 202 Dopamine-receptor-interacting protein 78 (DRIP78), 34 Dopamine-transporter knockout mice, 28 Double electron–electron resonance, 5 Double strand brakes (DSBs), 363 Double-stranded DNA (dsDNA), 258 Downstream promoter element (DPE), 262 Doxorubicin, 381 Drosophila, 240 Drosophila melanogaster, 356 Drp1 in mammals, 202 Drug, 11 DT40, 243 Dynamin, 36 Dysfunctional telomeres, 408

E Electronic biosensors, 17 ELK1, 279 ELL gene, 270 Elongation factor, 270 Embryonic stem (ES) cells, 417 Endocytosis, 28 Endogenous ligand, 11 Endoplasmic reticulum (ER), 26, 101

429 Endoplasmic reticulum associated degradation (ERAD), 207 Endoreduplication, 388 Endosomal-sorting complex required for transport (ESCRT) machinery, 88 Endothelial cell migration, 166 proliferation, 166 survival, 167 Endothelium-derived relaxing factor (EDRF), 337 Endotoxin, 291 Eosinophils, 290 Epiblast cells, 421 Epidermal growth factor (EGF), 119, 120 Epidermal growth factor receptor (EGFR), 83 Epstein-Barr virus (EBV), 10, 89 ERβ, 274 ErbB1 EGFRvIII mutation, 134, 137, 139 kinase domain mutation, 138 over-expression, 133, 134 ErbB2, over-expression, 133 ErbB receptors, 119, 120, 122 dimerization, 122 dimerization partners, 120 ligand production, 120 ligands, 122 negative regulation, 130 nuclear signaling, 129 structure, 121 ERK, 13 ERK-activation, 221 ERK1/2 MAPK, 105 ERK1/2 signaling pathways, 221 E2 signaling, 280 Estrogen 17β-estradiol, 274 receptor subtypes, 274 Estrogen response element (ERE), 273 Extracellular signal-regulated protein kinase (ERK), 5, 70, 221, 404, 300 Extracellular signal-regulated protein kinase 1/2 (ERK1/2) pathway, 105

F Fbx15 gene, 421 Fe-S clusters, 316 Fetal liver erythropoiesis, 359 Flk4, 422, 424 Flow cytometry, 17 Fluorescence resonance energy transfer (FRET), 9, 190, 245

430 Fluorescent DOR, 33 FNR, 319 Focal adhesion kinase (FAK), 172, 227 Forkhead Homology (FHA), 192 Forkhead transcription factor family (FKHR/Foxo1), 106 Four-Cys zinc motif, 321 Foxo1, 104, 167 Free E2F, 360 Frizzled/Taste2, 4 Full agonists, 11

G GADD45, 387 G1 arrest, 385, 388 G2 arrest, 387 Gatekeepers, 399 Gatekeeper tumor suppression, 404 G1 checkpoint, 386 Genotoxic stress, 385 Germinal center kinase (GCK), 229 G12 family, 86 Gi-protein mutations, 84 Glioblastoma, 134, 137 Global gene expression, 279 Glucagon-like peptide (GLP) receptor, 13 Glucocorticoid-induced leucine zipper protein (GILZ), 292 Glucocorticoid receptors (GR), 292 nitrosylation, 303 recognition sequences, 304 Glucocorticoid response elements (GRE), 292 Glucocorticoids, 289 Glucose uptake, 105 GLUT4, 105 Glutamate, 4, 5 Glutamate, Rhodopsin, Adhesion, Frizzled/ Taste2 and Secretin (GRAFS), 4 Glutathione, 316 Glycogen synthase kinase-3 (GSK3-β), 104 Glycogen synthase kinase 3 (GSK3) β inhibitor, 419 Glycogen synthesis and gluconeogenesis, 106 Glycosylation, 109 G2/M transition, 361 Golgi complex, 25 gp130, 417 GPCR30, 275 GPCR kinase 3 (GRK3), 31 GPCR kinase (GRK), 5, 36 G protein coupled receptor (GPCR), 3, 25, 83, 241 defective trafficking, 90

Index densensitization, 87 downregulation, 87 dysfunction, 4 endyocytosis, 90 gene, 89 internalization, 87 phosphorylation, 87 signaling, 65 G protein coupled receptor interacting proteins (GIPs), 8 G protein coupled receptor kinases (GRKs), 7, 65 G protein heterotrimer, 9 G protein-mediated pathways, 69 G protein signaling, 84 G protein signaling (RGS) proteins, 7 GPx4, 328 Granulocyte-macrophage colony stimulating factor (GM-CSF), 291 Gravin (AKAP250), 187 GRβ, 302 GRK phosphorylation sites, 66 Growth differentiation factor 6 (GDF6), 418 Growth factors, 290 GTPase activating proteins (GAP), 45 Guanine-nucleotide exchange factors (GEFs), 4, 9, 44, 84 Guanylyl cyclase activation, 339 membrane forms, 338 soluble forms, 338 Guanylyltransferases, 265

H Half-life of NO-sGC, 343 Hallmarks of cancer, 132 HAUSP, 379 γH2AX, 364 H2AXs, 364 HDAC1, 297 HDAC3, 297 Heat shock protein-90 (hsp90), 292 Heat shock proteins, 223 HEK293, 243 Helix movement model, 5 Hematopoietic protein kinase-1 (HPK1), 229 Hematopoietic stem and progenitor cell (HSPC), 359 Hereditary disorders, 363 Hereditary non-polyposis colorectal cancer (HNPCC), 402 Heterodimerization, 164 Heterologous expression system, 12

Index Heterotrimeric G proteins, 84 hFis1, 202 High throughput screening (HTS), 13 Histone acetyltransferase (HAT), 259, 291 Histone deacetylases, 291 Histone deacetylases (HDACs), 291 Histone H4, 294 Histone methyltransferases, 297 Histones acetylation, 259 deacetylation, 259 proteins, 290 History of the insulin receptor, 108 H2O2, 313 Holo-receptors, 110 HOMER, 247 Homologous recombination (HR), 363 Horizontal molecular networks, 16 Hsp33, 321 Ht31, 184 Human cytomegalovirus, 10 Human ES cell signal transduction, 420–421 Human iPS cells, 423 Human papillomavirus (HPV), 355, 366 Huntington-interacting protein 1 (HIP1), 95 HuR, 300 Hydroxylation of HIF, 162 Hydroxyl radical, 313 Hyperactivation of mitogenic signaling pathways, 410 Hyperbaric oxygen, 316 Hypermitogenic signaling, 408 Hypermitogenic stimulation, 410 Hypersensitivity to NO, 345 Hypertension, 178 Hypoxia inducible factor (HIF), 162

I Id proteins, 418 IGF-1, 102 IGF-1 receptor (IGF-1R), 389 Immunoglobulin repeats, 166 Immunohistochemistry, 26 Immunoprecipitation, 239, 297 Importin-13, 292 Importin-α, 292 Induced pluripotent stem (iPS) cells, 421 Inflammatory genes, 300 Inflammatory peptides, 290 Inhibition of VEGF, 177 Inhibitor of apoptosis (IAP) family, 389 Inositol-1,4,5-triphosphate (IP3), 8, 166, 238

431 Insulin, 101 Insulin/IGF-1 pathway, 401 Insulin-like growth factor-1 (IGF-1), 400 Insulin receptors (IR), 102 Insulin receptor substrate (IRS) proteins, 103 Insulin resistance, 107 Insulin signaling function, 105 Insulin signaling, retina and brain, 108 Integrins, 171 Interdomain interactions, 9 Interleukin(IL)-1β, 291 Intracellular mechanism, central leptin resistance, 148 Intrinsically disordered regions (IDRs), 268 Inverse agonists, 12, 44 Iron-response protein-1, 319 Irradiation-induced foci (IRIF), 364 IR regulation/function, 111 IRS1/PI3K/Akt/ NF-kB-dependent pathway, 152 IscR, 319 Islets of Langerhans, 101

J JAK/STAT pathway, 138 Janus-associated (JAK) tyrosine kinases, 417 JIP1, 225 JIP1b, 226 JLP, 227 JNK binding domain (JBD), 226 JSAP scaffold protein, 226 Jun-N-terminal kinase (JNK), 224 interacting protein, 225 pathway, 303

K Kaposi’s sarcoma-associated herpesvirus (KSHV), 10, 89 Karyopherin-β, 292 KEAP1, 325 KEAP1-NRF2, 325 Kinase Suppressor of Ras signaling (KSR), 222 Kinesin light chain 1 (KLC1), 228

L Latent p53, 383 Lck adaptor (LAD), 167

432 Leptin gene expression, 145 resistance, 144 sensitivity, 151 signaling, 143 surge, 146 Leukemia inhibitory factor, 417 Leutinizing hormone receptor (LHR), 88 “Lever-arm,” “gear-shift,” and “latch” models, 8 Li-Fraumeni syndrome, 375 LIF receptor, 417 Lipid mediators, 290 metabolism, 106 peroxidation, 328 rafts, 240 Lipopolysaccharides (LPS), 227 Lithium action, 69 Loss of p14ARF, 390 Lung cancer, 134, 137, 138 LXCXE-lacking proteins, 355 Lymphangiogenesis, 164, 168 Lysophosphatidic acid (LPA), 94 Lysosomal degradative pathway, 87

M Major depressive disorder, 63 MAP kinase kinase kinase kinase (MAP4K), 219 MAPKK kinase called MEK1/2, 125 MAPKKK kinase (Raf), 125 Matrix metalloprotease-1 (MMP1), 89 Matrix metalloproteinases (MMP), 162 Mdm2, 376 MdmX (or Mdm4), 378 Mediator, 268 Melanotan II, 151 Membrane receptor-mediated actions of E2, 275 mER signaling, 278 Messanger RNAs (mRNAs), 257 Metabotropic glutamate mGlu5 receptor, 36 Methionine, 318 Milton, 203 Miro, 203 Mismatch repair (MMR), 401 Mitochondrial dynamics, 209 fission, 202 fragmentation, 205 fusion, 200 motility, 203 plasticity, 205

Index Mitofusin homologues, 200 Mitogen-activated protein kinase (MAPK), 86, 105, 277, 300 cascades, 404 modules, 221 pathway, 279, 418 Mitogen activated protein kinase kinase (MKK), 70 Mitogen-activated protein kinase phosphatase-1 (MKP-1), 295 Mitosis, 385 Mitotic spindle, 385 Mixed lineage kinase-3 (MLK3), 230 M4 muscarinic ACh receptors, 27 Modifications, 280 Monoclonal antibody, 135 mechanism of action, 139 Mood disorders, 63 Mouse double minute 2 homolog (MDM2), 404 Mouse ES cell Signal Transduction, 417–420 Mouse iPS, 423 mTORC1 complex, 210 Multi-vesicular bodies (MVB), 32, 92 Muscarinic acetylcholine receptor (mAChR), 49 Mutagenic lesions, 400 Myc, 422–424 Myogenesis, 359

N NADPH oxidase, 314 Nanog, 420, 423, 424 Necrosis factor (TNF)-α, 291 NEDDylation of p53, 378 Negative regulatory loop, 382 Neoplastic transformation, 401 Neural stem cells (NSCs), 259 Neuropillins (NRP), 165 Neurotensin NTS1 receptor, 36 New Zealand (NZO) obese mice, 145 NFκB, 127 N-formylmethionyl-leucylphenylalanine (fMLP), 46 Nijmegen breakage syndrome (NBS), 402 Nitric oxide synthase (NOS), 240 Nitrosative, 316 NO-independent activators, 342 Non-homologous end joining (NHEJ), 363, 401 NOTCH, 168 Noxa, 388 N-phosphonoacetyl-L-aspartate, PALA, 381 NRF2, 325

Index Nuclear actions, 274 ER actions, 274 estrogen receptors, 273 Nuclear factor-κB (NF-κB), 290 Nucleosome, 258 Nucleotide excision repair (NER), 401 Nutlins, 380, 390

O Oct4, 420, 422 ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin1-one), 343 Oligodendrocyte differentiation, 360 Oligomerization, 16 Oncogenes, 401 Oncogenic protein Mdm2, 376 Opa1 (optical atrophy 1), 200 µ-Opiate receptor, 27 Orai1, 245 Orthosteric, 13 Osmosensing scaffold, 229 Oxidase A (MAO-A), 64 Oxidized/damaged proteins, 207 Oxotremorine, 32 OxyR, 314

P p53, 403 p107, 355, 367 p130, 355, 367 p63 and p73, 376 Parathyroid hormone (PTH) analog, 17 p14ARF, 378 PAR1, internalisation, 93 Parkinsons’s disease, 208 Partial agonists, 11 Pathophysiological changes in GPCR signaling, 54 Paxillin, 223 PCNA, 387 p53 deacetylation, 382 p21-deficient, 385 Pericyte loss, 175 Peripheral blood mononuclear cells (PBMCs), 405 Peripheral leptin resistance, 145 Permeability transition pore, 212 Peroxisomal fission, 202 PERP, 389 p53 family, 376–378 Pharmacocentric approach, 75

433 Pheromone signaling, 220 Phosducin, 71 Phosducin-like proteins (PhdLP), 65 Phosphatase and tensin homologue (PTEN), 104, 127, 139 Phosphatidylinositol (4,5)-bisphosphate (PIP2), 8, 90, 104 Phosphatidylinositol-3 lipid kinase (PI3K), 45, 278, 400 Phosphatidylinositol 3,4,5-triphosphate (PIP3), 104 Phosphoenolpyruvate carboxykinase (PEPCK), 106 Phospholipase C (PLC), 239 Phosphorylation, 274, 380 of pRb, 360 of Shc, 104 Phospho-tyrosine binding (PTB) domain, 226 PIDD, 389 PI3-K/Akt, 104–105 PI3K/AKT pathway, 105, 124, 138, 139 PI3K complex, 168 PI3K pathway, 279 PI3K-phosphodiestrase 3B-cyclic AMP pathway, 150 p16INK4A, 365 Pirh2, 379 PKGIα isoform, 329 p53-knockout mice, 376 Placental growth factor (PlGF), 164 Plasma membrane Ca2+ (PMCA), 239 Plasmenylethanolamine, 240 Platelet-derived growth factor (PDGF), 173 Platelet-derived growth factor receptor-β (PDGFR-β), 173 PLD-dependent endocytosis, 49 Pleckstrin homology (PH), 10, 103 Pleiotropic factor, 389 Plenty of SH3 (POSH), 228 Pluripotent, p38MAPK, 220 p38 MAP kinase, 295 Pocket domain, 355 Pocket proteins, 353 3'-Polyadenylation, 258 n-3 Polyunsaturated fatty acid (PUFA), 146 Post-receptor IR pathways, 110 Post-receptor pathways, 103 p21 overexpression, 385 p53 post-translational modifications, 379 pRb-mediated transcriptional repression, 365 pRb pathway, 355 p53 regulation, 378 Premature aging phenotypes, 407

434 Preproinsulin, 101 Pro-apoptotic proteins, 167, 405 Pro-inflammatory cytokines, 409 Proline-rich domain, 376 Promoter escape, 263 Promoters, 275 Proopiomelanocortin (POMC), 143 Prostacyclin, 244 Protean agonism, 14 Protease-activated receptor-1 (PAR1), 89 Protease-activated receptors (PARs), 93 Protein-1 (AP-1), 290 Protein kinase C (PKC), 105, 185 Protein phosphorylation, 183 Protein synthesis, 106–107 Protein tyrosine phosphatases, 327 Proteolysis, 33 PtdIns(4,5)P2, 240 p53 transcriptional activity, 382 p53 tumor suppressor, 326, 375 Pulmonary artery endothelial cells (PAEC), 244 PUMA, 388 Pyruvate dehydrogenase, 209

Q Quaternary complex model, 17

R Rab4, 53 Rab5, 36, 53 Rab7, 36 Rab GTPases, 51, 53 Rab mutants, 51 Radiolabeled ligands, 17 Radioligand binding assay, 13 Raf proteins, 277 Ral guanine nucleotide exchange factors (RalGEFs), 45 Ral subfamily, 46 Ras activation, 278 Ras-dependent activation, 45 RAS/Erk pathway, 124 Ras family members, 45 Ras pathways, 45 RAS-signaling pathway, 222 Rb gene, 353 Rb gene family, 353, 354 Reactive oxygen species (ROS), 313, 401 Receptor (IGF-1R), 400 Receptor for activated C-kinase 1 (RACK1), 240 Receptor/ligand interactions, 102, 109

Index Receptor tyrosine kinase domains, 165 Recruitment, 258 Redox mechanisms, 314 Regulation of mitochondrial function, 209 Regulation of receptor-coupled G protein signal transduction, 65 Regulators of G protein signaling (RGS), 63, 72 Regulatory cascades, 281 Replicative exhaustion, 407 Resensitization of protease receptors, 50 Restriction point, 360 Retinal pigmented epithelial (RPE) cells, 110 Retinoblastoma tumor suppressor, 353 Retroviruses, 390 RGS proteins, 84 Rhodopsin, 4, 5, 31 Rho family, 46, 48 Rho family GTPases, 44 Rho GTPases, 47 Ribonucleoside-tri-phosphates (NTP’s), 258 Ribosomal RNAs (rRNAs), 257 RII subunit, 184 RNA polymerase I (RNAP I), 257 RNA polymerase II (RNAP II), 257, 291 RNA polymerase III (RNAP III), 257 ROS receptor, 314 ROS scavengers, 314 Ryanodine receptors (RyRs), 190

S SAP90, 240 Sarcoendoplasmic reticulum Ca2+ (SERCA), 239 Sarcoma viruses, 45 SCID, 245 Second messengers, 43 Second messenger systems, 8 Secretin, 4, 5 Secretion coupling model, 238 Secretory leukoprotease inhibitor (SLPI), 294 Selenocysteine, 318 Self-renewal, 420 Senescence program, 409 Sequence element (Inr), 260 Serum response factor (SRF), 279 Seven transmembrane (7TM), 4 S-glutathionylation, 316 SH2-containing 5’-phophatase-2 (SHIP2), 104 SHP-2, 418 Signaling pathways, 277 Signal transduction, 420–421 siRNA, 245 Small GTPases, 44

Index Small-molecule inhibitors, 419 Small ribonucleoproteins (snRNPs), 265 S-nitrosylation, 318 SOCS3 pathway, 149 SOCS proteins, 108 Somatodendritic compartment, 26 Somatostatin sst2A receptors, 27 Son of sevenless (SOS), 221 Sorting nexin-1 (SNX1), 94 Sox2, 421, 422 SoxR, 314 Spacer region, 355 Spatiotemporal organization, 237 Sperm associated antigen 9 (SPAG9), 228 S phase, 365, 385 S-phase checkpoint, 386 Sphingolipids, 240 Squamous cell carcinoma of the head and neck, 133, 138 Src homology 2 (SH2), 417 Src homology 3 (SH3), 226 Stat family, 279 Steroid-receptor coactivator-1 (SRC-1), 294 STIM1, 245 STIM1/TRPC1/Orai1 complex, 247 Stimulus trafficking, 14 Store-operated calcium (SOC), 238 Store-operated calcium entry (SOCE), 238 Stromal interacting molecule 1 (STIM1), 245 α-Subunit, 110 Sulfenate, 317 Sulfenic acid, 323 Sulfinic, 327 Sumoylation, 209 Superoxide, 313 Suppressor of cytokine signaling (SOCS), 149 SV40 Large T Antigen, 355 SWI/SNF complexes, 267

T Targeted therapy, 138, 140 cetuximab, 135 erlotinib, 135 gefitinib, 135 lapatinib, 135 mechanisms of resistance, 139 panitumumab, 135 pertuzumab, 135 trastuzumab, 135 TATA box, 260 TATA box binding protein (TBP), 291 Termination, 264

435 Terminator region, 264 Terminator sequence, 258 Ternary complex model, 16 T98G human glioblastoma, 354 Therapeutic targets, 151 Thiolate anion, 317 Thioredoxin, 323 Third intracytoplasmic loop, 35 Third messengers, 281 Thyroid-stimulating hormone receptor (TSHR), 88 TIGAR, 390 Toll-like receptor (TLR), 227 Total Internal Reflection Flourescence Microscopy (TIRFM), 245 Transcript elongation, 264 Transcription coupled repair (TCR), 402 Transcription factors, 266, 282 Transcription initiation, 262 Transcription preinitiation complex, 266 Transcript termination, 264 Transferrin, 35 Transfer RNAs (tRNAs), 257 Transient receptor potential canonical (TRPC), 239, 242 Transmembrane (TM), 5 Triglycerides (TG), 146 Tristetraprolin, 300 TRP, 242 TRPC1, 242 TRPC4+TRPC1 heteromeric, 244 TRPC1–TRPC3 heteromers, 243 TRPC1–TRPC1 multimers, 243 Tryptophan hydroxylase (TPH), 64 Tumor cell proliferation, 92 Tumor progression, 86 Tumor suppressor functions, 399 Type I diabetes, 230 Type II PKA holoenzyme, 184 Type I PKA holoenzyme, 184 Tyrosine kinase inhibitors, 136, 139 Tyrosine phosphatase, 124, 130

U Ubiquitination, 88, 209 of p53, 381 Ubiquitin ligases, 379

V Vascular smooth muscle cells (VSMCs), 48 Vasodilator action, NO, 337

436

Index

W Wortmannin-sensitive pathway, 380

Xeroderma pigmentosum complementation group B (XPB), 266

X Xenobiotics, 316 Xeroderma pigmentosum (XP), 402

Y YC-1, 342