International Review of Cytology, Vol. 263 [1st ed.] 0123741793, 9780123741790, 9780080551623

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V O LU M E

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INTERNATIONAL REVIEW OF

CYTOLOGY A Survey of Cell Biology

INTERNATIONAL REVIEW OF CYTOLOGY Series Editors

GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN FRIEDLANDER JONATHAN JARVIK

1949–1988 1949–1984 1967– 1984–1992 1993–1995

Editorial Advisory Board

ISAIAH ARKIN EVE IDA BARAK PETER L. BEECH HOWARD A. BERN ROBERT A. BLOODGOOD DEAN BOK HIROO FUKUDA RAY H. GAVIN SIAMON GORDON MAY GRIFFITH WILLIAM R. JEFFERY KEITH LATHAM

WALLACE F. MARSHALL BRUCE D. MCKEE MICHAEL MELKONIAN KEITH E. MOSTOV ANDREAS OKSCHE THORU PEDERSON MANFRED SCHLIWA TERUO SHIMMEN ROBERT A. SMITH WILDRED D. STEIN NIKOLAI TOMILIN

V O LU M E

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INTERNATIONAL REVIEW OF

CYTOLOGY A Survey of Cell Biology EDITED BY

KWANG W. JEON Department of Biochemistry University of Tennessee Knoxville, Tennessee

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CONTENTS

Contributors

1. Mechanisms of Gradient Detection: A Comparison of Axon Pathfinding with Eukaryotic Cell Migration

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Anne von Philipsborn and Martin Bastmeyer 1. Introduction 2. Mechanisms of Gradient Detection in Selected Eukaryotic Cell Types 3. Common Grounds and Diversity 4. Concluding Remarks Acknowledgments References

2. Leptin and the Regulation of the Hypothalamic–Pituitary–Adrenal Axis

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Ludwik K. Malendowicz, Marcin Rucinski, Anna S. Belloni, Agnieszka Ziolkowska, and Gastone G. Nussdorfer 1. Introduction 2. Biology of Leptin and Its Receptors 3. Expression of Leptin and Its Receptors in the Hypothalamic–Pituitary–Adrenal Axis 4. Effects of Leptin on the Central Branch of the Hypothalamic–Pituitary–Adrenal Axis 5. Effects of Leptin on the Peripheral Branch of the Hypothalamic–Pituitary–Adrenal Axis 6. Involvement of Leptin in the Pathophysiology of the Hypothalamic–Pituitary–Adrenal Axis 7. Concluding Remarks Acknowledgments References

3. Focal Adhesion Kinase and p53 Signaling in Cancer Cells

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Vita M. Golubovskaya and William G. Cance 1. Introduction 2. Structure and Function of Focal Adhesion Kinase Protein

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3. FAK in Tumorigenesis 4. FAK and p53 Association 5. FAK and p53 Targeted Therapy 6. Summary Acknowledgments References

4. Cell and Molecular Biology of the Spindle Matrix

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Kristen M. Johansen and Jrgen Johansen 1. Introduction 2. Microtubule Spindle Dynamics and Force Production 3. Evidence for a Spindle Matrix 4. Concluding Remarks Acknowledgments References

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5. Mitochondrial Biology and Disease in Dictyostelium

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Christian Barth, Phuong Le, and Paul R. Fisher 1. Introduction 2. Mitochondrial Biology 3. Mitochondrial Disease 4. Concluding Remarks References

6. Oxytocin and the Human Prostate in Health and Disease

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Helen D. Nicholson and Kate Whittington 1. Introduction 2. The Human Prostate 3. Oxytocin and Oxytocin Receptor 4. Oxytocin and the Prostate 5. Possible Roles of Oxytocin in the Pathophysiology of Prostate Disease 6. Concluding Remarks References Index

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CONTRIBUTORS

Christian Barth Department of Microbiology, La Trobe University, Melbourne VIC 3086, Australia Martin Bastmeyer Department of Cell Biology and Neurobiology, University of Karlsruhe, D-76131 Karlsruhe, Germany Anna S. Belloni Department of Human Anatomy and Physiology, Section of Anatomy, School of Medicine, University of Padua, I-35121 Padua, Italy William G. Cance Department of Surgery, University of Florida School of Medicine, Department of Biochemistry and Molecular Biology, and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610 Paul R. Fisher Department of Microbiology, La Trobe University, Melbourne VIC 3086, Australia Vita M. Golubovskaya Department of Surgery, University of Florida School of Medicine, and UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610 Jørgen Johansen Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 Kristen M. Johansen Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 Phuong Le Department of Microbiology, La Trobe University, Melbourne VIC 3086, Australia Ludwik K. Malendowicz Department of Histology and Embryology, School of Medicine, Karol Marcinkowski University of Medical Sciences, PL-60781 Poznan, Poland

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Helen D. Nicholson Department of Anatomy and Structural Biology, University of Otago, New Zealand Gastone G. Nussdorfer Department of Human Anatomy and Physiology, Section of Anatomy, School of Medicine, University of Padua, I-35121 Padua, Italy Marcin Rucinski Department of Histology and Embryology, School of Medicine, Karol Marcinkowski University of Medical Sciences, PL-60781 Poznan, Poland Anne von Philipsborn Department of Cell Biology and Neurobiology, University of Karlsruhe, D-76131 Karlsruhe, Germany Kate Whittington Clinical Science South Bristol, University of Bristol, Bristol BS8 1TH, United Kingdom Agnieszka Ziolkowska Department of Histology and Embryology, School of Medicine, Karol Marcinkowski University of Medical Sciences, PL-60781 Poznan, Poland

C H A P T E R

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Mechanisms of Gradient Detection: A Comparison of Axon Pathfinding with Eukaryotic Cell Migration Anne von Philipsborn and Martin Bastmeyer

Contents 2

1. Introduction 2. Mechanisms of Gradient Detection in Selected Eukaryotic Cell Types 2.1. Chemotaxis of Dictyostelium 2.2. Chemotaxis of mammalian neutrophils 2.3. Chemotaxis of mammalian fibroblasts 2.4. Pathfinding of neuronal growth cones 3. Common Grounds and Diversity 3.1. Signaling pathways 3.2. Signal amplification 3.3. Adjustment of sensitivity/adaptation 3.4. Biological and functional context 4. Concluding Remarks Acknowledgments References

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Abstract The detection of gradients of chemotactic cues is a common task for migrating cells and outgrowing axons. Eukaryotic gradient detection employs a spatial mechanism, meaning that the external gradient has to be translated into an intracellular signaling gradient, which affects cell polarization and directional movement. The sensitivity of gradient detection is governed by signal amplification and adaptation mechanisms. Comparison of the major signal transduction pathways underlying gradient detection in three exemplary chemotaxing cell types, Dictyostelium, neutrophils, and fibroblasts and in neuronal growth cones, reveals conserved mechanisms such as localized PI3 kinase/PIP3 signaling and a common output, the regulation of the cytoskeleton by Rho GTPases.

Department of Cell Biology and Neurobiology, University of Karlsruhe, D-76131 Karlsruhe, Germany International Review of Cytology, Volume 263 ISSN 0074-7696, DOI: 10.1016/S0074-7696(07)63001-0

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2007 Elsevier Inc. All rights reserved.

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Local protein translation plays a role in directional movement of both fibroblasts and neuronal growth cones. Ca2þ signaling is prominently involved in growth cone gradient detection. The diversity of signaling between different cell types and its functional implications make sense in the biological context. Key Words: Gradient detection, Axon guidance, Cell migration, Cell polarization, Dictyostelium, Neutrophil, Fibroblast, Growth cone. ß 2007 Elsevier Inc.

‘‘There is a theory which states that if ever anyone discovers exactly what the Universe is for and why it is here, it will instantly disappear and be replaced by something even more bizarre and inexplicable. There is another theory which states that this has already happened.’’ —Douglas Adams, The Restaurant at the End of the Universe

1. Introduction Graded distributions of chemotropic factors (referred to as ‘‘gradients’’ herein) are essential guideposts in the development, regeneration, and function of multicellular organisms. They provide directional as well as positional information and steer migrating cells and neuronal growth cones. Detection of gradients allows Dictyostelium cells to aggregate and proceed in their life cycle, neutrophils to migrate to sites of infection, fibroblasts to invade and heal wounds, and neuronal growth cones to follow guidance cues and establish the complex connectivity of the nervous system. It should be noted, however, that directed movement can be established by cues other than graded distributions of guidance factors. Likewise, gradients can induce cellular responses different from directed movement. These topics will not be considered in this article. Gradient detection by eukaryotic cells differs fundamentally from the one in bacteria. Because bacteria are too small to sense a concentration gradient along their cell length, they use a temporal gradient-sensing mechanism. Dependent on temporal changes in the concentration of a chemoattractant or a chemorepellent, they regulate the frequency of random directional reorientation by changing the direction of flagellar rotation. This allows them to bias their overall direction of movement. Although highly efficient in detecting minimal concentration differences over a wide range by means of adaptation, a temporal gradient-sensing mechanism can be only employed by a moving cell and does not guide cells on a straight path toward or away from a chemotactic factor (Baker et al., 2005; Wadhams and Armitage, 2004). A migrating eukaryotic cell, on the other hand, can sense an external concentration gradient along the cell length and translate it into an internal signaling gradient. The internal gradient leads to a morphological polarization

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and, in some cases, an asymmetry of sensitivity. Once polarized, the cell initiates directed movement via cytoskeletal rearrangements. Similarly, a neuronal growth cone responding to an attractive or repulsive gradient activates differing signaling events at the side facing the higher concentration (gradient near side) and the side facing the lower concentration (gradient far side), while performing a turning response. To detect small concentration differences and translate them efficiently into a correlated turnaround, the external gradient has to be amplified by means of signal transduction. Furthermore, a cell or a growth cone migrating in a gradient possibly has to adjust its sensitivity and adapt to detect concentration differences over a broad range of absolute concentrations. In this chapter, we will exemplarily review these different aspects of gradient detection during chemotaxis in two amoeboid cell types (Dictyostelium and mammalian neutrophils) as well as in fibroblasts and compare them with gradient detection of neuronal growth cones. Necessarily, this focus on a few model systems excludes many other important cell types, which perform chemotaxis in response to gradients such as metastatic cancer cells (Condeelis et al., 2005), mesoderm cells during vertebrate gastrulation (Dormann and Weijer, 2006), germ line cells (Kunwar et al., 2006), or mammalian sperm (Eisenbach and Giojalas, 2006). To give a representative and detailed picture of eukaryotic chemotaxis, Dictyostelium cells and mammalian neutrophils are particularly suited, because they have been extensively studied in this regard and can serve as an exemplary model for chemotaxis of other eukaryotic cell types (Dormann and Weijer, 2006; Williams et al., 2006). Although evolutionary distant, Dictyostelium cells and neutrophils are morphologically similar and share common pathways for gradient detection (Charest and Firtel, 2006). Dictyostelium cells chemotax toward gradients of cAMP, meaning they move up a gradient of a single chemoattractant. Neutrophils are attracted by gradients of different chemotactic factors, which are released in the case of infection or inflammation. Both cell types detect external gradients of a chemoattractant with high sensitivity and perform a strong internal signal amplification by means of feedback loops comprising phosphatidylinositol-3 kinase (PI3K) and its catalytic product, phosphatidylinositide 3,4,5-trisphosphate (PIP3). Dictyostelium cells and neutrophils slightly differ, however, regarding the role of the cytoskeleton during gradient detection. Compared to amoeboid cells, fibroblasts are larger and have a different, more complex cytoskeletal architecture resembling the one in neuronal growth cones. Fibroblasts can sense and migrate up an attractant gradient of platelet-derived growth factor (PDGF) but are much slower than neutrophils. The intracellular signaling underlying gradient detection in fibroblasts shares key components with Dictyostelium and neutrophils. However, fibroblasts lack important feedback mechanisms and therefore have a low sensitivity and a strong dependence on the absolute PDGF concentration

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when navigating in attractant gradients (Schneider and Haugh, 2005). Interestingly, they employ local protein synthesis at the leading edge to promote directional growth (Shestakova et al., 2001), a principle that is also observed in growth cone guidance (Piper and Holt, 2004). At first view, neuronal growth cone guidance differs substantially from cell migration. Growth cones advance and navigate relatively independently from the neuronal cell body. Morphologically, growth cones are already polarized and have an intrinsic bias between the axon shaft and the protruding peripheral domain. In the nervous system, growth cones face attractive as well as repulsive gradients and respond to different classes of guidance factors, which can substantially differ in their downstream signaling. Nonetheless, many general features of eukaryotic gradient detection are conserved between migrating cells and growth cones. It is the aim of this review to point out these features and establish common ground between two related fields of research.

2. Mechanisms of Gradient Detection in Selected Eukaryotic Cell Types 2.1. Chemotaxis of Dictyostelium Dictyostelium cells detect and migrate up gradients of cAMP. In a cAMP gradient, the cells adopt a strong internal signaling polarity, which arises from localized activities of phosphatidylinositol-3 kinase (PI3K) and the PI3-phosphatase PTEN as well as a corresponding internal gradient of phosphatidylinositide 3,4,5-tris-phosphate (PIP3) along the cell membrane. As a result, the cytoskeleton is rearranged, morphological polarization becomes apparent, and the cell starts to move in the direction of the gradient source based on actin polymerization and myosin-mediated contraction. 2.1.1. Chemotaxis of Dictyostelium relies on spatial gradient sensing The myxameba Dictyostelium discoideum is unicellular in its vegetative cycle. When the food supply is exhausted, the cells start to aggregate to form a multicellular structure (pseudoplasmodium). The aggregation is triggered by cAMP, which is secreted by the cells themselves and functions as a chemoattractant. Thousands of individual cells thus move up a gradient of cAMP and converge at a central point (Strmecki et al., 2005). The response of Dictyostelium cells to cAMP can be easily studied in vitro with soluble gradients emanating from micropipettes and has served as an excellent model system for eukaryotic chemotaxis and gradient detection (Van Haastert and Devreotes, 2004). From early experiments, it was deduced that Dictyostelium cells employ a spatial gradient-sensing mechanism

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because they are able to migrate up a gradient on a more or less straight line. However, because cells experience an overall increase of the surrounding cAMP concentration when they move up a gradient, one could still speculate about a temporal gradient-sensing mechanism. Tani and Naitoh (1999) carefully tested the controversial concepts about spatial and temporal gradient sensing. Strong support for a spatial-sensing mechanism comes from the fact that cells still move up a cAMP gradient when the overall concentration of cAMP decreases over time (Fig. 1.1). A Dictyostelium cell can measure about 1% of concentration difference over its total length (10–20 mm) in a spatial cAMP gradient (Mato et al., 1975) and move with a speed up to 20 mm/min (Swanson and Taylor, 1982). The efficiency of gradient detection depends on both gradient slope and absolute cAMP concentration. In cAMP gradients with the same slope, chemotaxis is optimal for an intermediate gradient midpoint concentration, that is, there is a biphasic dependence of the efficiency of chemotaxis on the absolute cAMP concentration. Very high or very low cAMP concentrations are suboptimal for chemotaxis even when the gradient slope is optimal. In gradients with the same (optimal) gradient midpoint concentration, cells chemotax more efficiently in steeper gradients. A shallow gradient with

Figure 1.1 Spatial gradient sensing in Dictyostelium. At time point t1, the cell is at position x1 on its way up the gradient and is surrounded by the cAMP concentration c1. It faces a steep gradient along its length. Over time, the steepness of the gradient is decreasing while the cell still continuously encounters higher cAMP concentration at its leading edge than at its trailing edge. At time t2, after having crawled a distance Dx up the gradient, the cell is surrounded by the cAMP concentration c2, which is smaller than c1, although x2 is closer to the cAMP source than x1. Successful chemotaxis in the direction of the gradient in the sketched scenario is only possible for cells employing a spatial gradient sensing mechanism.

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optimal midpoint concentrations therefore possibly elicits a weaker chemotactic response than a steeper gradient with suboptimal gradient midpoint concentration. This means the cell measures relative and not absolute differences in concentration (Fisher et al., 1989). 2.1.2. Establishment of internal polarity: Feedback loops lead to signal amplification For spatial gradient sensing, the cell has to translate the external gradient into an internal signaling gradient. Corresponding to the direction of the external gradient, polarity is adopted. In chemotaxing Dictyostelium cells, this polarity is morphologically evident. A pseudopod is formed at the leading edge, and a uropod marks the trailing edge. Once polarized, the cell shows an asymmetry in sensitivity toward cAMP: The front is more sensitive than the rear (Swanson and Taylor, 1982). The strong and persistent morphological polarization and the correlated bias in cAMP sensitivity suggest the external concentration gradient is internally amplified. When does this amplification happen? Extensive studies have revealed the intracellular signaling cascade downstream of cAMP during chemotaxis. In short, cAMP binds to the 7-transmembrane-spanning receptor cAR1, which activates heterotrimeric G proteins. The Gbg subunit activates the Ras family of small G proteins, which subsequently recruits phosphatidylinositol-3 kinases (PI3K) to the plasma membrane. PI3K converts membrane-residing phosphatidylinositide 4,5-bis-phosphate (PIP2) to phosphatidylinositide 3,4,5-tris-phosphate (PIP3). The produced PIP3 mediates the membrane translocation and activation of a number of proteins containing PIP3-binding plextrin-homology (PH) domains. Among these are protein kinase B (PKB/Akt) and guanine nucleotide exchange factors for Rac (Rac GEFs). The latter are main regulators of the cytoskeletal rearrangements required for cell migration (Affolter and Weijer, 2005). Studies with a functional cAR1-GFP construct revealed that the receptor is uniformly distributed over the cell surface in polarized cells migrating in a cAMP gradient (Xiao et al., 1997). Measurement of G protein activation by FRET further shows that the extent of G protein activation in different regions of the cell reflects the extracellular cAMP concentration of the cAMP gradient faced by the cell (Jin et al., 2000; Xu et al., 2005b). Regarding receptor occupancy and G protein activation, the internal signal output is thus proportional to the input, and there is no strong asymmetric distribution of signaling molecules. The gradient amplification which accounts for a strong and persistent morphological polarization and a bias in sensitivity therefore has to occur downstream of G proteins. Using a GFP fusion of the PH-domain containing protein cytosolic regulator of adenyl cyclase (CRAC) as a sensor for PIP3, Parent et al. (1998) showed that PIP3 accumulates in a highly polarized fashion at the cell front and exclusively marks the site of pseudopod formation in cells facing a cAMP gradient. The internal PIP3

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gradient is up to sevenfold steeper than the external cAMP gradient ( Janetopoulos et al., 2004; Xu et al., 2005b). At the level of PIP3, small concentration differences of the external signal are translated into strong cell internal signaling polarity. How is this signal amplification achieved? As mentioned earlier, PIP3 is produced by PI3K. Dictyostelium has three PI3Ks, of which PI3K1 and PI3K2 are important for chemotaxis. Both PI3K1 and PI3K2 translocate rapidly to the cell membrane after a cAMP stimulus and are localized at the leading edge of chemotaxing cells. PI3K signaling is antagonized by the PI3-phosphatase PTEN, which breaks down PIP3 to PIP2. Dictyostelium cells lacking PTEN have defects in chemotaxis toward cAMP due to a rapid, erratic extension of multiple pseudopods and reduced polarity. In resting cells, a small portion of PTEN is membrane associated. Membrane association and function of PTEN are dependent on its PIP2 binding site. In polarizing cells, PTEN dissociates along the leading edge into the cytosol and accumulates at the cell rear: Its distribution becomes reciprocal to the one of PI3K. The inverse PTEN gradient shows little amplification with respect to the external cAMP gradient. However, PI3K and PTEN distributions taken together account for nearly all observed amplification on the level of PIP3 (i.e., PIP3 levels parallel the PI3K/PTEN ratio). PI3K/ PTEN/PIP3 participate in a feedback loop system. At the leading edge, recruitment of PI3K enhances local PIP3 production and the simultaneous decrease of PIP2, which is a decrease in PTEN binding sites. This causes PTEN to translocate to the rear. At the rear, PTEN lowers the PIP3 levels while increasing PIP2, creating in this way its own membrane-binding sites. The opposite enzyme activity of PI3K and PTEN sharpen the internal PIP3 gradient and stabilize the signaling polarity of the cell (Fig. 1.2). cAMP molecules binding at the cell rear only lead to a very attenuated signal because high levels of PTEN antagonize PI3K signaling (Funamoto et al., 2002; Iijima and Devreotes, 2002; Janetopoulos et al., 2004). The signal amplification arising from self-enhanced segregation of PI3K and PTEN potentiates the efficiency of gradient detection. It leads to persistent chemotaxis and makes the system robust for small concentration fluctuations of the attractant cAMP gradient. A bias in sensitivity explains why cells tend to maintain their once-established polarity even when the direction of the gradient is altered. As observed by Swanson and Taylor (1982), chemotaxing cells respond with an L-shaped turn of the pseudopod instead of reorganizing their morphological polarization, when the cAMP-loaded microneedle producing the gradient is shifted to the side of the cell. A characteristic of systems containing feedback loops is their selforganizing property. Small stochastic fluctuations in the environment can lead to stable changes of the system. The PI3K/PTEN/PIP3 feedback loop may therefore explain the transient, spontaneous polarization and random migration of Dictyostelium cells in the presence of a uniform

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PI3K External cAMP PIP2 PI3K activity

PTEN activity

PTEN

PAK inactive MHC kinase

PIP3 Uropod

Pseudopod

PTEN

PI3K PIP3

PIP3

MyosinII-mediated contraction Uropod

Rac GEFs Rac WASP

Actin polymerization Pseudopod

f-actin Myosinll

Directional movement

Figure 1.2 Signal amplification in Dictyostelium gradient sensing. An external cAMP gradient triggers a slightly amplified gradient of PI3K signaling and a nonamplified, inverse gradient of PTEN signaling inside the cell. A feedback loop comprising PI3K and PTEN builds up a highly amplified gradient of PIP3 at the cell membrane. PIP3 recruits PH domain containing proteins such as Rac GEFs to the membrane at the pseudopod. Activation of Rac and cytoskeletal regulators such as WASP drives actin polymerization and finally leads to directional movement. At the uropod, the inactivation of MHC by PAK enhances myosinII-mediated contraction.

cAMP stimulus. Cells treated with a uniform dose of cAMP show first a rapid (1–10 s) and uniform rise of PIP3 at the cell membrane, which is associated with a peak of unbiased actin polymerization. Subsequently, between 30–60 s after the stimulus, there is a lower second peak of PIP3 accumulation and actin polymerization, which is localized at the sites of randomly emerging pseudopods. For a certain period of time, cells move around in the absence of a cAMP gradient (Chen et al., 2003). Postma et al. (2003) further analyzed how the uniform accumulation of PIP3 at the membrane after a uniform cAMP stimulus subsequently organizes in small and distinctly restricted patches. In contrast to the PIP3 accumulation in a gradient, where PIP3 always rises at the gradient near side, PIP3 patches appear randomly at the cell membrane after a uniform cAMP stimulation. At the site of these PIP3 signaling patches, pseudopods are likely to form (Fig. 1.3). After low cAMP doses, the probability of the patches decreases, but their size, intensity, and lifetime is the same as after high cAMP doses. The signal output is partially decoupled from the input. This observation

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Uniform cAMP

cAMP gradient

cAMP gradient, latrunculinA

0s

10 s

30 s

40 s

Figure 1.3 Polarization and gradient sensing in Dictyostelium. After a uniform dose of cAMP, a GFP-PH domain sensor for PIP3 (shown in gray) is rapidly recruited from the cytoplasm to the cell membrane, indicating a first phase of uniform increase of PIP3 at the cell membrane (10 s). After approximately 30 s, the PIP3 signal has segregated into distinct patches, which precede the formation of pseudopods. In a gradient of cAMP (the source of cAMP is indicated by an arrow), only one PIP3 signaling patch forms after the first uniform rise of PIP3 at the membrane. It points at the uphill direction of the gradient and marks the side of the pseudopod. Cells treated with latrunculinA, which inhibits actin polymerization, are rounded up and cannot form pseudopods, meaning they cannot morphologically polarize. However, the sequence of PIP3 accumulation and polarization at the membrane in response to a cAMP gradient is the same as in untreated cells, indicating gradient sensing precedes morphological polarization.

strongly suggests self-organizing properties of the patches (Postma et al., 2004). In accordance with the notion that this self-organization emerges from the PI3K/PTEN feedback loop, Chen et al. (2003) found that abrogation of either PI3K or PTEN affects the localized PIP3 accumulation after a uniform cAMP stimulus. 2.1.3. Cytoskeletal readout: Morphological polarization is not required for gradient detection Morphological polarization and directed movement during chemotaxis are performed by the cytoskeleton. The pseudopod protrudes through localized actin polymerization, whereas the uropod contracts in a myosinII-dependent fashion, thus enabling the cell to move forward. Actin polymerization at the leading edge is controlled among others by Dictyostelium Rac (Chung et al., 2000) and the PIP3 binding adaptor Wiskott-Aldrich syndrome protein (WASP) (Myers et al., 2005). In cells facing a cAMP gradient, myosinII translocates to the cell rear and myosin heavy chain (MHC) kinase to the leading edge. Active MHC kinase at the leading edge is thought to phosphorylate cortical MHC, causing the disassembly of myosinII filaments,

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which subsequently reassemble at the cell rear (Rubin and Ravid, 2002). At the cell rear, MHC kinase is inhibited by the ser/thr kinase PAKa. PAKa is activated by Akt after a cAMP stimulus. Although the level of Akt activation is highest at the cell front, active PAKa is predominantly found at the cell rear, where it colocalizes with myosinII. So far, it is not completely understood how activated PAKa translocates to the cell rear (Chung and Firtel, 1999; Chung et al., 2001). Knockout or inhibition of the mentioned cytoskeletal regulators was shown to impair morphological polarization and chemotaxis in Dictyostelium cells. However, one has to be careful to distinguish the difference among gradient detection, morphological polarization, and directed movement (Devreotes and Janetopoulos, 2003). Gradient detection depends on the translation of an external gradient to an amplified internal gradient. This signaling asymmetry leads to an asymmetry in cell shape accomplished by the cytoskeleton. Morphological polarization may be only a passive readout and not a requirement for gradient detection, so that the lack of morphological polarization does not necessarily suggest failing gradient detection. Similarly, the inability to move in a given direction tells nothing about the capability to sense this direction. Signaling events generally required for cell motility are likely to disrupt directional movement, although they may not be required specifically for sensing of direction or maintenance of directionality during movement. Indeed, experiments with latrunculinA have shown that morphologically unpolarized and immobile Dictyostelium cells can still sense a cAMP gradient. LatrunculinA blocks actin polymerization and causes cells to round up. The formation of pseudopods and the general motility of the cells are inhibited. However, in a gradient of cAMP, PIP3 still accumulates in a polarized fashion at the gradient near side of the cell (see Fig. 1.3). The internal PIP3 gradient is an amplified copy of the external cAMP gradient and the extent of amplification is only a little smaller in unpolarized latrunculinA treated cells than in untreated cells (Janetopoulos et al., 2004; Parent et al., 1998). The PI3K/ PTEN feedback loop seems to be intact in unpolarized cells because they display the same self-organized formation of PIP3 patches after a uniform cAMP stimulus as it is seen in cells competent to polarize (Postma et al., 2004). Taken together, gradient sensing and internal signal amplification in Dictyostelium is relatively independent of the cytoskeletal rearrangements, which are mainly required for morphological polarization and cell motility.

2.2. Chemotaxis of mammalian neutrophils Gradient detection in mammalian neutrophils is substantially similar to the one in Dictyostelium cells. Neutrophils attracted by gradients of chemotactic factors translate the external gradients into amplified internal gradients of

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PI3K activity and PIP3 accumulation. The feedback loops leading to signal amplification are comparable to Dictyostelium feedback loops but differ somewhat in their orchestration. Furthermore, neutrophils are specialized to navigate in several superimposed gradients of different chemotactic factors and have developed mechanisms to integrate a complex signal environment. 2.2.1. Gradients guide neutrophils to sites of infection In the mammalian immune system, an elaborate network of chemotactic factors guides neutrophil granulocytes (polymorphonuclear leukocytes) to sites of infection and inflammation, where they engulf invading bacteria, dead cells, and foreign particulate matter by phagocytosis. Chemotactic factors are either bacterial (e.g., formyl-MetLeuPhe [f MLP]) or host derived (e.g., interleukin 8 [IL-8], leukotriene B4 [LTB4], complement factor C5a). Chemotaxis of neutrophils toward sources of chemotactic factors has been extensively studied in vitro with either primary neutrophils or differentiated human promyelocytic leukemia cells (HL-60 cells), which look and behave like neutrophils. Although evolutionarily distant, mammalian neutrophils and Dictyostelium cells share many signaling mechanisms in gradient detection. When exposed to a uniform dose of chemotactic factor neutrophils morphologically polarize and develop a leading pseudopod and a trailing uropod. They start random migration (chemokinesis) with typical amoeboid movement. Ranking among the fastest moving mammalian cells known so far, neutrophils can reach speeds up to 20 mm/min, which is about the same velocity reached by aggregating Dictyostelium cells (Niggli, 2003). Like other chemotaxing eukaryotic cells, neutrophils employ a spatial mechanism of gradient sensing (Zigmond, 1974). In a concentration gradient, they quickly orient their pseudopod toward the source of a chemotactic factor and initiate chemotaxis. Neutrophil orientation in a gradient depends on both steepness of the gradient and the mean concentration of the chemotactic factor. Steeper gradients orient cells more efficiently. For all gradient slopes, orientation is optimal for a medium mean concentration of the chemotactic factor. When the overall concentration of a chemotactic factor gets too high, cellular motility is inhibited. In optimal gradients, neutrophils can detect down to 1% difference in concentration of a chemotactic factor over their length (10 mm) (Lin et al., 2004; Zigmond, 1977). Similar to Dictyostelium cells, morphologically polarized neutrophils also show a polarity in sensitivity with a more responsive leading edge. Due to this asymmetry in sensitivity, neutrophils following a gradient frequently make U-turns when the direction of the gradient is reversed instead of reversing their polarity (Zigmond et al., 1981). Each neutrophil can respond to multiple chemotactic factors and many different chemotactic factors are simultaneously released from a single site

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of inflammation. In vivo, neutrophils thus have to navigate in a complex environment of superimposed, graded distributions of chemotactic factors. To fulfill their role efficiently, they have to assess a combination of chemotactic factors in a meaningful way. Among chemotactic factors, there is a dominance of so-called ‘‘end target chemotactic factors’’ (f MLP and C5a) over ‘‘intermediary endogenous chemotactic factors’’ (IL-8 and LTB4). Neutrophils can migrate down the concentration gradient of an intermediary chemotactic factor by responding to a distant end target chemotactic factor, but not vice versa. This is true for a wide range of concentration combinations and may be an important mechanism to respond to several sites of inflammation in a sequential way without being trapped in the middle of two opposed gradients. On the other hand, the hierarchy of chemotactic factors can become fatal for septic patients, which have increased levels of f MLP and other dominant chemotactic factors. The concentration of dominant chemotactic factors is far above the optimal midpoint concentration for gradient detection, and neutrophils fail to detect sites of infection because they are inhibited in chemotaxis. In the normal situation, however, neutrophils can very well migrate beyond a saturating concentration of one chemotactic factor (which alone would stop the cell) in response to a second gradient of a different chemotactic factor. Such multistep navigation allows neutrophils to be attracted over a greater distance and arrive at multiple target areas dependent on their expression pattern of receptors for chemotactic factors (Foxman et al., 1997, 1999). The sophisticated integration of several chemotactic signals is based on the resistance of certain, but not all, receptors for chemotactic factors to heterologous desensitization (Richardson et al., 1995) and the employment of different signaling pathways in response to different chemotactic factors (Heit et al., 2002). 2.2.2. PI3 kinase/PIP3 signaling builds up internal polarity All chemotactic factors involved in neutrophil chemotaxis bind to G protein-coupled receptors (GPCRs). As shown for the receptor for Ca5 (Ca5R), chemotactic factor receptors are homogenously distributed in the plasma membrane of unstimulated as well as gradient stimulated, polarized chemotaxing neutrophils (Servant et al., 1999). Like in Dictyostelium cells, the internal amplification of the external concentration gradient takes place on the level of PIP3 accumulation at the cell membrane and can be visualized by the recruitment of PH-domain containing, PIP3 binding sensors such as a construct of the PH-domain of Akt and GFP (PHAkt-GFP). PHAkt-GFP gets locally recruited to the pseudopod of neutrophils stimulated with an external gradient of a chemotactic factor and forms an intracellular gradient. This PHAkt-GFP gradient is approximately sixfold steeper than the external concentration gradient (Servant et al., 2000), which is about the same amount of amplification observed for Dictyostelium cells.

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Is this signal amplification achieved by the same mechanisms that act in Dictyostelium? The relevant isoform of PI3K, which is expressed in neutrophils and signals in chemotaxis is PI3Kg. Neutrophils from PI3Kg null mice fail to produce PIP3 after stimulation with various chemotactic factors. The chemotaxis of neutrophils lacking PI3Kg is impaired because the cells are less motile and cannot stabilize and maintain their leading edge (Hannigan et al., 2002; Hirsch et al., 2000; Li et al., 2000; Sasaki et al., 2000). Interestingly, PTEN, which is crucial for gradient detection in Dictyostelium, does not seem to play a major role in neutrophils. Antibody stainings as well as different PTEN-GFP constructs failed to reveal an asymmetric PTEN localization in polarized and chemotaxing neutrophils (Lacalle et al., 2004; Xu et al., 2003). Recent research strongly suggests that the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1) fulfills the role of breaking down PIP3 and restricting PI3K activity to the pseudopod in neutrophils. Neutrophils lacking SHIP1 have a phenotype reminiscent of Dictyostelium cells lacking PTEN, suggesting that SHIP1 and PTEN have similar functions in gradient detection and polarization in the two different cell types (Nishio et al., 2007). 2.2.3. Feedback loops involving f-actin and Rho amplify the signaling asymmetry Neutrophils respond to chemotactic factors with rapid polymerization of actin. Subsequently, the filamentous actin (f-actin) partially depolymerizes and redistributes focally into the emerging pseudopod while the rounded cells acquire a polarized morphology (Howard and Oresajo, 1985). The microtubule network rearranges in a way that the majority of microtubules gets oriented toward the uropod and is excluded from the actin-rich pseudopod. This rearrangement depends on an intact actin cytoskeleton and activated myosinII. It takes place without local microtubule disassembly (Eddy et al., 2002). The microtubule network was found to stabilize the directionality during neutrophil chemotaxis (Xu et al., 2005a). Approximately 50% of the neutrophils that get morphologically polarized in response to a uniform dose of a chemotactic factor also display an asymmetric recruitment of the PIP3 sensor PHAkt-GFP to the pseudopod, supporting the notion that they have an intrinsic capacity to build up a signaling polarity in the absence of a polarized external stimulus (Servant et al., 2000). Indeed a uniform dose of membrane permeable PIP3 can mimic a chemotactic factor and induce its own asymmetric accumulation as well as polarized polymerization of actin. PIP3 fails to induce polarization in the presence of PI3K or Rho family kinase inhibitors. This indicates that PI3K signals downstream as well as upstream of PIP3 and thus participates in a positive feedback loop which also includes members of the Rho GTPases (Niggli, 2000; Weiner et al., 2002).

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Which members of the Rho family kinases are involved and what are their individual roles? Trimeric G proteins downstream of the chemotactic factor receptors activate Rac via Gi and RhoA via G12/G13. Activation of Rac has been shown to mediate the so-called ‘‘frontness signal,’’ that is, PIP3 accumulation, actin polymerization, and the formation of pseudopods at the leading edge. The stabilization of a single pseudopod depends on the additional activation of cdc42 (Srinivasan et al., 2003). The effect of Rac on the actin cytoskeleton is mediated among others via WAVE (WiskottAldrich syndrome protein family Verprolin-homologous protein) protein complexes and other leading-edge complexes scaffolded by hematopoietic protein-1 (Hem-1) (Weiner et al., 2006). Activated RhoA, on the other hand, translocates to the trailing edge and sets up a ‘‘backness signal,’’ which is characterized by activation of Rho kinase (Rock), accumulation of myosinII, and the formation of a uropod (Xu et al., 2003). Frontness (Rac) and backness (RhoA) signals mutually inhibit each other, enhancing their own polarity. The activation of RhoA at the back depends on PIP3induced activation of cdc42 at the leading edge. The molecular mechanisms, which connect cdc42 to RhoA across the cell’s diameter are not yet understood and seem to require intermediate effectors (Van Keymeulen et al., 2006). The exclusion of RhoA signaling from the pseudopod and its segregation to the uropod seem to be partially monitored by the actin cytoskeleton. Actin polymerization suppresses and localizes RhoA activity by a yet-unknown mechanism, corroborating the notion that actin dynamics are not only a passive readout of the system, but also are rather actively integrated into the feedback loop (Wong et al., 2006). Further evidence indicates that the PIP3 feedback loop in neutrophils depends on f-actin. Inhibition of actin polymerization or depolymerization by latrunculinA or jasplakinolide, respectively, leads to a markedly attenuated and transient translocation of PH-sensor constructs to the membrane, indicating that PIP3 accumulation fails to get stabilized at the leading edge in neutrophils with disturbed f-actin organization (Wang et al., 2002). In summary, signaling pathways during neutrophil chemotaxis (Fig. 1.4) include positive feedback loops between PIP3 accumulation, the activation of Rac, and actin polymerization at the pseudopod. At the uropod, RhoA activity enhances myosinII-mediated contraction.

2.3. Chemotaxis of mammalian fibroblasts In comparison to amoeboid cells, fibroblasts chemotaxing in attractive gradients of PDGF do not employ a sophisticated and highly specialized signaling network of positive feedback loops. The amplification of the external gradient is mainly based on morphological polarization. In addition to PI3K signaling, localized protein synthesis of b-actin, a mechanism not observed in Dictyostelium or neutrophils, affects directional movement in the gradient.

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Figure 1.4 Signal transduction during neutrophil gradient sensing. The binding of a chemotactic factor to its G protein-coupled receptor leads to the activation of PI3K and Rho GTPases. At the pseudopod, signaling is dominated by positive feedback loops of PI3K, PIP3, Rac, and f-actin, which mutually enhance their activities. Rac signaling at the pseudopod inhibits RhoA signaling, which is thus restricted to the uropod and there drives myosinII-mediated contraction through activation of Rock.

2.3.1. A gradient of PDGF attracts fibroblasts during wound healing One step in the complex process of wound healing and tissue repair is the migration of fibroblasts toward the wound. This migration is triggered by PDGF, which is released at the site of injury and forms a concentration gradient in the surrounding tissue (Deuel and Kawahara, 1991). Like Dictyostelium cells or neutrophils, fibroblasts have to detect an external concentration gradient and respond to it with directed locomotion. With respect to morphology and cellular architecture fibroblasts differ markedly from the cell types discussed previously. They are larger (50–250 mm compared to 10–20 mm for amoeboid cells) and migrate at a much lower speed (approximately 2 mm/min compared to 20 mm/min). Fibroblast

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polarity and motility depends not only on the asymmetrical distribution of f-actin and myosinII, but also on the microtubule network and local substrate adhesion (Kole et al., 2005; Small et al., 2002; Vasiliev, 1991). In short, forward migration is driven by localized actin polymerization, which leads to the extension of broad and flattened lamellipodia in the direction of movement. During the protrusion of the leading edge, so-called ‘‘ruffles’’ are formed by the bending of the cell membrane (Abercrombie et al., 1970). Addition of PDGF to fibroblasts in vitro causes rapid lamellipodial actin polymerization and the appearance of membrane ruffles (Mellstro¨m et al., 1988). In the presence of a PDGF gradient, fibroblasts chemotax toward the PDGF source. This chemotactic response is optimal for an intermediate mean PDGF concentration and is inhibited at high concentrations (Seppa¨ et al., 1982). The PDGF signaling cascade is as follows: PDGF binds to the PDGF receptor (PDGFR), a receptor tyrosine kinase (RTK). Phosphorylated PDGFR binds and activates among others PI3K, phosphatidylinositolspecific phospholipase C-g (PLC-g), and Ras-GTPase–activating protein (ras GAP) (Kazlauskas and Cooper, 1990; Kundra et al., 1994). Although PDGF binds to a different receptor type (receptor tyrosine kinase) than the attractants mediating chemotaxis in Dictyostelium and neutrophils (GPCRs), it activates the same downstream effector PI3K. 2.3.2. PI3 kinase/PIP3 and Rho GTPase signaling transduces the external gradient PI3K/PIP3 signaling plays a well-established role in fibroblast gradient sensing. The response of fibroblasts toward PDGF is abolished in the presence of PI3K inhibitors (Derman et al., 1997). A shallow external PDGF gradient triggers a steeper PIP3 gradient in the fibroblast membrane, which can be visualized by a PHAkt-GFP sensor. The region with the highest GFP-AktPH signal exhibits lamellipodia spreading toward the PDGF source (Haugh et al., 2000). The effect of PDGF on fibroblasts can be mimicked by exogenous addition of membrane permeant PIP3. Interestingly, PIP3 does not activate or enhance PI3K signaling like it was shown in Dictyostelium cells or neutrophils. In fibroblasts, PIP3 exclusively signals downstream of PI3K and does not seem to be integrated in a positive feedback loop (Derman et al., 1997). In this context, it is noteworthy that different PI3K isoforms are activated by GPCRs (i.e., the receptors for chemotactic factors in neutrophils) and by PDGFR (Vanhaesebroeck et al., 2001). This could explain the differences in the PIP3 signaling cascade in fibroblasts and neutrophils. The opponent of PI3K, PTEN, is certainly involved in PDGF-induced chemotaxis. However, there is little data about PTEN distribution in chemotaxing fibroblasts and its participation in the development and the maintenance of PIP3 polarity. In unstimulated cells, PTEN is uniformly

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distributed (Tamura et al., 1998). Upon PDGF stimulation, PTEN is recruited to the cell membrane by PDGFR via the adaptor protein NHERF (Naþ/Hþ exchanger regulatory factor) into a ternary complex. Fibroblast with disrupted PTEN signaling display prolonged PI3K pathway activation, increased cell motility, and enhanced cytoskeletal rearrangements after PDGF stimulation (Liliental et al., 2000; Takahashi et al., 2006). These findings suggest fibroblast PTEN antagonizes PI3K signaling. It may not exclusively localize to the cell rear like in Dictyostelium but rather may have the same distribution as PDGFR. PIP3 produced by PI3K activates the Rho GTPases Rac and cdc42. Activated Rac stimulates actin polymerization, the characteristic membrane ruffling, and lamellipodia formation in fibroblasts. When microinjected, Rac mimics the effects of PDGF. The action of Rac in fibroblasts is exclusively downstream of PIP3 because the Rac-induced cytoskeletal rearrangements are not prevented by PI3K inhibitors (Nobes et al., 1995). In migrating fibroblasts, a gradient of Rac activation and colocalized actin polymerization peaks near the leading edge, meaning it is correlated with the direction of cell movement (Kraynov et al., 2000). The increase of actin polymerization during ruffle formation is triggered by the Rac-dependent activation of WAVE proteins, which activate, in turn, actin-related protein 2/3 (Arp2/3) complex (Miki et al., 1998; Suetsugu et al., 2003). Rac-null mouse fibroblasts have a dramatically changed morphology. Whereas wildtype cells respond to PDGF with the formation of f-actin rich dorsal and lateral ruffles, Rac-null cells lack this response and only generate small filopodia-like protrusions. However, they show activation of PI3K and PIP3 accumulation to the same extent as wild-type cells. Mutant cells still chemotax in response to PDGF, but they do not translocate via lamellipodia and ruffles, but rather via finger-like protrusions, and their average velocity is reduced. In the absence of Rac, a PDGF gradient can thus still be correctly sensed. Rac is therefore required for the formation of lamellipodia and ruffles mediating efficient cell translocation, but not for gradient detection itself (Vidali et al., 2006). Interestingly, the spatiotemporal dynamics of RhoA activity differ in randomly migrating fibroblasts and fibroblasts chemotaxing toward PDGF. Pertz et al. (2006) used a recombinant sensor with intramolecular FRET that responds to RhoA activation. In randomly migrating cells, RhoA activity is concentrated in a sharp band at the edge of protrusions and in peripheral ruffles, but it is very low in the cell body. In cells migrating toward a gradient of PDGF, however, the PDGFinduced protrusions display low RhoA activity. This can be explained by the fact that PDGF activates Rac and activated Rac downregulates RhoA activity. Taken together, spatial gradient detection in fibroblasts is mediated by PI3K/PIP3, but a positive feedback loop involving PI3K and/or downstream Rho family kinases is apparently missing.

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As pointed out by Schneider and Haugh (2005), the absence of positive feedback loops leads to decreased sensitivity and stronger dependence on the midpoint PDGF concentration in a gradient. According to a simple model, fibroblast gradient detection differs depending on the PDGF concentration. At low PDGF concentrations, the gradient sensing is absolute, meaning the level of PI3K activation is directly proportional to the receptor activation because cytosolic PI3K is not depleted. At midpoint PDGF concentrations, the gradient sensing is relative; front and rear of the cell have to share the common PI3K pool according to their receptor activation. At high PDGF concentrations, gradient sensing fails because all receptors are saturated: The PI3K activity at the leading edge is actually lower than in nonsaturating concentrations. Although the classical PIP3 feedback loop is missing, the morphological polarity of the cell, namely, protruding membrane structures at the leading edge may provide an enhancement of PI3K signaling. Once properly aligned with the gradient, the cell displays an intrinsic bias—it is more sensitive at the leading edge. To overcome the strong dependence on the midpoint concentration of PDGF gradients, fibroblasts may partially decay the encountered PDGF. Haugh (2006) proposes a model in which PDGFR activation is coupled to PDGF endocytosis and intracellular proteolysis. PDGF consumption by the migrating cells might thus allow them to maintain an optimal gradient during the invasion of a dermal wound, where PDGF concentrations are likely to span a large scale. 2.3.3. Local protein translation at the leading edge mediates directional movement In early studies, inhibition of protein translation was reported to inhibit fibroblasts chemotaxis (Seppa¨ et al., 1982). More recent data show that this inhibition is not a rather unspecific global effect, but point out the importance of localized and controlled protein translation of defined mRNAs at the leading edge of migrating fibroblasts. Most importantly, b-actin mRNA gets located to the protruding pseudopod via a 30 UTR zip-code sequence. The translocation of b-actin mRNA to the leading edge correlates with the magnitude and direction of cell translocation. When the correct localization of b-mRNA is blocked, cells have collapsed lamellipodia and no leading edge; they lose their morphological polarity (Kislauskis et al., 1994, 1997). More precise analysis shows that directionality and persistence of migration are decreased in fibroblasts with delocalized b-actin mRNA without a decrease in the rate of locomotion. This suggests that mRNA localization is not required in general for cell migration, but rather for directional movement (Shestakova et al., 2001). The localization of b-actin mRNA requires the zip-code binding protein 1 (ZBP-1). ZBP-1 binds mRNA in the nucleus, prevents premature translation initiation, and regulates its transport. At the destination point, ZBP-1

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gets phosphorylated by src, which disrupts RNA binding and activates translation. Local translation can therefore be regulated by spatial restricted activity of src (Hu¨ttelmaier et al., 2005). Notably, src is activated in fibroblasts upon PDGF stimulation. Src family kinases (SFK) can directly bind to activated PDGF receptor and phosphorylate various targets proteins involved in cytoskeletal regulation (Shah and Vincent, 2005). Based on these data, it is conceivable that ZBP-1 is phosphorylated by src upon PDGF stimulation and subsequently triggers the local translation of b-actin at the fibroblast leading edge. Newly synthesized b-actin monomers are a preferential substrate for actin polymerization and therefore establish nucleation sites. Local translation of b-actin may therefore be a more efficient mechanism for driving f-actin accumulation than the usage of the already existing pool of actin monomers in the cell (Shestakova et al., 2001). Local protein translation is required for fibroblast gradient detection, because it sets up the morphological polarization, which plays a crucial role in the establishment and maintenance of the internal signaling gradient. Taken together, morphological polarity and directional movement of fibroblasts in a PDGF gradient is the result of several signaling pathways converging at the cytoskeleton. Local protein translation as well as the activation of PI3K/PIP3 signaling promotes actin polymerization and membrane ruffling at the leading edge, whereas the trailing edge is dominated by RhoA/myosinII activation (Fig. 1.5).

2.4. Pathfinding of neuronal growth cones Axon guidance is either attractive or repulsive, and guidance factors fall into distinct classes, such as the netrins, semaphorins, and ephrins, which differ with respect to receptors and downstream signaling (Guan and Rao, 2003). Looking at three major responses of a growth cone to a gradient, attractive turning, repulsive turning, and collapse, it becomes clear that, in part, alternative signaling pathways can execute theses responses. Depending on the guidance factor, signaling during turning or collapse differs and corresponds to different aspects of gradient detection and chemotaxis of migrating cells. 2.4.1. Growth cones respond to gradients of axon guidance cues Gradients of axon guidance molecules are essential for wiring up the developing nervous system. For example, an attractive gradient of netrin guides vertebrate commissural axons ventrally to the floorplate in the developing spinal cord (Kennedy et al., 2006). After reaching the floorplate, commissural axons cross the midline and are subsequently directed anteriorly toward the brain by a combination of an attractive Wingless 4 (Wnt4) and a repulsive sonic hedgehog (Shh) gradient (Charron and TessierLavigne, 2005). A repellent gradient of slit regulates midline crossing and defines the distance of longitudinal axon tracts to the midline in Drosophila

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Figure 1.5 Signal transduction in fibroblasts chemotaxing toward PDGF. Downstream of the PDGF receptor, parallel pathways control actin polymerization at the leading edge. Activation of PI3K and subsequent polarized accumulation of PIP3 at the membrane lead to the dominance of Rac versus RhoA signaling. Rac activates among others WAVE and thus enhances the formation of a stable, f-actin^rich leading edge. Mutual inhibition of Rac and RhoA may sharpen the internal signaling polarity between leading and trailing edge. Additional to the action of PI3K, local phosphorylation of ZBP1, which is likely to happen via src downstream of the PDGF receptor, increases the availability of b-actin mRNA for translation. Newly translated b-actin at the leading edge supports actin polymerization.

(Simpson et al., 2000). Besides providing directional information, gradients in the nervous system also confer positional information during topographic mapping. The development of the retinotopic map is controlled by the graded distribution of several different molecules, most prominently ephrinAs, which act as repellents during anterior–posterior mapping (McLaughlin and O’Leary, 2005).

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Important insights into how growth cones read and respond to these in vivo gradients have mainly come from in vitro assays. Chemotactic turning of growth cones was first shown in vitro in response to nerve growth factor (NGF). NGF not only has a growth promoting effect, but can also orient growth direction when applied in a soluble gradient emanating from a pipette into the medium. When the pipette is repositioned, growth cones can rapidly follow (in 10 min) the direction of the NGF source. The chemotactic response is seen over a range of concentrations but gets saturated at high NGF levels (Gundersen and Barrett, 1979; Letourneau, 1978). A neuronal growth cone responding to a graded distribution of a guidance molecule is at a different starting point compared to the previously discussed cell types. A neuron with a growing axon is already polarized, and the growth cone has the intrinsic property to advance on a more or less straight path. Gradients of guidance factors alter this path by causing attractive or repulsive turning, by stimulating further outgrowth, eliciting sidebranching, stopping the growth cone at a specific position, or causing collapse and subsequent retraction. These manifold and partially interrelated reactions are specialized readouts of gradient detection. For the sake of clarity, we will focus on gradient detection in the context of growth cone turning, which has been studied extensively with the help of simple in vitro assays. In addition, we will briefly discuss gradient detection during topographic mapping. Like in chemotaxing cells, signaling during gradient detection in growth cones has to establish a polarized distribution of intracellular effectors mirroring the external gradient and finally rearranges the cytoskeleton in a localized fashion to trigger directional movement. For the proper establishment of the neuronal connectivity, different subsets of axons have to react differently to the same guidance factors. Moreover, reactions to attractive and repulsive gradients have to be distinguished. Depending on the context, the same growth cone may react in a different way to the same guidance factor. Signaling cascades elicited by gradients of axon guidance factors therefore have to be versatile and amenable to modulations. Consequently, different intracellular signaling pathways can elicit the same morphological and mechanistic event: a turning reaction of the growth cone. The most important of these pathways will be introduced in the following section. The growth cone advances by means of actin polymerization, which drives the protrusion of filopodia and lamellipodia. Filopodia play an important role for the turning response: Just before turning, an increasing number of filopodia forms on the side of the growth cone facing the (attractive) gradient. Elimination of filopodia with cytochalasinB inhibits turning without inhibiting axon extension (Zheng et al., 1996). Growth cone turning could thus be interpreted as a localized and biased promotion of filopodia. Alternatively, turning can be conceptualized as a localized

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collapse at the side of the growth cone, which points away from the direction of the turn (Fan and Raper, 1995). Local disruption of actin bundles induces a partial growth cone collapse and subsequent repulsive turning. During this process, the local loss of f-actin causes microtubules to disappear and rearrange in the direction of the induced turn. The translocation of f-actin further leads to the appearance of asymmetric lamellipodial protrusions (Zhou et al., 2002). Both attractive and repulsive external gradients have to elicit intracellular signaling gradients. Theoretically, one can postulate two extreme possibilities: (1) The internal signaling gradients mediating attraction and repulsion differ completely with respect to the components, meaning they activate actin polymerizing and actin depolymerizing factors, respectively. (2) The internal signaling gradients are similar or identical except for their orientation. As exemplified in the following, these concepts do not mutually exclude each other and can both be supported by experimental data. 2.4.2. Signaling events during growth cone turning PI3 kinase/PIP3 signaling: Polarity versus gradient detection PI3K/PIP3 signaling is essential for polarization and gradient detection in Dictyostelium and neutrophils and is also implicated in fibroblast chemotaxis. It is therefore tempting to investigate the role of PI3K/PIP3 signaling in neuronal polarization and growth cone guidance. In neuronal cells, there are several levels of polarization during differentiation. First, a rounded cell initiates the outgrowth of neurites. Subsequently, one of the neurites specifies as an axon, which becomes distinct from the dendrites. Once the axon has adopted its identity and developed a growth cone, which turns in response to guidance factors, polarity can be also found inside the growth cone. Interestingly, PI3K/PIP3 signaling in neuronal cells is involved in all of these cell polarity decisions. NGF stimulation of PC12 cells, a neuronal cell line, leads to neuronal differentiation and neurite outgrowth. Neurite outgrowth and maintenance depends on PI3K activation and a subsequent rise of PIP3, or the injection of activated PI3K into PC12 cells is sufficient for the formation of neurites (Carter and Downes, 1992; Jackson et al., 1996; Kimura et al., 1994; Kita et al., 1998). Interestingly, PI3K/PIP3 signaling during neurite outgrowth closely resembles the formation of a leading edge in Dictyostelium cells or neutrophils. NGF stimulation of PC12 cells results in an early phase of global PI3K activation (0–10 min). PI3K activity subsequently gets restricted to the protruding neurites. The local accumulation of PIP3 leads to activation of Rac1/cdc42, thus promoting process outgrowth. Rac1/cdc42 further activates PI3K at the outgrowing protrusions and establishes a positive feedback loop similar to the one observed in Dictyostelium cells and neutrophils (Aoki et al., 2005).

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The same feedback loop functions again in the later step of axon specification. In hippocampal neurons, PI3K activity and PIP3 are selectively localized at the tip of the axon and act upstream of the polarity protein mPar3. The specification of the axon is strengthened by a positive feedback via Rac1/cdc42. A small rise in PIP3 in one neurite induced by a stochastic fluctuation or an external cue such as laminin will be strongly enhanced and can thus specify the axon (Me´nager et al., 2004; Shi et al., 2003). PI3K signaling causes phosphorylation and inactivation of GSK-3. In the absence of active GSK-3, APC can bind to the plus end of microtubules, trigger microtubule polymerization, and thus enhance fast axonal growth. In the dendrite, in contrast, PTEN signaling dominates, increasing the ratio of activated GSK-3 and attenuating the rate of elongation (Jiang et al., 2005; Zhou et al., 2004). The described development of polarity during neuronal differentiation is not necessarily linked to gradient detection. However, it can be regarded as a step toward the establishment of the axonal growth cone, a cellular structure specialized for the detection of guidance cues during axonal pathfinding. Once the axon is specified, PI3K signaling plays a role in growth cone collapse as well as growth cone turning. In an advancing axon of chick dorsal root ganglion cells, active PI3K keeps GSK-3 inactive in the f-actin–rich peripheral domain of the growth cone. PTEN is normally sequestrated to the microtubule-rich central domain of the growth cone. Stimulation with the repulsive cue semaphorin3A (Sema3A) causes an accumulation of PTEN at the membrane of the peripheral domain of the growth cone, where it antagonizes PI3K signaling and activates GSK-3. Active GSK-3 leads to decreased microtubule polymerization via interactions with microtubule binding proteins and induces growth cone collapse (Fig. 1.6A). The Sema3A-dependent collapse is prevented by either inhibition of GSK-3, stimulation of PI3K, or knockdown of PTEN. Contrariwise it can be mimicked by inhibitors of PI3K (Chadborn et al., 2006; Eickholt et al., 2002). Because Sema3A-induced collapse is mediated by PTEN/PI3K signaling, repulsive turning away from a Sema3A gradient could possibly be realized by localized recruitment of PTEN to the peripheral membrane at the side of the growth cone facing the repulsive gradient and a subsequent partial growth cone collapse. Attractive turning toward NGF gradients, on the other hand, was shown to depend on the activation of PI3K downstream of trkA receptor tyrosine kinase (Ming et al., 1999). Furthermore, attractive gradients of brain-derived neurotrophic factor (BDNF) or netrin-1 induce an accumulation of PIP3 on the gradient near side of the growth cone in Xenopus spinal neurons. Addition of membrane permeant PIP3 to the medium can induce intracellular PIP3 accumulation and chemoattraction. This PIP3-induced chemoattraction depends on Akt and PI3K activity, indicating a possible positive feedback loop reminiscent of the one in Dictyostelium and neutrophils.

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Figure 1.6 PI3K signaling in growth cone collapse and growth cone turning. (A) In an advancing growth cone, PI3K activity is localized to the f-actin^rich peripheral domain of the growth cone, where it inhibits GSK-3 signaling. PTEN resides mainly in the central domain of the growth cone and is thought to be sequestered to the microtubule network. During Sema3A-induced collapse, PTEN moves into the peripheral domain of the growth cone, antagonizes PI3K signaling, and activates GSK-3. Active GSK-3 acts on microtubules and positively affects growth cone collapse and retraction. (B) During attractive turning toward a gradient of netrin-1 or BDNF, PI3K signaling gets elevated at the gradient near side of the growth cone and supports asymmetric accumulation of PIP3. PIP3 signaling may elevate local Ca2þ via Akt dependent opening of TRPC channels in the cell membrane and thus trigger attractive turning. Because PI3K and PIP3 engage in a positive feedback loop, an external gradient of membrane permeant PIP3 can mimic netrin-1^ or BDNF-induced attraction.

Moreover, it seems that PIP3/Akt signaling can activate transient receptor potential channels (TRPCs), meaning it is located upstream of Ca2þ signaling (Fig. 1.6B) (J. Henely, personal communication). As detailed in the next section, high local Ca2þ elevations trigger attractive growth cone turning.

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Taken together, it seems a modulation of PI3K/PIP3 signaling in the peripheral domain of the growth cone can account for turning or collapse (see Fig. 1.6). In line with this, the peripheral domain of the growth cone might be comparable to the leading edge of a neutrophil or a Dictyostelium cell, whereas the central domain might correspond to the uropod. During Sema3A-induced growth cone collapse, the peripheral domain of the growth cone basically abandons its leading edge equivalence, becomes dominated by ‘‘uropod signaling,’’ and retracts. In an attractive gradient, PI3K/PIP3 signaling seems to be elevated on the gradient near side of the growth cone. In this way, the orientation of the peripheral domain with respect to the axon shaft is possibly shifted toward the source of the gradient and predates the turning of the whole growth cone. Although the picture of PI3K/PIP3 signaling in a growth cone responding to guidance cues fits well with models explaining polarity and chemotaxis in Dictyostelium and neutrophils, there are still many open questions. Strangely, it was reported that inhibition of PI3K reduces the collapse caused by ephrinA5 or slit2 in chick retinal ganglion cell axons (Wong et al., 2004). Furthermore, PI3K activity seems to be required for repulsive turning in a gradient of slit-2 (Ming et al., 1999). In contrast to the previous mentioned findings that stimulation of PI3K signaling prevents Sema3Adependent growth cone collapse, rather these data suggest that PI3K signaling positively affects growth cone collapse or repulsive turning. Further experiments are needed to elucidate these seemingly contradictory results. Ca2þ and cyclic nucleotides On the search for intracellular effectors of the observed turning in response to guidance molecules, it was discovered that growth cones also turn toward external gradients of Ca2þ plus Ca2þ ionophore or membrane permeant analogs of cAMP (Gundersen and Barrett, 1980; Lohof et al., 1992). cAMP and Ca2þ play the role of an intracellular messenger in the growth cone during detection and interpretation of gradients of many axonal guidance factors. Focal laserinduced photolysis (FLIP) of caged intracellular Ca2þ or caged intracellular cAMP leads to an attractive growth cone turn toward the release site of the caged component (Munck et al., 2004; Zheng, 2000). Attractive gradients of neurotransmitters such as acetylcholine (ACh) and glutamate (Zheng et al., 1994, 1996) cause an asymmetric Ca2þ influx in the growth cone, which is higher at the gradient near side. This asymmetric influx creates a local Ca2þ elevation and an internal gradient of Ca2þ in the growth cone with its high side pointing in the direction of the turn. As shown for the guidance factors netrin-1 and BDNF, Ca2þ influx during growth cone attraction is mediated by transient receptor potential channels (TRPCs), a class of Ca2þ permeable receptor-operated channels (Li et al., 2005; Shim et al., 2005; Wang and Poo, 2005).

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Intriguingly, a local Ca2þ elevation can mediate not only attractive, but also repulsive turning. Thus, FLIP of caged Ca2þ triggers repulsion away from the site of Ca2þ release, when the resting intracellular Ca2þ concentration is lowered by removal of Ca2þ from the extracellular medium and the local Ca2þ elevation generated by FLIP is accordingly lower. The amplitude of the local Ca2þ elevation relative to the global Ca2þ level determines the directionality of the response: A high local Ca2þ elevation causes attractive turning, and a low local Ca2þ elevation causes repulsive turning (Zheng et al., 2000). In line with this, the turning response of a growth cone toward Ca2þ dependent guidance factors can be switched from attraction to repulsion by manipulation of intracellular Ca2þ. Netrin-1 triggered attraction depends on influx of external Ca2þ as well as Ca2þ release from internal stores. If one of these sources is blocked, that is, if the induced Ca2þ elevation is decreased, attraction is switched to repulsion (Hong et al., 2000). The extend of a Ca2þ elevation caused by a guidance factor is crucially influenced by cAMP signaling. cAMP levels act like a switch determining whether the response to a Ca2þ-dependent guidance factor is attractive or repulsive. BDNF, Ach, or netrin-1 gradients, which normally induce attractive turning of Xenopus growth cones by high local Ca2þ elevation, lead at the same time to an intracellular increase of cAMP. If cAMP signaling is blocked by competitive analogs of cAMP or inhibitors of its downstream effector protein kinase A (PKA), attraction is converted to repulsion (Ming et al., 1997; Song et al., 1997). Gradients of myelinassociated glycoprotein (MAG), on the other hand, which normally trigger repulsive turning, become attractive upon pharmacological activation of cAMP signaling pathways (Song et al., 1998). A repulsive gradient of MAG induces an intracellular Ca2þ elevation in the shape of a gradient with the highest Ca2þ on the gradient near side of the growth cone. However, this graded elevation of Ca2þ is about half of that associated with the attractive turning induced by a netrin-1 gradient. Elevation of cAMP signaling enhances the MAG-induced Ca2þ signals up to the level induced by netrin-1 and thus switches repulsion to attraction. The different amplitudes of Ca2þ signaling may be explained by the fact that netrin-1 triggers an internal Ca2þ release as well as Ca2þ influx from the outside, whereas MAG causes only internal Ca2þ release. The moderate Ca2þ elevation induced by MAG is achieved without cAMP signaling (Henley et al., 2004). In summary, attractive and repulsive turning are mediated by intracellular Ca2þ gradients of the same polarity but with differing magnitudes. The magnitude of the intracellular Ca2þ gradient is positively affected by cAMP signaling. Ca2þ signals of different magnitude are sufficient to trigger bidirectional turning independently of cAMP signaling, but turning induced by a cAMP gradient needs downstream Ca2þ signals (Henley et al., 2004). Indeed cAMP regulates Ca2þ levels by activating via PKA L-type Ca2þ channels

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(Nishiyama et al., 2003). These findings suggest cAMP is upstream of Ca2þ signaling. However, Ca2þ can activate the Ca2þ-dependent adenyl cyclase and thus increase cAMP levels (Song et al., 1997). It therefore cannot be excluded that cAMP and Ca2þ act in a feedback loop and possibly amplify the external gradient of a guidance factor through their concerted action. This could be the case during attractive turning, where both cAMP and Ca2þ levels are high at the gradient near side of the growth cone. In repulsive turning, however, cAMP and Ca2þ levels might not reach the threshold required for the proper functioning of the feedback loop. Notably, the described Ca2þ/cAMP switch was proven to be physiologically relevant and regulates different responses to a single guidance factor in vivo. In the nervous system, the cAMP level of a neuronal growth cone depends on substrate-specific signals and/or the developmental stage. When growing on poly-D-lysine or fibronectin, Xenopus retinal growth cones have high intracellular cAMP levels and show attractive turning toward netrin-1. On a laminin substrate, however, netrin-1 is repulsive because laminin lowers the cAMP levels (Ho¨pker et al., 1999). The bifunctionality of MAG is developmentally regulated in rat dorsal root ganglion (DRG) neurons. Although MAG promotes neurite outgrowth in cells from early postnatal animals, it is repulsive for neurites from DRGs from slightly older animals, which display a correlated developmental decrease in endogenous cAMP levels (Domeniconi and Filbin, 2005). Downstream of Ca2þ signals, there are alternative pathways mediated by two different Ca2þ-regulated kinases. High local Ca2þ elevations preferentially activate Ca2þ-calmodulin–dependent protein kinase II (CaMKII) and induce attraction; lower local Ca2þ elevations activate calcineurinphosphatase-1 (CaN-PP1) and induce repulsion. Elevated cAMP levels may have a dual role in effectively converting repulsion to attraction. First, they amplify the Ca2þ signal in the suggested feedback loop. Second, they lead to activation of PKA, which inhibits CaN-PP1 and blocks the repulsion pathway. Upon activation of the repulsion pathway, on the other hand, PP1 acts negatively on CaMKII and thus blocks attraction (Han et al., 2007; Wen et al., 2004). The mutual exclusion of attraction and repulsion signaling pathways may explain why the growth cone is either attracted or repulsed at borderline conditions and does not display an intermediate response (Ming et al., 1997). CaMKII and CaN-PP1 regulate cytoskeletonassociated proteins, tubulin and Rho GTPases, which effect the cytoskeletal rearrangements required for growth cone turning ( Jin et al., 2005). Figure 1.7 summarizes the alternative signaling downstream of an intracellular Ca2þ gradient of different magnitude leading to either attraction or repulsion. Data suggests that attractive but not repulsive Ca2þ elevations in the growth cone also mediate asymmetric transport and exocytosis of membrane vesicles. The vesicle transport toward the side of the growth cone

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Figure 1.7 Intracellular Ca2þ gradients mediate both attractive and repulsive growth cone turning. (A) A high Ca2þ elevation leads to growth cone attraction toward the site of the elevation. High Ca2þ levels support a positive feedback between Ca2þ and cAMP via PKA and activate CaMKII while inhibiting CaN-PP1. CaMKII regulates the cytoskeleton in a way that affects attractive turning. This first scenario is triggered by the attractive guidance cues netrin-1 and BDNF. It can be also induced by the typical repellent MAG, when MAG is applied in combination with activators of cAMP signaling. (B) A low local Ca2þ elevation, on the contrary, leads to repulsive turning away from the site of the Ca2þ elevation. In this situation, there is no activation of a positive feedback loop between Ca2þ and cAMP, and CaN-PP1 wins over CaMKII signaling. As a result, the cytoskeleton is differently regulated and directs the growth cone to gradient far side. This second situation dominates in a gradient of MAG or in a gradient of netrin-1 or BDNF under conditions of suppressed cAMP signaling.

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generates an asymmetric expansion of the plasma membrane and a subsequent attractive turn (Tojima et al., 2007). Taken together, Ca2þ signaling plays a prominent role in growth cone turning. In combination with cAMP signaling, it can affect attraction as well as repulsion, thus conferring context-dependent bifunctionality to axon guidance molecules. It should be noted, however, that not all guidance molecules recruit Ca2þ in their downstream signaling. Growth cone turning induced by semaphorin (Li et al., 2005; Song et al., 1998), neurotrophin 3 (NT-3) (Song et al., 1997), or ephrinA (Lo¨schinger et al., 1997), for example, is independent of Ca2þ and its modulator cAMP. Local protein translation and degradation The neuronal growth cone has its own local protein translation and protein degradation machinery. Protein levels can thus independently be regulated from the cell soma and axonal protein transport (Piper and Holt, 2004). Growth cone turning or collapse in response to many guidance cues requires either local protein translation or degradation in the growth cone or both. For example, turning in response to netrin-1 requires translation and degradation, Sema3A-triggered collapse requires translation, and lysophosphatidic acid (LPA)-triggered collapse requires degradation. The translational activation in response to netrin-1 or Sema3A is mediated by the following signal cascade: activation of MAP kinases leads to the phosphorylation of the eukaryotic initiation factor 4B binding protein (eIF-4EBP), which subsequently releases eIF-4E and allows its binding to mRNAs present in the growth cone. Protein degradation after stimulation with netrin-1 or LPA is partially mediated by activation of caspase-3 downstream of MAP kinases (Campbell and Holt, 2001, 2003). So far, it is not known which proteins get degraded in response to specific guidance cues (Campbell and Holt, 2003). More progress has been made in the identification of specific mRNAs translated in the growth cone. Like in fibroblasts, local translation of b-actin mRNA is an important mechanism to enhance local actin polymerization. An attractive gradient of netrin-1 or BDNF was shown to trigger asymmetric activation of eIF-4E and a subsequent rise in b-actin on the gradient near side. Local protein translation is linked to Ca2þ signaling because a high local and therefore attractive Ca2þ elevation induces b-actin synthesis (Leung et al., 2006; Yao et al., 2006). There is some controversy, however, regarding how b-actin translation is regulated in a repulsive gradient of netrin-1 or BDNF. Although Leung et al. (2006) reported that repulsive turning in a netrin-1 gradient is independent of b-actin translation, Yao et al. (2006) suggested a repulsive gradient of BDNF or a low local (repulsive) Ca2þ elevation creates a local decrease of b-actin translation. This local decrease on the gradient

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near side of the growth cone can be seen as a relative increase of b-actin at the gradient far side, that is, as a kind of inverted attractive turning. Turning away from a repulsive gradient, however, can also be induced by an increase of proteins negatively affecting growth cone advance. Sema3A causes local translation of RhoA mRNA and a rise in RhoA, which counteracts actin polymerization. This translation is necessary and sufficient for the Sema3A-induced growth cone collapse (Wu et al., 2005). Besides increasing the protein level of RhoA, semaphorin receptors (plexins) can also regulate Rho GTPases via GEFs (Kruger et al., 2005). Local translation of RhoA and local activation of newly synthesized RhoA by specific GEFs can be thus envisaged to cooperate during the Sema3A signaling. Sema3A, as well as the repulsive cue slit-2, was furthermore found to induce a protein translation dependent rise of the actin-depolymerizing protein cofilin, which is likely due to translation of cofilin mRNA in the growth cone. An increase of cofilin causes a simultaneous decrease in f-actin, accounting in part for the growth cone collapse (Piper et al., 2006). Although the local translation of RhoA and cofilin was originally observed during growth cone collapse (Fig. 1.8A), it seems plausible that repulsive turning away from Sema3A or slit-2 is triggered by a more localized activation of protein translation leading to a partial collapse coupled with a reorientation of the growth cone. It was demonstrated that the homeodomain transcription factor engrailed-2 (En-2), in addition to regulating the transcription of ephrins, can also directly guide retinal axons. When applied in an external gradient, En-2 is internalized by the growth cone and regulates local translation by activation of eIF-4E by mechanisms similar to the ones observed downstream of netrin-1, BDNF, or Sema3A (Brunet et al., 2005). It is left to be investigated whether transcription factors other than En-2 can act as secreted cues and adopt a dual role in controlling transcription in the nucleus as well as translation in the growth cone (Butler and Tear, 2007). Because both repulsive and attractive guidance cues regulate translation via eIF-4E and there are a number of different mRNAs present in the growth cone, it is a crucial question how a specific mRNA gets translated in response to a specific cue. Axonal mRNA is incorporated in RNA granules, which contain mRNA, ribosomes, and RNA binding proteins. The latter play a role in mRNA transport as well as in translational regulation and may therefore be involved in the specificity and localization of translation in a turning or collapsing growth cone. In Xenopus growth cones, b-actin mRNA colocalizes with ZBP1 in granules. A graded netrin-1 or BDNF stimulus induces the movement of the b-actin mRNA containing ZBP-1 granules into the filopodia of the gradient near side by a yet unknown mechanism. As known from fibroblasts, ZBP-1 releases b-actin mRNA for translation upon phosphorylation by src. Because attractive BDNF gradients trigger a graded activation of src in the growth cone, the release

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Figure 1.8 Growth cone collapse and turning in response to several guidance cues is mediated by local protein translation. (A) Growth cone collapse triggered by the repulsive cues Sema3A or slit-2 were shown to induce local translation of RhoA and cofilin mRNAs via the activation of eIF-4EBP downstream of MAP kinases. RhoA and cofilin activity lead to cytoskeletal rearrangements causing collapse and retraction. (B) Attractive gradients of netrin-1 or BDNF cause local translation of b-actin. The asymmetry of b-actin translation is achieved by at least three mechanisms. First, RNA granules containing b-actin mRNA bound to ZBP-1 are transported preferentially to the gradient near side of the growth cone. Gradients of src and eIF-4E activation (depicted by gray shading) lead to graded release of b-actin mRNA from its binding partner and graded protein translation, respectively. Newly synthesized b-actin serves as a nucleation site for f-actin and shifts the growth cone toward the source of the gradient.

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of b-actin mRNA from ZBP-1 RNA granules is supposed to be graded as well. The asymmetry of b-actin synthesis is thus ensured by directed transport of the RNA granules, graded release of mRNA from translational silencing, and asymmetric activation of eIF-4E (Fig. 1.8B) (Leung et al., 2006; Yao et al., 2006). Both transport of RNA granules and release of mRNA from binding proteins are supposedly differentially regulated dependent on the type of mRNA and the extracellular trigger, which could thus account for different translational readouts in response to different guidance cues. Rho GTPases and the cytoskeleton: A common signaling output Interestingly, the signaling events during growth cone turning described in the previous sections are selectively activated in different combinations in response to different guidance cues (Table 1.1) and build up a cascade in some cases. For example, Ca2þ signaling, PI3K/PIP3 pathways, and local protein translation are all implicated in growth cone turning in response to netrin-1 or BDNF. Akt activation at the sites of local PI3K activity and accumulation of PIP3 was suggested to lead to an increase of intracellular Ca2þ via regulation of TRPC channels. Ca2þ signaling, in turn, is upstream of local protein translation of b-actin. b-actin levels directly affect the rate of actin polymerization and the advance of the growth cone toward the gradient source. Aside from possibly acting upstream of protein translation, both PI3K and Ca2þ signaling can directly affect the cytoskeleton. PI3K regulates the microtubule network via GSK-3 and PIP3 may, as in neutrophils, influence the balance of Rho GTPases. Ca2þ signaling affects CaMKII and CaN-PP, which regulate Rho GTPases as well as cytoskeletonassociated proteins. Rho GTPases, as elaborated in the following, are additionally activated by Rho GEFs acting immediately downstream of guidance cue receptors. Taken together, the turning of a growth cone toward netrin-1 or BDNF seems to be achieved by the cumulative action of several signaling pathways on the cytoskeleton, each of which can be essential or even sufficient for turning. The exact interrelation of these signaling pathways, however, is not completely clear. Based on present data, they could be either independently activated by the external cue or rather arranged in a strict hierarchy (Fig. 1.9). Signaling downstream of Sema3A, on the other hand, is independent of Ca2þ but involves the modulation of PI3K/PTEN signaling as well as local protein translation of cofilin and RhoA mRNA. Whereas local protein translation is essential for the response of growth cones to netrin-1, BDNF, or Sema3A, it seems not to be required in ephrinA-triggered collapse (our own unpublished results). However, no matter how the signaling pathways vary dependent on the guidance cue causing growth cone turning or collapse, they commonly lead to rearrangements of the cytoskeleton.

Table 1.1

Signaling events in the growth cone vary dependent on the guidance factor

Guidance cue (receptor)

Netrin-1 (Dcc)

BDNF (trkB)

Response of the growth cone

Intracellular signaling PI3K/PIP3

Ca2þ

Local protein translation

Attractive turning Repulsive turning Attractive turning Repulsive turning Repulsive turning Collapse

Enhanced PI3K activity, PIP3 accumulation Not known

High elevation leading to activation of CaMKII Low elevation leading to activation of CaN-PP1 High elevation leading to activation of CaMKII Low elevation leading to activation of CaN-PP1 Low elevation leading to activation of CaN-PP1 Not involved

Increase of b-mRNA translation Not involved?

Enhanced PI3K activity, PIP3 accumulation Not known

MAG (Nogo receptor) Sema3A (plexinAþ neuropilin1) Slit (Robo)

Collapse

PI3K activity required

Not known

EphrinA (EphA)

Collapse

PI3K activity required

Not involved

For references, see text.

PI3K activity required PTEN translocation and activation

Increase of b-mRNA translation Decrease of b-mRNA translation? Not known Increase of RhoA and cofilin mRNA translation Increase of RhoA and cofilin mRNA translation Not involved

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Figure 1.9 During attractive turning toward netrin-1 or BDNF, different interrelated signaling pathways concertedly act on the cytoskeleton. Attractive gradients of netrin-1 or BDNF activate PI3K/PIP3 signaling, Ca2þ signaling, and local protein translation of b-actin mRNA in the growth cone.These signaling pathways all act on the cytoskeleton via different downstream effectors. PI3K/PIP3 signals upstream of Ca2þ, and Ca2þ levels supposedly influence local protein translation. It is not yet known whether the signaling cascade is strictly hierarchical (A) or if the external cues directly activate each pathway (B).

Rho GTPases are probably the best-studied cytoskeletal regulators ( Jaffe and Hall, 2005). They play an instructive role in growth cone guidance and are implicated at some point of the signaling cascade in the response to practically every known axon guidance molecule. In general, Rac and cdc42 stimulate f-actin assembly in lamellipodia and filopodia, respectively, whereas RhoA leads to increased contraction of the actin-myosin network. According to a simplified model, Rac/cdc42 signaling dominates in attraction and RhoA signaling in repulsion. Because RhoA and Rac inhibit each other, activation of one Rho GTPase is likely to affect the balance of all three. Asymmetries in the Rho GTPase activities may therefore reinforce and efficiently direct growth cone turning (Dickson, 2001). By doing so, Rho GTPases are not only the output of gradient detection, but also participate in the setup of internal signaling polarity. Rho GTPases can be regulated via the PI3K pathway, by intracellular Ca2þ, or on the translational level as in case of RhoA during stimulation with Sema3A. Moreover, most guidance cue receptors also activate Rho GTPases via Rho GEFs. The netrin-1 receptor DCC is thought to interact with GEFs such as Trio during attractive signaling to activate both cdc42 and Rac1. Cdc42 and Rac1 stimulate an increase of the number of filopodia and the enlargement of the lamellipodial area (Barallobre et al., 2005). TrkB, when stimulated with its ligand BDNF, binds and activates the Rac1 specific GEF Tiam1 (Miyamoto et al., 2006). In the case of ephrinA5, EphA receptors phosphorylate and activate the GEF ephexin, which activates

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RhoA and its downstream effector Rock but does not have any effect on Rac/cdc42 (Sahin et al., 2005; Shamah et al., 2001; Wahl et al., 2000). Similarly, RhoA/Rock is activated during Sema3A-induced collapse and retraction (Gallo, 2006) and LPA-induced chemorepulsion (Yuan et al., 2003). Interestingly, there is evidence that growth cone collapse and subsequent retraction is mediated by RhoA activity in the axon shaft rather than in the peripheral domain (Nakamura et al., 2005). This indicates that the effect of RhoA activity depends on its site of action. Repulsive guidance molecules may therefore not globally activate RhoA but lead to a spatially restricted activation pattern. The Rac-specific GEF FARP2 (FERM domain-containing guanine nucleotide exchange factor) is activated immediately downstream of the Sema3A receptor complex. Its activity is required for Sema3A-induced collapse of DRG growth cones (Toyofuku et al., 2005). Because Rac activity has been correlated so far with growth cone extension and attraction, this result is confusing on the first sight. There is evidence that Rac mediates endocytosis of the growth cone plasma membrane rather than promoting actin polymerization during Sema3A-mediated collapse ( Jurney et al., 2002). The detailed differences of Rac signaling during collapse versus growth cone extension or attraction await further investigation. 2.4.3. Adaptation in growth cone gradient detection Adaptation of sensitivity is a common cellular phenomenon in response to a large number of biological stimuli. Adaptation seems especially reasonable during gradient detection of chemotaxing cells or growing axons because they move relative to the external gradient and are thus exposed to changing concentration ranges of the guidance factor. It is therefore not astonishing to observe adaptation under various conditions during axonal guidance. Adaptation, meaning the readjustment of sensitivity according to the strength of the signal, may be assumed based on different observations. First, a high concentration of a guidance cue may cause a strong response, but a longer exposure to the constantly high concentration may weaken or completely abolish this response. This attenuation of the normal response of a ‘‘naive’’ growth cone is referred to as desensitization. Following a period of desensitization, the growth cone may regain its sensitivity (resensitization)—either in the presence of the stimulus or in its absence (Fig. 1.10). Second, a low, subthreshold concentration of a guidance cue, which does not elicit any morphologically detectable response, may be nonetheless sufficient to attenuate the detectable response to a subsequent higher dose of a guidance cue. In short, adaptation can explain why the same growth cone may react differently to the same guidance cue concentration dependent on its ‘‘history.’’ Ming (2002) showed a uniform bath concentration of netrin-1 or BDNF leads to a dose-dependent desensitization of the growth cone, which becomes apparent when it is faced immediately afterward to a standard

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Figure 1.10 Adaptation in gradient detection by growth cones. The turning response toward a gradient of chemoattractant such as netrin-1 (shaded triangle) can be abolished by the application of a uniform low concentration of the attractant (gray rectangle), which leads to desensitization. The growth cone no longer responds to a subsequently applied gradient. If the uniform attractant concentration is maintained during a prolonged time span, the growth cone undergoes a period of resensitization and is again attracted by the gradient. Resensitization can also occur in the absence of the initially desensitizing stimulus.

gradient of chemoattraction, which is no longer able to elicit a turning response under these conditions. The desensitization is reversible, that is, the growth cone regains sensitivity within 90–120 min and seems to readjust its sensitivity to the new basal concentration of chemoattractant. Moreover, when the course of a growth cone in a soluble gradient of netrin-1 is followed over several hours, the extension toward the gradient source shows a ‘‘zigzag’’ pattern. This was interpreted as an indication of alternating periods of attraction and repulsion (Ming et al., 1997). The zigzag course is likely to arise from the higher level of desensitization at the gradient near

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side of the growth cone, which results in a higher relative signal at the gradient far side of the growth cone during periods of apparent repulsion. The average interval for the zigzag is approximately 20 min in a netrin-1 or BDNF gradient. In high concentrations of attractant, the desensitization is heterologous, in low concentrations homologous. Desensitization goes along with persistent Ca2þ elevation in the growth cone. After resensitization, a gradient can induce a further Ca2þ elevation superimposed on top of the elevated basal Ca2þ level. Resensitization is dependent on MAPK activity and local protein synthesis (Ming et al., 2002). In the context of growth cone collapse, desensitization and resensitization can occur very rapidly. The collapse of Xenopus retinal axons in response to either netrin-1 or Sema3A is markedly reduced when growth cones are pretreated for 1–2 min with a low dose of the collapse-inducing factor, which itself produces minimal collapse. This desensitization depends on endocytosis. However, if the low dose is maintained for approximately 5 min, the high collapse-inducing dose has the same effect as without pretreatment; namely resensitization occurs in the presence of the low dose after the initial desensitization. This resensitization depends on protein synthesis. During desensitization, the receptors for Sema3A and netrin-1, respectively, disappear from the cell surface in a endocytosis-dependent manner. During the subsequent resensitization, receptors reappear. This reappearance is only partially dependent on protein synthesis (Piper et al., 2005). It would be interesting to know whether proteins other than the receptors are locally translated during resensitization. Are they the same, which are also translated during the initial response of the growth cone toward the guidance factor? Notably, desensitization and resensitization also occur in growth cones treated with ephrinA5, the action of which seems independent of protein synthesis (von Philipsborn et al., 2006; our own unpublished results). Are there different mechanisms of adaptation depending on the guidance cue? From a more conceptual point of view, it is also tempting to speculate about the interrelation of signal amplification and adaptation. Signal amplification, which might be crucial for the detection of a shallow external gradient, could have the consequence that a stimulus of intermediate strength already triggers maximal internal signaling in the growth cone and blinds it for further increase of the external stimulus. If desensitization and subsequent resensitization is tightly coupled to signal amplification, such restrictions could be overcome. 2.4.4. Gradient detection during topographic mapping Gradient detection not only directs growth cone turning, but also is essential in the formation of topographic neural maps. A topographic map consists of a population of projecting neurons, whose axonal connections in the target region reflect their original spatial order. As first proposed by Sperry (1963),

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topography can theoretically be established by the graded distribution of a guidance cue in the target region and the graded expression of the corresponding receptor on the ingrowing axons. For example, topography is found in the thalamo-cortical, the hippocampo-septal, and the vomeronasal projection in the nervous system (McLaughlin and O’Leary, 2005). One of the best-studied neural topographic maps is the connection between the vertebrate eye and the brain, the retino-tectal/collicular projection that is mainly set up by ephrins and Eph receptors. Numerous in vivo studies such as ephrinA/EphA knockout or knockin mice have consolidated the notion that temporal retinal ganglion cells (RGCs) axons, which express high levels of EphA receptors, are confined to the anterior tectum/superior colliculus containing low ephrinA levels, whereas nasal RGC axons with less EphA receptor invade the ephrinA gradient in the target region up to the posterior part of the tectum/superior colliculus (Brown et al., 2000; Du¨tting et al., 1999; Feldheim et al., 2000, 2004; Nakamoto et al., 1996). Topographic mapping has been extensively reviewed (Flanagan, 2006; McLaughlin and O’Leary, 2005) and explained by different theoretical models (Gierer, 1983; Goodhill and Urbach, 1999; Honda, 2004; Koulakov and Tsignakov, 2004; Lo¨schinger et al., 2000; Reber et al., 2004; Yates et al., 2004). We intend to focus here on a few principal questions with respect to gradient detection during mapping and selected experiments addressing them. So far, it is still not completely understood how a RGC growth cone reads a repulsive gradient of ephrinA. The process of anterior-posterior mapping in the visual system is far more complicated than growth cone turning. First, growth cones have to follow a repulsive gradient up to a certain point, instead of avoiding it at all and performing a negative turning response as soon as they detect the gradient. Second, growth cones should be able to read not only the directional, but also the positional information of the gradient. Intuitively, one might assume that the sensing of position requires sensing of absolute concentration, which could be in conflict with the concept of signal amplification and adaptation. Because mapping is accomplished by graded distributions of substratebound molecules and implicates the stop of a growth cone rather than its turning, the classical turning assay with soluble gradients can only give limited information. Moreover, ephrinAs require membrane attachment and/or clustering for proper EphA activation (Davis et al., 1994; Egea et al., 2005). Uniform addition of soluble clustered or dimeric ephrinA causes temporal RGC growth cones to collapse. Xenopus RGC growth cones were also observed to turn in response to a soluble gradient of dimeric ephrinA5. However, this turning occurs at a high background level of growth cone collapse (Weinl et al., 2003). The action of substrate bound guidance molecules such as ephrinA has been studied with the so-called stripe-assay, an array of ephrinA-covered

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lanes alternating with lanes devoid of ephrinA. EphrinA-sensitive axons avoid the ephrinA-containing lanes and display a striped outgrowth on the permissive lanes (Monschau et al., 1997; Vielmetter et al., 1990; Walter et al., 1987). The stripe assay can impressively demonstrate preference for or avoidance of guidance factors, but provides no information about the behavior of growth cones in gradients of these factors. EphrinA is a repulsive cue when applied in a nongraded form. However, a growth cone obviously invades ephrinA gradients in vivo and switches from advance to stop in the target area. This switch could either result from an inherent, concentration-dependent bifunctionality of ephrinA or from the counteraction of a second, superimposed attractive guidance force. There is experimental data supporting both notions. Indeed, temporal RGC axons can invade substrate bound gradients in vitro and switch at a certain point of the artificial gradient to growth inhibition or avoidance reactions. Early studies using repellent membrane material from chick posterior tectum or ephrinA-overexpressing cells to create gradients of various shapes come to differing conclusions about the question, which parameters of the gradient confer its repulsive properties. Baier and Bonhoeffer (1992) suggest retinal axons stop in gradients depending on the gradient slope. Only sufficiently steep gradients caused a growth inhibition. Rosentreter et al. (1998), however, found in a similar assay that temporal retinal growth cones avoid a certain threshold concentration of repellent membrane material when they invade a gradient. This postulated threshold concentration is independent of the gradient slope. When growing out on basal levels of repellent membranes, growth cones travel up the gradient for an additional fixed increment of concentration, indicating a certain adaptation mechanism. In contrast to diffusible gradients, substrate-bound gradients are far more complicated to fabricate in vitro. In gradients of membrane material, it is difficult to quantify the exact amount of a single protein and exclude the influence of other components. Progress has been made by generating concentration gradients in three-dimensional (3D) gels, which are comparatively stable for several hours (Rosoff et al., 2005). To obtain highly reproducible and long-lasting graded distributions of purified ephrinA5, microcontact printing proved to be a useful technique. Gradients fabricated by microcontact printing are discontinuous on a microscale, that is, they consist of a geometric pattern of protein dots or lines, which vary with respect to sizes and spacing. As shown by von Philipsborn et al. (2006), temporal retinal growth cones can read discontinuous gradients of ephrinA produced by microcontact printing and stop at a certain point in the gradient. By testing different gradient slopes and concentrations, the influence of gradient steepness versus a threshold concentration on growth cone stop was explored in detail. Because growth cones also stop in nongraded patterns of ephrinA5, they apparently do not solely measure a fixed threshold

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concentration but rather employ a kind of summation mechanism to determine their stop position. Interestingly, growth cones encountered higher total amounts of ephrinA5 and stopped at higher local ephrinA5 concentrations in steep gradients than in shallow gradients. Shallow gradients may be more efficient in causing growth cone stop with less repellent material because they cause less desensitization. The authors hypothesize that the growth cone’s tendency to stop may increase with the amount of encountered ephrinA5, whereas the local ephrinA5 concentration counteracts this tendency by leading to a constant readjustment of sensitivity. The growth cone stops when both parameters reach a certain ratio. Such an integrative mechanism would be advantageous for the detection of a target zone in a gradient in the in vivo situation because it is rather insusceptible to variances in ephrinA expression levels. Moreover, it could explain how growth cones stop in a repellent gradient, although they are able to adapt to the repellent. Substrate-bound gradients of ephrinA alone can thus be permissive up to a certain point for temporal RGC axons and confer a repulsive stop or avoidance signal beyond this point. Intriguingly, temporal growth cones react in the gradient assays as a uniformly sensitive population, and all nasal axons display the same extend of insensitivity. Along the naso-temporal axis of the retina, there is no graded sensitivity to ephrinA gradients, as one would expect based on the expression pattern of EphA receptors, but rather a sharp binary split in a responsive temporal and an unresponsive nasal half. A certain graded response to ephrinA across the retina was observed by Hansen et al. (2004). When used in a uniform distribution as a substrate, ephrinA2 stimulates neurite outgrowth at low concentrations and inhibits outgrowth at high concentrations. Furthermore, the transition point from outgrowth promotion to inhibition is dependent on the naso-temporal position of the RGCs tested in this assay. Although these results give no direct information about gradient detection, they indicate that ephrinA is not only permissive, but also stimulates axon outgrowth at low concentration. This acts as a bifunctional cue, at least in initial neurite outgrowth, which may be governed by different mechanisms than growth cone guidance. By no means can it be excluded that the invasion of the ephrinA gradient in the tectum/superior colliculus is also accomplished by other attractive guidance cues. Candidates are EphA receptors, which signal via ephrinAs (‘‘reverse signaling’’) and BDNF signaling via trkB (Marotte et al., 2004; McLaughlin and O’Leary, 2005). Taken together, gradient detection during topographic mapping implicates a higher complexity than gradient detection during a turning response because it not only requires the processing of directional, but also of positional information. Further research is needed to investigate if and how the sensing of direction and position differs with respect to the underlying signaling events.

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3. Common Grounds and Diversity 3.1. Signaling pathways The signal transduction during eukaryotic gradient detection shares a number of conserved pathways (Table 1.2). Dictyostelium cells and neutrophils have been frequently compared in terms of signaling, and their mode of gradient detection has been explained based on common models (Charest and Firtel, 2006; Skupsky et al., 2005; Van Haastert and Devreotes, 2004). Fibroblast gradient detection is accomplished by similar signaling pathways, although it is much simpler as in amoeboid cells and restricted with respect to signal amplification, sensitivity, and adaptation (Schneider and Haugh, 2005). Dictyostelium cells, neutrophils, and fibroblasts all move toward the source of the gradient. The picture is more complex in the growth cone because it detects and reacts to both attractive and repulsive graded cues, which moreover fall within several different classes of molecules. The combination of signaling pathways downstream of different guidance cues differs, but the core components are shared with other eukaryotic cell types. Signaling downstream of attractant and repellent gradients are remarkably similar. A switch in the directionality of the response is achieved by modifications of a signaling system rather than by completely novel mechanisms. In chemotaxing cells, signaling events at the leading edge/pseudopod facing the source of the attractive gradient is generally antagonistic to the ones at the trailing edge/uropod. Attractive growth cone turning is often effected by signaling events characteristic for the leading edge of chemotaxing cells. Signaling during repulsive turning or collapse in response to some axon guidance cues, on the other hand, resembles trailing edge signaling. However, ‘‘front’’ and ‘‘back’’ signaling are not completely independent pathways but have to interact and balance each other during detection of and response to a gradient. In line with this, the activity of many ‘‘front’’ signaling components is essential for repulsive turning and growth cone collapse and vice versa. PI3K activity, for example, is required for attractive turning as well as for growth cone collapse in neurons, whereas it is exclusively coupled to the advance of the leading edge in other cell types. Rac is activated predominantly at the leading edge/pseudopod in migrating cells. Despite its front signaling role, it is also activated during growth cone collapse triggered by Sema3A, possibly fulfilling a distinct function. In summary, growth cones employ the common signaling pathways of eukaryotic gradient detection in a sophisticated and context-dependent manner to respond in various ways to different guidance cues. In specific cases, signaling during growth cone turning or collapse can be directly compared to the scenario in other eukaryotic cells.

42 Table 1.2

Signaling components in eukaryotic gradient detection and their role in different model systems Dictyostelium

Neutrophils

Fibroblasts

Growth cones

PI3 kinase signaling

PIP3 is the first amplified readout of the external gradient. PI3K at the cell front and PTEN at the cell rear participate in a feedback loop to strengthen PIP3 accumulation at the leading edge.

PIP3 is the first amplified readout of the external gradient. PI3K is localized at the cell front; PTEN is distributed uniformly in polarized cells.

Active PI3K and PIP3 are concentrated at the leading edge, but they do not engage in a positive feedback loop.

Ca2þ signaling

cAMP causes transient intracellular back to rear Ca2þ gradients. Ca2þ enhances myosinII-mediated contraction.

Chemotactic factors induce an increase in intracellular Ca2þ. Ca2þ enhances myosinII-mediated contraction.

PDGF induces Ca2þ influx. Ca2þ enhances myosinII contraction.

PIP3 accumulates in attractive gradients of netrin-1 or BDNF at the gradient near side of the growth cone. Active PTEN in the peripheral domain of the growth cone is implicated in Sema3A-induced collapse. Local Ca2þ elevations can induce both attractive and repulsive turning dependent on their magnitude. Not all guidance factors signal via Ca2þ.

Local protein synthesis

Required for cell motility, but not for gradient sensing.

Not known.

Rho family kinase signaling

Rac activity is concentrated at the pseudopod.

Rac signaling at the pseudopod contrasts with RhoA signaling at the uropod.

For references, see text.

Local translation of b-actin mRNA at the leading edge is required for directionality and persistence of movement during chemotaxis. Rac signaling at the leading edge contrasts with RhoA signaling at the trailing edge.

Turning toward or away from several guidance factors depends on asymmetric protein synthesis in the growth cone.

RhoA activity is implicated in growth cone collapse. Rac/ cdc42 can mediate attractive turning.

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3.1.1. PI3 kinase/PIP3 signaling and events at the plasma membrane PI3K signaling plays a significant role in all cell types discussed here but clearly shows differences with respect to its integration into the whole signaling machinery mediating gradient detection. After stimulation with an external gradient, PI3K rapidly translocates to the cell membrane of Dictyostelium cells and neutrophils. Its product, PIP3, accumulates in a polarized fashion and sets up an amplified internal signaling gradient. PIP3 represents binding sites for a great number of PH domain containing signaling proteins, among those Akt and Rho GEFs. In both cell types, PIP3 enhances its own production by PI3K and thus establishes a positive feedback loop, which has been elucidated in great detail. Polarized PI3K signaling at the leading edge is sharpened by the signaling of its antagonist PTEN at the trailing edge in Dictyostelium, whereas in neutrophils the role of PTEN seems to be accomplished by SHIP1. Only found to be important for polarization and chemotaxis, the cellular distribution of SHIP has not yet been investigated. In neutrophils, the accumulation of PIP3 at the leading edge is further sustained by actin polymerization. Inhibition of actin polymerization in Dictyostelium cells does not impair PIP3 patterns during signaling polarization. In fibroblasts chemotaxing toward PDGF, PI3K generates an amplified PIP3 gradient along the membrane as well. In contrast to amoeboid cells, PIP3 cannot enhance its own production by PI3K, indicating the lack of a PI3K/PIP3 feedback loop. PTEN is involved in the chemotactic response of fibroblasts toward PDGF, most likely as a negative regulator. So far, there is no data about its distribution in chemotaxing fibroblasts and its detailed influence on polarized PI3K signaling and local PIP3 accumulation. It is thus not completely understood how the amplified gradient of PIP3 is generated in chemotaxing fibroblasts. PI3K activity is generally required for axon elongation and growth cone advance. A prevalence of PTEN over PI3K in the peripheral domain of the growth cone plays a role in Sema3A triggered growth cone collapse. In this context, growth cone collapse and retraction is correlated with signaling events typical for the trailing edge/uropod of migrating cells. PI3K signaling during growth cone turning has been less investigated so far. Asymmetries in PI3K activity and local PIP3 accumulation may lead to attractive turning of the growth cone toward netrin-1 or BDNF. Although PI3K is a positive regulator of axonal elongation, its activity is required for growth cone collapse in response to MAG, slit-2, or ephrinA. The exact role and localization of active PI3K during the collapse response is not known but is likely to differ from the one during attractive turning. An important feature about PI3K and PIP3 signaling is its localization to the plasma membrane. The establishment and maintenance of locally restricted membrane-linked signals is possibly further enhanced by cholesterol-enriched

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microdomains or ‘‘lipid rafts.’’ Membrane microdomains have been correlated with the development of cell polarity in neutrophils (Gomez-Mouton et al., 2004; Seveau et al., 2001) and were shown to enhance uropod and restrict pseudopod signaling (Bodin and Welch, 2005). Disruption of membrane microdomains can abolish the response of the growth cone to guidance factors such as BDNF, netrin-1, and Sema3A, most likely because of defective association of the respective receptors with microdomains (Guirland et al., 2004). 3.1.2. Ca2þ signaling Ca2þ signaling regulates both attractive and repulsive growth cone turning in response to some, but notably not all axon guidance factors. The magnitude of a local Ca2þ elevation determines the direction of the turn and is interrelated with internal cAMP levels. Ca2þ levels in the growth cones regulate numerous cytoskeleton interacting proteins (Gomez and Zheng, 2006; Henley and Poo, 2004) and can additionally influence local protein translation required for the response to some guidance factors (Leung et al., 2006; Yao et al., 2006). Ca2þ signaling is far less involved, or at least far less considered during gradient detection of other cell types. Among migrating cells, chemotaxing fibroblasts probably come closest to navigating growth cones with respect to the impact of Ca2þ signaling. In fibroblasts migrating toward PDGF, Ca2þ influx was shown to signal upstream of calmodulin, an MLC kinase. The phosphorylation of MLC activates myosinII and thus regulates trailing edge contraction. This mechanism is crucial for cell motility and migration (Yang and Huang, 2005). Ca2þ elevations after PDGF stimulation activate CaMKII, an activator of the Rac1-specific GEF Tiam1 and cause Rac1dependent membrane ruffling (Buchanan et al., 2000). As described earlier, CaMKII is an important mediator of attractive growth cone turning toward Ca2þ (i.e., guidance cues triggering attractive intracellular Ca2þ elevations). In Dictyostelium cells and neutrophils, Ca2þ signaling mainly regulates myosin-dependent contraction at the cell rear. When Dictyostelium cells respond to a cAMP gradient, internal Ca2þ levels transiently increase at the uropod. An internal rear to front Ca2þ gradient, which is apparent in some but not all cells, may support the accumulation of contractile myosinII and/or actin depolymerization at the uropod (Nebl and Fisher, 1997; Yumura et al., 1996). The stimulation of neutrophils with chemotactic factors also causes an increase in intracellular Ca2þ, whose polarity has not been investigated. This increase modulates integrin cell adhesion molecules and seems to be crucial for neutrophil migration on adhesive substrates. Like in Dictyostelium, Ca2þ in neutrophils might furthermore regulate myosinII-dependent contractile forces (Niggli, 2003).

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3.1.3. Local protein translation Localized protein translation is implicated in fibroblast chemotaxis and axon guidance. Synthesis of b-actin and subsequent increase in actin polymerization governs the advance of the fibroblast leading edge and growth cones turning toward attractive gradients of netrin-1 and BDNF. In both systems, src phosphorylates ZBP-1 and leads to the release of b-actin mRNA, which is required for its translation. Local translation of RhoA and cofilin mRNA, which is associated with Sema3A- and slit-2–dependent growth cone collapse, was so far only observed in growth cones. In the future, it will be interesting to investigate whether these mRNAs are also translated during repulsive turning. Neither Dictyostelium (Clotworthy and Traynor, 2006) nor neutrophil gradient detection is substantially dependent on local translation of specific mRNAs. The role of local protein translation during gradient detection may have exclusively emerged in fibroblasts and neurons and is probably correlated with the more complex cytoskeletal organization in these cells. In neuronal growth cones, local translation is the most important way to control protein levels rapidly and independently from axonal transport (Piper and Holt, 2004). This might be the reason why local protein translation is particularly relevant for gradient detection in growth cones. Although local protein translation is required for the response of the growth cone to guidance cues such as BDNF, netrin-1, and Sema3A, the very same responses (i.e., turning or collapse) can be performed by the growth cone without local protein translation after stimulation with ephrinA or LPA. Local protein translation is also important for adaptation mechanisms in growth cones. However, local protein translation is not linked to adaptation in chemotaxing fibroblasts, whose ability to adapt to increasing PDGF concentrations seems to be far less prominent (Haugh, 2006). 3.1.4. Rho GTPases Rho GTPases are common cytoskeletal regulators in migrating cells and steering axons. In neutrophils and fibroblasts, Rac activity at the pseudopod contrasts with RhoA activity at the uropod, leading to f-actin–based protrusion and myosinII-based contraction, respectively. In Dictyostelium, RhoA homologs have so far not been identified. The pattern of active Rac, however, resembles the one in neutrophils and fibroblasts. In all three cell types, Rac was shown to promote actin polymerization (Charest and Firtel, 2007). Rac and cdc42 have been associated with the advance of growth cone filopodia and lamellipodia. Most attractive axonal guidance cues indeed lead to the activation of Rac and cdc42. Two GEFs specific for Rac1, Trio and Tiam1, which are essential for the membrane ruffling of fibroblasts

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stimulated with PDGF (Buchanan et al., 2000; Debreceni et al., 2004; Sander et al., 1999), signal also downstream of netrin-1 and BDNF, respectively (Barallobre et al., 2005; Miyamoto et al., 2006). During attractive growth cone turning, Rac and cdc42 seem to play a role in actin polymerization comparable to the one in migrating cells (Dickson, 2001; Yuan et al., 2003). However, there are also reports that repulsive cues such as Slit (Wong et al., 2001; Yang and Bashaw, 2006), Sema3A (Toyofuku et al., 2005), and ephrinA2 (Jurney et al., 2002) activate Rac and/or cdc42. Whether Rac generally mediates membrane endocytosis during growth cone collapse instead of actin polymerization, as it was proposed for the collapse triggered by Sema3A and ephrinA2 ( Jurney et al., 2002), has not been fully investigated. Distinct effects of Rac on growth cone advance versus collapse could also arise by distinct localization of Rac activation in the growth cone or a different intracellular context. RhoA, which signals predominantly at the back of chemotaxing cells, plays a well-established role in growth cone collapse and repulsive turning. RhoA and Rock signal downstream of ephrinA5 (Wahl et al., 2000), Sema3A (Gallo, 2006), and LPA (Yuan et al., 2003). They enhance myosinII-mediated contraction and thus establish a signaling pathway which is also found in the trailing edge/uropod of migrating cells.

3.2. Signal amplification Signal amplification is a prevalent property of signaling cascades. During eukaryotic gradient detection, not only the given signal, but also rather small signal differences have to be amplified. There has to be biased signal amplification across the cell to amplify the absolute as well as the relative concentration differences of an external gradient (Fig. 1.11). In the cell types discussed here, this is accomplished to different extents and by slightly differing mechanisms. Efficient signal amplification arises from feedback loops, which are integrated at different levels into the major signal transduction pathways mediating gradient detection. In Dictyostelium and neutrophils, a small local initial rise in PIP3 at the gradient near side of the cell leads to further accumulation of PIP3 via the activation of PI3K. A similar feedback loop is likely to be present in turning growth cones. In fibroblasts, however, PIP3 does not stimulate the activity of PI3K but signals exclusively downstream of PI3K. Positive feedback loops can lead to the amplification of absolute concentration differences, but they are not sufficient to explain the amplification of relative concentration differences. For this, signaling downstream of the chemotactic cue has to be selectively enhanced at the gradient near side of the cell and/or selectively inhibited at the gradient far side. As modeled by

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Figure 1.11 Signal amplification during gradient detection. An external gradient of a chemotactic cue is defined by the concentration Ce(b) at the back of the cell and the concentration Ce(f) at the front of the cell. If the extracellular signal gets equally amplified at the back and the front of the cell, the proportion of the concentrations of intracellular effectors at the front Ci(f) and the back Ci(b) stays the same as the proportion of the external concentrations Ce(f) and Ce(b), although the absolute difference of the concentrations increases. To amount to an amplification of the relative difference of the concentrations, there has to be an asymmetry in the signal amplification at the back and the front. In the most extreme case, the signal is highly amplified at the front and not transduced at all at the back.

Meinhardt (1999), a self-enhanced reaction coupled to competing antagonistic reactions is sufficient to establish polarized patterns within a cell. This process, which has been referred to as local activation/global inhibition mechanism (Devreotes and Zigmond, 1988; Parent and Devreotes, 1999; Skupsky et al., 2005), can be achieved by the segregation of signaling domains mutually excluding each other. In Dictyostelium cells, the signaling antagonism between front and rear arises mainly from the reciprocal distribution of PI3K and PTEN activity. Taking the internal gradient of PIP3 as a reference for the internal signaling, it was determined that the relative external cAMP gradient gets approximately sevenfold amplified (Janetopoulos et al., 2004; Xu et al., 2005b). Notably, this amplification is accomplished independently from cytoskeletal rearrangements and morphological polarization. In neutrophils and fibroblasts, sharpening of the internal signaling gradient seems to also occur at the level of Rho GTPases. Because Rac and RhoA mutually inhibit each other, an initially small bias in their spatial activity pattern can finally establish a strong intracellular signaling asymmetry.

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This signaling asymmetry causes asymmetries in cytoskeletal architecture, meaning morphological polarization, which again enhances the signaling polarization. Whereas neutrophils are equal with Dictyostelium in their efficiency to detect and amplify a gradient, fibroblasts are less specialized in responding to gradients independently of the absolute PDGF concentration. Compared with amoeboid cells, which were reported to detect relative concentrations gradients down to 1% across the cell length, a growth cone can only detect a minimal external cAMP concentration gradient of 10% across its length (Lohof et al., 1992). This value may vary for different guidance molecules and crucially depends on the involved signaling pathways. Although it was not directly measured like in Dictyostelium cells, an amplification of the relative external gradient is likely to happen in growth cones as well through a number of feedback loops in the different signaling pathways. Preliminary data suggests PIP3 can stimulate its own accumulation in growth cones as it does in chemotaxing cells ( J. Henley, personal communication). During attractive growth cone turning caused by high local Ca2þ elevations, Ca2þ stimulates its own rise via a positive feedback loop between Ca2þ and cAMP. A robust asymmetry in local protein translation of b-actin mRNA in gradients of netrin-1 or BDNF is established by the synergistic action of mRNA transport and cooperating gradients of src and eIF-4E activation. The antagonistic signaling events at the gradient near versus the gradient far side of a turning gradient have been less well described than the frontrear signaling asymmetry in migrating cells.

3.3. Adjustment of sensitivity/adaptation Signal amplification allows cells to detect minimal concentration gradients. However, as soon as the signal input partially uncouples from the internal signal output, absolute sensing is no longer possible. If the external signal is not amplified proportionally to its strength but rather in a switchlike fashion, the cells respond within certain range to low concentration gradients the same way as to high concentration gradients. A similar behavior results from adaptation. Adaptation may partially depend on the modulation of signal amplification but can also result from a number of other mechanisms such as the downregulation of signaling at various levels during a strong stimulus. Because a number of phenomena result from or are linked to adaptation, it is helpful to define adaptation as the adjustment of the sensitivity according to the signal strength to prevent conceptual confusion. This adjustment comprises desensitization during strong stimulation as well as sensitization during weak or absent stimulation. Furthermore, resensitization may also occur after a period of desensitization, that is, after the shutdown or absence of an internal signal in the persisting presence of a strong external signal.

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Notably, adaptation introduces a temporal sensing element in eukaryotic gradient detection, which is principally based on a spatial-sensing mechanism. Adaptation has been observed in migrating eukaryotic cells as well as in growth cones, but it is certainly best studied and probably also most prominent in growth cones (Section 2.4.3). After prolonged stimulation with cAMP, Dictyostelium cells display a decreased sensitivity for the chemoattractant, which is coupled to a reduction of cAMP affinity and a loss of ligand binding of the cAMP receptor cAR1 (Caterina et al., 1995). Similarly, neutrophils can adapt to different chemoattractant concentrations and display a transient loss of responsiveness after rapid concentration changes (Zigmond and Sullivan, 1979). The sensitivity of a neutrophil toward a chemotactic factor and the cell’s ability to adapt can also be influenced by the presence of other factors (Foxman et al., 1997; Lin et al., 2005). As mentioned in Section 2.2.1, the balancing of adaptation to a whole set of chemotactic factors is crucial for proper guidance of neutrophils to different sites of infection as well as for the guidance over long distances. From this point of view, neutrophils face a similar complexity as growth cones, which often have to navigate in superimposed distributions of different guidance cues. Highly suggestive by the in vivo expression patterns in the nervous system, the integration of different guidance cues by growth cones has been demonstrated in vitro. For example, combined gradients of NGF and NT-3 are synergistic and can guide axons over a longer distance than one gradient alone (Cao and Shoichet, 2003). Furthermore, NGF can counteract repulsive signaling. When applied to axons before treatment with Sema3A, NGF reduces Sema3A triggered growth cone collapse (Dontchev and Letourneau, 2003). Adaptation mechanisms thus have to be considered not only in response to a single chemotactic factor, but also during cross talk of several factors.

3.4. Biological and functional context The varieties in eukaryotic gradient detection make sense in the biological context of the different cell types. The gradient of cAMP attracting solitary Dictyostelium cells is relatively simple in regard to its function: It serves to guide the cells to a central point of aggregation. Because the cAMP gradient emitted by the cells serving as an aggregation center oscillates in its concentration (Dormann et al., 2000), the chemotaxing cells have to detect the external gradient over a wide range of concentrations. Dictyostelium has developed strong signal amplification and responds to gradients with a substantial internal polarization, which allows maintaining persistently the directionality of migration during changes in the external gradient. There is no requirement for sensing absolute cAMP concentrations or to charge the cAMP signal against other chemoattractants.

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Mammalian neutrophils are equally sensitive to gradients as Dictyostelium cells and detect the latter by very similar mechanisms. Additionally, they are able to integrate several guidance cues to coordinate the invasion of rivaling infection sites in the organism. Fibroblasts attracted by PDGF, however, detect gradients in a simpler way than amoeboid cells. Migrating through the tissue and a 3D array of extracellular matrix, their speed of chemotaxis is much slower than the one of neutrophils. The invasion of a dermal wound by fibroblasts takes several days during which the PDGF gradient may adopt an optimal shape for chemotaxis with the help of successive PDGF degradation by the progressing fibroblasts. In addition to the attracted fibroblasts, wound healing is supported by PDGF triggered proliferation of already present cells (Haugh, 2006). In terms of function, gradients guiding growth cones differ in two major aspects from the discussed gradients relevant for cell migration. First, they are attractive as well as repulsive. Repulsive gradients can either direct the growth cone away from the gradient or allow the axon to proceed up to a certain point, like in the case of the ephrinA gradient in the developing tectum/superior colliculus. Second, gradients in the nervous system provide not only directional, but also positional information because they are implicated in topographic mapping. The requirement to detect positional information entails restrictions in signal amplification and adaptation, which in turn may limit the growth cones sensitivity.

4. Concluding Remarks Chemotaxis is an important task for eukaryotic cells in different biological and functional contexts. Migrating cells as well as neuronal growth cones are specialized to detect gradients of chemotactic factors by a spatial gradient sensing mechanism. During spatial gradient sensing, the external gradient has to be translated into an internal signaling gradient across the length of the cell or the growth cone, respectively. This signaling asymmetry or internal polarization normally results in a morphological polarization and the establishment of a cytoskeletal architecture capable of directional movement. Signal amplification, and more specifically, biased processing of the external signal at the gradient near side and the gradient far side of the cell provide for a high sensitivity in the detection of shallow and/or low concentrated gradients. Furthermore, adaptation during gradient sensing allows for maintaining this sensitivity over a broad range of concentrations. Gradient detection during eukaryotic cell migration and axon pathfinding meets similar demands. It is therefore plausible that the underlying signaling is conserved in many aspects and shares common mechanistic features, for example, the employment of feedback loops during signal

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amplification and internal signal polarization. Signaling pathways such as the PI3K/PIP3 pathway function in all systems discussed here. Localized protein translation of specific mRNAs plays a role in gradient detection and chemotaxis of fibroblasts as well as neuronal growth cones. The focus on common ground and diversities during eukaryotic gradient detection tells more about the singularity of gradient sensing in a certain cell type and, at the same time, can fill the gaps in the knowledge about one system with the help of experimental findings from a related system. In the future, it is promising to further explore the similarities and differences in cell migration and axon guidance for new impulses in both fields of research.

ACKNOWLEDGMENTS We thank F. Weth and C. Gebhardt for critical reading and suggestions on the manuscript. This work was supported by the DFG (grant 1034/14-1 to M.B.). A. P. received a stipend from the German National Scholarship Foundation.

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C H A P T E R

T W O

Leptin and the Regulation of the Hypothalamic–Pituitary–Adrenal Axis Ludwik K. Malendowicz,* Marcin Rucinski,* Anna S. Belloni,† Agnieszka Ziolkowska,* and Gastone G. Nussdorfer†

Contents 64 65 65 68

1. Introduction 2. Biology of Leptin and Its Receptors 2.1. Leptin 2.2. Leptin receptors 3. Expression of Leptin and Its Receptors in the Hypothalamic–Pituitary–Adrenal Axis 3.1. Hypothalamus 3.2. Anterior pituitary 3.3. Adrenal gland 4. Effects of Leptin on the Central Branch of the Hypothalamic–Pituitary–Adrenal Axis 4.1. Hypothalamus and CRH secretion 4.2. Anterior pituitary and ACTH secretion 5. Effects of Leptin on the Peripheral Branch of the Hypothalamic–Pituitary–Adrenal Axis 5.1. Adrenal cortex 5.2. Other steroid-secreting cells 5.3. Adrenal medulla 6. Involvement of Leptin in the Pathophysiology of the Hypothalamic–Pituitary–Adrenal Axis 6.1. Response to stresses 6.2. Pituitary adenomas 6.3. Adrenocortical tumors and pheochromocytomas 6.4. Macronodular adrenal hyperplasia 6.5. Hyperreninemic hypoaldosteronism

69 69 70 73 76 76 77 78 78 83 84 85 85 86 86 87 87

*Department of Histology and Embryology, School of Medicine, Karol Marcinkowski University of Medical Sciences, PL-60781 Poznan, Poland Department of Human Anatomy and Physiology, Section of Anatomy, School of Medicine, University of Padua, I-35121 Padua, Italy

{

International Review of Cytology, Volume 263 ISSN 0074-7696, DOI: 10.1016/S0074-7696(07)63002-2

#

2007 Elsevier Inc. All rights reserved.

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7. Concluding Remarks Acknowledgments References

88 89 89

Abstract Leptin, the product of the obesity gene (ob) predominantly secreted from adipocytes, plays a major role in the negative control of feeding and acts via a specific receptor (Ob-R), six isoforms of which are known at present. Evidence has been accumulated that leptin, like other peptides involved in the central regulation of food intake, controls the function of the hypothalamic–pituitary– adrenal (HPA) axis, acting on both its central and peripheral branches. Leptin, along with Ob-R, is expressed in the hypothalamus and pituitary gland, where it modulates corticotropin-releasing hormone and ACTH secretion, probably acting in an autocrine–paracrine manner. Only Ob-R is expressed in the adrenal gland, thereby making it likely that leptin affects it by acting as a circulating hormone. Although in vitro and in vivo findings could suggest a glucocorticoid secretagogue action in the rat, the bulk of evidence indicates that leptin inhibits steroid-hormone secretion from the adrenal cortex. In keeping with this, leptin was found to dampen the HPA axis response to many kinds of stress. In contrast, leptin enhances catecolamine release from the adrenal medulla. This observation suggests that leptin activates the sympathoadrenal axis and does not appear to agree with its above-mentioned antistress action. Leptin and/or Ob-R are also expressed in pituitary and adrenal tumors, but little is known about the role of this cytokine in the pathophysiology. Key Words: Leptin, Leptin receptor (Ob-R), Hypothalamus, Anterior pituitary, Adrenal gland, Corticotropin-releasing hormone (CRH), ACTH, Steroid hormone, Catecholamine. ß 2007 Elsevier Inc.

1. Introduction Leptin (from the Greek lEpto´B, thin) is a 147-amino acid residue peptide, first described by Zhang et al. (1994). It is the product of the obesity gene (ob) and is predominantly secreted by adipocytes and stomach (Myers, 2004; Zhang et al., 1994, 2005). Leptin plays a role in the control of feeding, acting to decrease caloric intake and to increase energy expenditure (Ahima and Flier, 2000; Mantzoros and Moschos, 1998; Myers, 2004; Remesar et al., 1997; Unger, 2000; Zhang et al., 2005). Compelling evidence indicates that peptides involved in the regulation of food intake (e.g., beacon, cholecystokinin, galanin, neuropeptide-W, neuropeptide-Y, and orexins) (Baker et al., 2003; Bedecs et al., 1995; Cerda-Reverter and Larhammar, 2000; Collier et al., 2000; Crawley and

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Corwin, 1994; Wolf, 1998) control the function of the hypothalamic– pituitary–adrenal (HPA) axis, acting on both its central and peripheral branch (Andreis et al., 2005, 2007; Hocho´l et al., 2007; Krysiak et al., 1999; Malendowicz et al., 1994, 2003b; Mazzocchi et al., 1998, 2005; Nussdorfer et al., 2005; Rucinski et al., 2005a,b; Spinazzi et al., 2005, 2006). Accordingly, leptin also regulates neuroendocrine axes, including the HPA one (Ahima et al., 2000; Bates and Myers, 2003; Casanueva and Dieguez, 1999; Sahu, 2003; Wauters et al., 2000). The interactions of peptides regulating food intake, and especially leptin, with the HPA axis are of great relevance, inasmuch as glucocorticoid hormones are known to be involved in the control of energy homeostasis and adipogenesis (Jeong et al., 2004; Mastorakos and Zapanti, 2004). At low concentrations, glucocorticoids exert anabolic effects and stimulate feeding, adipocyte differentiation, and normal fat deposition (Campfield et al., 1996; Dallman et al., 1993; Freedman et al., 1986; Hauner et al., 1987). The permissive role of glucocorticoids in the development of obesity is suggested by experiments showing that adrenalectomy prevents the progression of obesity in genetically obese Zucker rats (Freedman et al., 1986) and high doses of glucocorticoids cause excessive fat storage (Davenport et al., 1989). On the other hand, glucocorticoids have been reported to enhance leptin expression in and secretion from adipocytes (Slieker et al., 1996; Zakrzewska et al., 1997), an effect that could dampen their anabolic action. Despite the large number of investigations carried out in the past 12 years and the physiological relevance of the matter, only two short survey articles have been published on the role of leptin in the regulation of the HPA axis (Glasow and Bornstein, 2000; Wauters et al., 2000). Thus, after a brief account of the biology of the leptin system, we will review findings indicating that leptin and/or its receptors (R) are expressed in the anatomical components of the HPA axis and that leptin plays a role in the functional regulation of the HPA axis under both physiological and pathological conditions.

2. Biology of Leptin and Its Receptors 2.1. Leptin 2.1.1. Biosynthesis and secretion The human ob gene is located on chromosome 7q31.3, has more that 15,000 base pairs, and consists of three exons and two introns. It encodes for the leptin precursor, peptides of 167 amino acids including the 21 residues of the signal peptide (Fig. 2.1). The tertiary structure of the leptin molecule resembles that of the members of the growth hormone (GH) four-helical cytokine subfamily (Zhang et al., 2005). There is considerable homology in

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1 H2N-

22 26 SP

39

56

93

105 116 130 138 150

167 COOH

Figure 2.1 Scheme illustrating the human leptin precursor. Signal peptide (SP) and leptin fragments used in experimental works (see Section 5) are shown in gray and/or dashed-gray.

the leptin sequence among the various mammalian species and astonishingly also among mammals, birds, and fish (Table 2.1). Leptin synthesized in the rat adipose tissue is secreted into the bloodstream probably via a constitutive mechanism (Barr et al., 1997; Hardie et al., 1996). However, in humans leptin secretion is pulsatile, with 32 pulses every 24 h and a pulse duration of 33 min (Licino et al., 1997). The biological half-life of circulating leptin has been reported to be 25–70 min in humans (Hill et al., 1998; Klein et al., 1996), about 100 min in monkeys, 6–7 min in rats, and about 50 min in mice (Ahren et al., 2000; Vila et al., 1998). 2.1.2. Structure–function relationships There is a remarkable disagreement on this matter. According to Imagawa et al. (1998), the N-terminal region of the human leptin molecule (94amino acid residues), but not the C-terminal loop region (51-amino acid residues), is essential for both R-binding and biological activities. Also the region between the N-terminal and C-terminal regions does not possess biological activity because synthetic leptin(109–133)S-S(159–166) was shown to be ineffective. In contrast, Grasso et al. (1997) localized the active domain of the murine leptin molecule in the 106–140 amino acid sequence and Rozhavskaya-Arena et al. (2000) localized it in the 116–122 sequence. In vivo experiments identified the 85–119 sequence, which includes the end of a-helix C and the intervening C/D loop with helix E and is outside the region where leptin contacts its R (i.e., the interface of a-helices A and C), as critical for appetite suppression and weight loss in obese mice (Grasso et al., 1997, 1999). These investigators also showed that the leptin fragment between amino acids in the 21 and 105 positions is deprived of functional epitopes connected with feeding regulation and that the administration of leptin fragments 106–120, 116–130, and 126–140 causes body weight loss in female obese mice. Of interest, Tena-Sempere et al. (2000) reported that in the rat pituitary gland and ovary leptin fragment 116–130 exerts actions both similar to and distinct from those of the native molecule. The implications of this observation in explaining the role of leptin in adrenal gland regulation will be discussed in Section 5.1.1.

Table 2.1 Leptin sequences among various organismsa Homo sapiens

a

Rattus norvegicus

Mus musculus

Species

% identity

% positives

% identity

% positives

% identity

% positives

Homo sapiens (M) Rattus norvegicus (M) Mus musculus (M) Pongo pygmaeus orangutan (M) Pan troglodytes (M) Gorilla gorilla (M) Bubalus bubalis (M) Bos taurus (M) Ursus thibetanus japonicus (M) Sus scrofa (M) Capra hircus (M) Ovis aries (M) Canis familiaris (M) Felis catus (M) Halichoerus grypus (M) Anas platyrhynchos (A) Gallus gallus domesticus (A) Ctenopharyngodon idella (P) Cyprinus carpio (P) Megalobrama amblycephala (P) Silurus asotus (P) Channa argus (P) Aristichthys nobilis (P)

– 83.7 85.0 97.3 99.3 98.6 87.1 87.1 83.7 87.1 87.1 87.1 82.3 86.4 66.0 84.4 81.6 85.0 84.4 83.7 83.7 83.0 82.3

– 90.5 92.5 98.6 100 99.3 93.9 93.9 92.5 93.9 93.2 93.2 89.8 92.5 78.2 91.8 89.1 92.5 91.8 91.8 91.2 90.5 90.5

83.7 – 95.9 81.0 83.0 82.3 85.7 85.7 81.6 83.7 85.0 85.0 79.6 82.3 63.3 95.9 92.5 95.9 95.2 94.6 93.9 93.9 93.2

90.5 – 98.0 89.1 90.5 89.8 91.2 91.2 89.1 90.5 90.5 90.5 86.4 87.8 73.5 98.0 94.6 98.0 97.3 97.3 96.6 95.9 95.9

85.0 95.9 – 82.3 84.4 83.7 85.0 85.0 81.0 83.0 84.4 84.4 78.9 81.6 62.6 99.3 95.9 100 99.3 98.6 98.0 98.0 97.3

92.5 98.0 – 91.2 92.5 91.8 91.2 91.2 89.1 90.5 90.5 90.5 86.4 87.8 74.1 99.3 96.6 100 99.3 99.3 98.6 98.0 98.0

Leptin sequence homology of different species of mammals (M), aves (A), and pisces (P) in relation to leptin molecule of Homo sapiens, Rattus norvegicus, and Mus musculus. The alignment was performed in AlignX software.

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2.2. Leptin receptors Leptin exerts its biological effects through the activation of a specific R, the Ob-R, the product of the db gene (Tartaglia et al., 1995). The human Ob-R gene contains 18 exons and 17 introns and encodes for six R isoforms (see following), the longest of which is composed of 1162 amino acid residues. The Ob-R structurally belongs to the Class-I cytokine R family. The extracellular part of the human Ob-R contains at least seven structural domains (Fong et al., 1998): domains 1 (62–178 residue) and 2 (235–327 residue) possess a fibronectin type III fold and together form the cytokine R homology module 1; domain 3 (328–427 residue) has an Ig-like fold; domains 4 (428–535 residue) and 5 (536–635 residue) again display a fibronectin type III fold and together form the cytokine R homology module 2; and domains 6 and 7 also adopt a fibronectin type III fold. Multiple splice variants of the Ob-R mRNA encode for at least six isoforms (from Ob-Ra to Ob-Rf ), which all share a common extracellular ligand-binding domain. Ob-Re does not contain transmembrane and intracellular domains and circulates as soluble R (Friedman and Halaas, 1998). The other isoforms possess intracellular domains, but only the longest one, Ob-Rb (also called Ob-Rl), contains all domains required to activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway (Ahima et al., 2000; Tartaglia, 1997). The short isoforms may serve as leptin-binding poteins, and play a role in leptin transport across the blood–brain barrier and clearance from circulation. Ob-Rb is primarily expressed in the hypothalamus (see Section 3.1.2), but, along with other isoforms, also in peripheral tissues, including lungs, kidneys, liver, pancreas, thyroid gland, adrenal glands (see Section 3.3.2), and gonads (see Section 5.2) (Ahima et al., 2000; Baldelli et al., 2002; Bendinelli et al., 2000; Lloyd et al., 2001; Malendowicz et al., 2004a; Mantzoros, 1999; Nowak et al., 1998, 2002a; Seufert, 2004; Tena-Sempere and Barreiro, 2002; Zhang et al., 2005). Ob-Rb not only activates the JAK/STAT cascade, but, along with Ob-Ra, also mitogen-activated protein kinase (MAPK) p42/p44 and p38 signaling pathways, as well as stress-activated protein kinase (PK) c-Jun N-terminal kinase (JNK). Ob-Rb has also been reported to signal via phosphoinositide-3-kinase (PI3K)/phosphodiesterase-3B/cyclic-adenosine 30 ,50 -monophosphate (cAMP) and 50 -AMP-activated PK cascades. PI3K products may in turn stimulate PKB (Akt) and PKC isoforms and endothelial nitric oxide (NO) synthase (NOS). The extremely complex and not yet settled signaling mechanism of Ob-Rb may also involve its cross talk with other R, for example, insulin and insulin-like growth factor (IGF) R (Ahima and Osei, 2004; Bjorbaek et al., 1997; Fru¨hbeck, 2006; Hegyi et al., 2004; Murakami et al., 1997; Peelman et al., 2006; Yamashita et al., 1998).

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3. Expression of Leptin and Its Receptors in the Hypothalamic–Pituitary–Adrenal Axis 3.1. Hypothalamus 3.1.1. Leptin Available data on ob gene expression in the hypothalamus are few and rather conflicting. Reverse transcription (RT)-polymerase chain reaction (PCR) and immunocytochemistry (ICC), but not Western blotting, detected leptin mRNA and protein in the rat hypothalamus (Morash et al., 1999). In subsequent studies, semiquantitative PCR analysis did not evidence agerelated changes in leptin mRNA expression in the female rat hypothalamus from day 2 to day 28 of postnatal life (Morash et al., 2001). Double-label fluorescent ICC showed that in the rat paraventricular and supraoptic nuclei (PVN and SON, respectively) most oxytocin- and vasopressinimmunoreactive neurons also contained leptin immunoreactivity (ir) (Ur et al., 2002). Leptin expression was not detected in the hypothalamus of calves (Chelikani et al., 2003) and sheep (Dyer et al., 1997). In the female pig, leptin gene expression in the medial basal hypothalamus was found to be higher in the mid- than in the late-luteal phase and at days 30–32 than days 14–16 of pregnancy (Kaminski et al., 2006). 3.1.2. Leptin receptors After the pioneeristic demonstration of the presence of high-affinity leptinbinding sites in the rat hypothalamus (Stephens et al., 1995), PCR, Northern blotting, in situ hybridization, Western blotting, and ICC studies consistently showed the expression of the Ob-R, and especially of the long isoform Ob-Rb, in the hypothalamus of humans (Burguera et al., 2000; Couce et al., 1997), monkeys (Finn et al., 1998; Hotta et al., 1998), cows (Chelikani et al., 2003; Ren et al., 2002), sheep (Iqbal et al., 2001; Muhlhausler et al., 2004; Williams et al., 1999), pigs (Czaja et al., 2002; Kaminski et al., 2006; Lin et al., 2000; Smolinska et al., 2004; Zhou et al., 2004), rats (Hakansson and Meister, 1998; Schwartz et al., 1996a,b; Zamorano et al., 1997), mice (Fei et al., 1997; Mercer et al., 1996; Raber et al., 1997), and fowls (Taouis et al., 2001). There is general consensus that Ob-R is primarily in the PVN, and colocalization of Ob-Rb with corticotropin-releasing hormone (CRH) or proopiomelanocortin (POMC) in many hypothalamic neurons has been demonstrated in the sheep (Iqbal et al., 2001) and rat (Hakansson and Meister, 1998). Evidence has also been provided that the level of Ob-R expression does vary according to the age, sex, and strain of animals. In the pig hypothalamus, Ob-Rb mRNA was higher in the Large White than in the Erhualian

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strain. The expression in the former strain was very low after birth, increased gradually until weaning, and then decreased with age. In the Erhualian pigs, Ob-Rb expression displayed a net rise from day 120 to day 180 of postnatal life (Zhou et al., 2004). Elevated Ob-R expression was detected in the gilt hypothalamus at days 14–16 and 30–32 of pregnancy (Kaminski et al., 2006; Smolinska et al., 2004). In the rat hypothalamus, Ob-Rb mRNA was undetectable at day 4 of postnatal life, then rose significantly from day 4 to days 14–22. However, ICC detected Ob-Rbir also at day 4 (Morash et al., 2003). Ob-Rb mRNA was elevated at day 7 of pregnancy, but returned to the prepregnancy level by midgestation and then remained stable during lactation (Seeber et al., 2002). In contrast, it has been recently reported that hypothalamic Ob-R mRNA expression decreased in rats during pregnancy and then rose after delivery (Szczepankiewicz et al., 2006). Leptin expression has been firmly demonstrated only in the pig and rat hypothalamus. In contrast, abundant evidence showed the expression of Ob-R (especially the Ob-Rb isoform) in the hypothalamus of all mammalian species so far examined. Ob-R expression occurs in the PVN, and the localization of the Ob-Rb in many hypothalamic CRH- and POMC-positive neurons has been reported.

3.2. Anterior pituitary 3.2.1. Leptin Leptin expression, as mRNA and protein, has been detected in the anterior pituitary of humans (Isono et al., 2003; Jin et al., 1999; Korbonits et al., 2001a,b; Lloyd et al., 2001; Vidal et al., 2000), cows (Yonekura et al., 2003), pigs (Kaminski et al., 2006; Smolinska et al., 2004), rats ( Jin et al., 2000; Morash et al., 1999, 2001, 2003; Yonekura et al., 2003), and mice ( Jin et al., 2000). Probably due to the different primers used, Chelikani et al. (2003) failed to demonstrate by PCR leptin mRNA in bovine (Holstein strain) anterior pituitary. In the rat, pituitary leptin mRNA levels were maximal during postnatal days 7–14 and then decreased gradually to adulthood (Morash et al., 2001, 2003). Marked interspecies differences were observed in leptin-ir localization in the various anterior-pituitary cell types. ICC showed leptin-ir in 25–50% of cells of the human anterior pituitary, mostly in hormone-secreting ones (Isono et al., 2003; Jin et al., 1999; Lloyd et al., 2001; Vidal et al., 2000). Leptinpositive cells were corticotrophs, 70–80%; somatotrophs, 10–21%; thyreotrophs, 20–32%; gonadotrophs, 25–33%; lactotrophs, 3%; and folliculostellate cells, 64%. Immunoelectron microscopy showed the colocalization of leptin with the respective hormone in the secretory granules, suggesting its intracellular storage. Of interest, confocal ICC analysis of a murine ACTH-secreting

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AtT20 cell line expressing an epitope-tagget human leptin (FLAG-leptin) demonstrated that leptin-ir was colocalized with endogenus ACTH at the tips of the cytoplasmic processes, where regulated secretory granules accumulate (Chavez and Moore, 1997). In contrast with humans, ICC revealed the presence of leptin-ir only in a small fraction of rodent anterior-pituitary cells (about 5% and 7% in rats and mice, respectively) and showed that less than 1% of rat corticotrophs expressed leptin ( Jin et al., 2000). These observations may suggest different regulatory functions of leptin in the human and rodent pituitary during its evolutionary development. 3.2.2. Leptin receptors Ob-R expression, as mRNA and protein, has been demonstrated in the anterior pituitary of humans (Dieterich and Lehnert, 1998; Jin et al., 1999; Korbonits et al., 2001a,b; Shimon et al., 1998), monkeys (Finn et al., 1998), cows (Chelikani et al., 2003; Yonekura et al., 2003), sheep (Dyer et al., 1997; French et al., 2006; Iqbal et al., 2000), pigs (Kaminski et al., 2006; Lin et al., 2000, 2001, 2003; Siawrys et al., 2005), rats ( Jin et al., 2000; Morash et al., 1999, 2003; Sone et al., 2001; Szczepankiewicz et al., 2006; Yonekura et al., 2003; Zamorano et al., 1997), and mice (Cai and Hyde, 1998; Raber et al., 1997). Interesting findings have been reported on the splice variant of the Ob-R expressed, which can be summarized as follows. Ob-Ra and Ob-Rb have been detected in the anterior pituitary of humans (Dieterich and Lehnert, 1998), cows (Chelikani et al., 2003), and rats ( Jin et al., 2000; Morash et al., 1999); however, according to Shimon et al. (1998), both isoforms were expressed only in the adult human pituitary, the fetal gland expressing only Ob-Rb. Only Ob-Ra expression was found in cultured bovine and rat anterior-pituitary cells (Yonekura et al., 2003), and no Ob-Rb mRNA was detected in the ovine pituitary by in situ hybridization (Williams et al., 1999). Pituitary Ob-Ra mRNA levels were high in neonatal rats (day 4) and then declined, paralleling the fall in leptin expression, whereas Ob-Rb mRNA was stable from day 4 to day 22 of postnatal life (Morash et al., 2003). Ob-Rb expression in the gilt and rat pituitary has been reported to decrease in the late phases of pregnancy (Kaminski et al., 2006; Szczepankiewicz et al., 2006). ICC revealed that Ob-R was present in about 97% of rat somatotrophs and less than 1% of lactotrophs, thyreotrophs, gonadotrophs, and corticotrophs (Sone et al., 2001). In contrast, Iqbal et al. (2000) by double-label immunofluorescence observed Ob-R in about 27% of corticotrophs of the sheep pituitary. The concomitant expression of leptin and Ob-R in the anterior pituitary may suggest that autocrine–paracrine loops are operative, mediating leptin effects on the secretion of pituitary hormones. In contrast with other

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Figure 2.2 Ethidium bromide-stained 2% agarose gel showing cDNA amplified with rat leptin (Ob) and Ob-R-isoform-specific primers from ZG, ZF/R, and adrenal medulla (AM) of adult rat adrenals. Primer sequences were leptin sense (190^209), 50 GACATTTCACACACGCAGTC-30 and leptin antisense (366^384), 50 -GAGGAGGTCTCGCAGGTT-30 (195 bp; accession number NM 013076); Ob-Ra sense (110^131), 50 -CACTGTTAATTTCACACCAGAG-30 and Ob-Ra antisense (323^344), 50 -GTCATTCAAACCATAGTTTAGG-30 (235 bp; accession number AF 304191); Ob-Rb sense (2635^2653), 50 -TGCTCGGAACACTGTTAAT-30 and Ob-Rb antisense (2785^2805), 50 GAAGAAGAGGACCAAATATCA-30 (171 bp; accession number U52966); Ob-Rc sense (35^53), 50 -TGCTCGGAACACTGTTAAT-30 and Ob-Rc antisense (172^195), 50 -ATAGAGTATCTAAACTGCAACCTT-30 (161 bp; accession number AF 007818); Ob-Re sense (595^614), 50 -TCCTGGACACTGTCACCTAA-30 and Ob-Re antisense (759^778), 50 -ATCAGGATTGCCAATTTACA-30 (184 bp; accession number AF 007819); and Ob-Rf sense (2676^2696), 50 -GCTGCTCGGAACACTGTTAAT-30 and Ob-Rf antisense

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mammalian species so far examined, only a very low percent of corticotrophs of rodents express leptin and Ob-R, which could cast doubt on the relevance of the leptin system in the regulation of ACTH secretion in this order of animals.

3.3. Adrenal gland 3.3.1. Leptin RT-PCR did not detect leptin mRNA either in the cortex or the medulla of human (Glasow et al., 1998), calf (Chelikani et al., 2003), and rat adrenals (see Figs. 2.2 and 2.3), as well as in the human carcinoma-derived NCIH295 cell line and primary human adrenal cultures containing both cortical and medullary cells (Glasow and Bornstein, 2000). Personal ICC data confirmed the absence of leptin-ir in the cortex but evidenced the presence of leptin-positive cells in the medulla (Fig. 2.4). The location of these cells near the blood vessels and their positivity to tryptase (not shown) could suggest that they are mast cells. It must be noted that colocalization of leptin with tryptase has been recently demonstrated in mast cells of human myometrium (Ribatti et al., 2007). 3.3.2. Leptin receptors Adrenal cortex RT-PCR demonstrated Ob-Rb mRNA expression in the adrenal cortex of humans (Glasow and Bornstein, 2000; Glasow et al., 1998, 1999; Pralong et al., 1998), cows (Chelikani et al., 2003), pigs (Lin et al., 2000), rats (Malendowicz et al., 2003a, 2004b; Pralong et al., 1998; TenaSempere et al., 2000), and mice (Hoggard et al., 1997), as well as in NCIH295 cells (Biason-Lauber et al., 2000; Glasow and Bornstein, 2000). Noteworthy, Northern blot analysis only partially confirmed the presence of Ob-R mRNA in human and rat adrenal cortex (Pralong et al., 1998; Zamorano et al., 1997). Ob-Ra mRNA expression, in addition to Ob-Rb expression, was found in cow and rat adrenal cortex (Chelikani et al., 2003; Malendowicz et al., 2003a, 2004b; Tena-Sempere et al., 2000). Semiquantitative PCR showed the predominant expression of the Ob-Ra and Ob-Rb isoforms over that of the Ob-Rc and Ob-Rf isoforms in the rat adrenal cortex, Ob-Re expression being negligible (Tena-Sempere et al., 2000). Personal unpublished real-time PCR findings demonstrated the expression of Ob-Ra, Ob-Rb, Ob-Rc, Ob-Re, and Ob-Rf in dispersed (2806^2826), 50 -ACGGCATCCACTCTATATCCT-30 (151 bp; accession number D84125). The PCR program was denaturation step (95 C for 10 min), followed by 35 cycles of three step amplification (denaturation, 95 C for 10 sec; annealing, 58 C for 5 sec; and extension,72 C for 10 sec). Lane1was loaded with Roche MarkerVIII (200 ng).

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Figure 2.3 Real-time PCR semiquantitative analysis of rat leptin and Ob-R gene expression in the ZG, ZF, and adrenal medulla (AM) of adult rat adrenals. Primer sequences for Ob-Rwere those indicated in the legend to Fig. 2.2, and that for GAPDH was sense (18^27), 50 -TTCTAGAGACAGCCGCATCT-30 and antisense (104^123), 50 TGGTAACCAGGTGTCCGATA-30 (106 bp; accession number X02231). The program, as described in the legend to Fig. 2.2, was a total of 45 cycles, followed by a melting curve (60^90 C with a heating rate of 0.1C/sec). All samples were amplified in duplicate and the GAPDH gene was used as reference to normalize data.

rat zona glomerulosa (ZG) and zona fasciculata-reticularis (ZF/R) cells, the level of expression being Ob-Rf > Ob-Ra ¼ Ob-Rc > Ob-Rb > Ob-Re. Except for Ob-Re, the level of expression of other Ob-R isoforms was lower in the cortex than in the medulla (see Figs. 2.2 and 2.3). ICC showed intense Ob-Rb immunostaining in the human adrenal cortex (Glasow and Bornstein, 2000; Glasow et al., 1998). In contrast, earlier studies did not detect Ob-R protein in the rat adrenal cortex (Cao et al., 1997).

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Figure 2.4 ICC demostration of leptin-ir in exemplary cryosections of human adrenal gland. Leptin-ir is absent in the capsule (c), subcapsular ZG, and ZF (A), while in adrenal medulla few leptin-positive cells (arrowheads), either isolated (B) or grouped in small clusters (C) near blood vessels (*), can be observed. Negative controls (D) were obtained by incubating cryosections with primary antibodies preabsorbed with leptin. Sections were incubated with the primary rabbit antibody to human leptin (Phoenix Pharmaceuticals, Belmont, CA) (1:500 dilution) at 37 for 60 min and then with the secondary peroxidase-conjugated antirabbit IgG goat antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) (1:50 dilution) at 37 for 40 min. After rinsing, the reaction was developed for 5 min with Sigma Fast 30,30 -diaminobenzidine 0.7-mg tablets (Sigma-Aldrich Corp., St. Louis, MO). Magnification 750.

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Adrenal medulla The expression of Ob-Rb mRNA has been demonstrated in the adrenal medulla of humans (Glasow and Bornstein, 2000; Glasow et al., 1998), pigs (Takekoshi et al., 1999), and mice (Hoggard et al., 1997). In cultured bovine adrenomedullary cells only the expression of Ob-Ra was found (Yanagihara et al., 2000). Personal unpublished real-time PCR data showed that Ob-R expression was markedly higher in the medulla than in the cortex of rat adrenals, the level of expression of the various Ob-R isoforms being that described in the cortex (Fig. 2.3). ICC revealed the presence of Ob-R protein in the adrenal medulla of humans, where Ob-Rb immunostaining was weaker than in the cortex (Glasow and Bornstein, 2000; Glasow et al., 1998), and rats (Cao et al., 1997). Consistent evidence indicates that at variance with the hypothalamus and anterior pituitary, leptin is not expressed in the adrenal gland. In contrast, Ob-R is expressed in both adrenal cortex and medulla, leading to the view that leptin can modulate the secretion of both corticosteroid hormones and catecholamines, acting as a circulating hormone.

4. Effects of Leptin on the Central Branch of the Hypothalamic–Pituitary–Adrenal Axis 4.1. Hypothalamus and CRH secretion 4.1.1. CRH expression and biosynthesis Earlier studies showed that the intracerebroventricular (icv) injection of leptin raised by about 40% CRH mRNA in the PVN of normal rats, but not leptin-resistant Zucker animals (Schwartz et al., 1996a), as well as induced c-fos protein in the parvocellular division of PVN (Elmquist et al., 1998; Masaki et al., 2003; Van Dijk et al., 1996). There is also an indication that icv leptin administration specifically activated CRH sympathetic neurons giving rise to descending autonomic transmission (Elmquist et al., 1997; Okamoto et al., 2000). Leptin, either icv or systemically administered, was found to increase within 2–6 h the hypothalamic CRH concentration (Uehara et al., 1998) and CRH mRNA in the rat PVN, as revealed by in situ hybridization (Nishiyama et al., 1999). However, subcutaneous (sc) leptin infusion for 5 days did not change CRH expression (Nishiyama et al., 1999). The leptin-induced increase in CRH mRNA in rat PVN was prevented by pretreatment with a V1a-R antagonist, suggesting that the effect was at least partly mediated by arginin-vasopressin (AVP) (Morimoto et al., 2000). Daily leptin administration for 5 days did not alter CRH mRNA expression in PVN of obese ob/ob mice (Schwartz et al., 1996a), but leptin prolonged infusion (for 7 days) prevented the starvation-induced CRH biosynthesis and c-fos protein induction in PVN of ob/ob male animals (Huang et al., 1998).

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Accordingly, leptin was found to downregulate CRH expression in mouse PVN and to prevent the bilateral adrenalectomy-induced increase in CRH mRNA (Arvaniti et al., 2001). To conclude, we wish to mention that continuous infusion with leptin for 6 h failed to alter CRH mRNA expression in the fowl hypothalamus (Dridi et al., 2005). 4.1.2. CRH release Evidence has been provided that leptin, but not the heat-inactivated peptide, enhanced (from 10 10 to 10 7M) CRH release from male rat hypothalamic explants, the maximal effective concentration being 10 8M (Costa et al., 1997). Similar findings have been obtained in mice (Raber et al., 1997). Although leptin has been reported to inhibit hypoglycemia-induced CRH release from perfused male rat hypothalamic explants (Heiman et al., 1997), more recent studies confirmed the stimulating action of leptin (Jethwa et al., 2006). In contrast to CRH, AVP release was not affected by leptin in mice (Raber et al., 1997); however, it was increased in rabbits and rats (Matsumura et al., 2000; Yamamoto et al., 1999). Some data suggest that the effect of leptin on CRH secretion may depend on the presence of glucocorticoid hormones ( Jang et al., 2000). Leptin icv administration did not change within 1–3 h either CRH concentration in the PVN of lean and obese mice or CRH release from hypothalamic explants of animals with intact adrenal glands. Conversely, it raised by about 50% CRH secretion from hypothalamic preparations of adrenalectomized animals. Taken together, these findings indicate that leptin acutely stimulates hypothalamic CRH biosynthesis in rats, while its prolonged administration exerts an inhibitory effect in mice. It remains unsettled whether these apparently conflicting observations depend on the different modalities of treatment (acute versus chronic) or interspecies differencies (rat versus mouse). In contrast, there is large consensus that leptin enhances hypothalamic CRH release in rodents, the secretagogue action being perhaps restrained by the presence of endogenous glucocorticoids.

4.2. Anterior pituitary and ACTH secretion 4.2.1. ACTH expression and biosynthesis Northern blot analysis revealed that acute leptin icv administration raised POMC mRNA expression in the rat anterior pituitary. The effect was partially reversed by pretreatment with an antagonist of the V1a-R, thereby suggesting the involvement of AVP (Morimoto et al., 2000). The prolonged (from 2 to 16 days) systemic administration of leptin increased pituitary ACTH concentration in both adrenalectomized female rats (Malendowicz et al., 2001) and intact animals (Nowak et al., 2002b). In contrast, leptin has been reported to decrease POMC and PC2 (a POMC processing enzyme)

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mRNAs in the anterior pituitary of obese mice (where the expression was elevated), but not in primary cultures of anterior pituitary cells from young female C57BL/6J mice (Renz et al., 2000). 4.2.2. ACTH release Leptin did not affect either basal or CRH-stimulated ACTH release from primary cultures of rat pituitary cells (Heiman et al., 1997; Pralong and Gaillard, 2001). In contrast, it enhanced ACTH release from superfused mouse pituitary slices, and the effect was specific because it was blocked by antileptin antibodies (Raber et al., 1997). In light of the rather scanty investigations herein surveyed, it seems legitimate to conclude that leptin exerts opposite effects on the anterior pituitary of rats and mice. Leptin appears to enhance ACTH biosynthesis in rats, and to inhibit (or not to affect) it in mice, and to raise ACTH release in mice, without affecting it in rats.

5. Effects of Leptin on the Peripheral Branch of the Hypothalamic–Pituitary–Adrenal Axis 5.1. Adrenal cortex 5.1.1. Steroid-hormone secretion In vitro studies Humans Pralong et al. (1998) reported that the 24- (but not 6-) h exposure to leptin inhibited ACTH-stimulated, but not basal, cortisol secretion from primary cultures of human adrenocortical cells, and subsequent studies confirmed this observation (Glasow and Bornstein, 2000; Glasow et al., 1998). It was shown that leptin lowered ACTH-stimulated aldosterone secretion by about 30% and lowered cortisol and dehydroepiandrosterone (DHEA) yield by about 15% and that the drop in cortisol secretion was associated with a 50% decrease in cytochrome P450 (CYP) 17a mRNA expression. Completely different results were obtained using the human adrenocortical carcinoma-derived NCI-H295 cell line, which is commonly used as a reliable model of normal human steroid synthesis (Rainey et al., 2004). Earlier studies did not show any effect of leptin on basal and forskolinstimulated cortisol secretion (Lado-Abeal et al., 1999). However, further investigations evidenced a twofold effect of leptin on CYP17, which combines 17a-hydroxylase and 17,20-lyase activities, in NCI-H295 cells expressing Ob-R (Biason-Lauber et al., 2000). Leptin (3  10 8M) exposure for 24 h stimulated 17,20-lyase activity and DHEA production, without markedly changing 17a-hydroxylase activity. Conversely, shorter incubations raised 17a-hydroxylase activity and 17-hydroxyprogesterone

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synthesis, the effect becoming manifest within 30 min and disappearing within 4 h. Cows Bornstein et al. (1997) reported that within 24 h recombinant murine leptin inhibited basal and ACTH-stimulated cortisol release (about 20–50% decrease) from cultured bovine adrenocortical cells, as well as reduced CYP17 mRNA expression. Further investigations also showed the inhibition of the expression of CYP11A and CYP21A, which possess cholesterol side-chain cleaving and 21C-hydroxylase activity, respectively (Kruse et al., 1998). Rats Investigations on the direct effect of leptin on adrenocortical steroidogenesis in the rat gave intriguing and controversial results. The first studies showed that within 60 min recombinant murine leptin enhanced basal, but not ACTH-stimulated, aldosterone (10 8 and 10 7M, but not lower and higher concentrations) and corticosterone output (from 10 9 to 10 6M) from dispersed ZG and ZF/R cells, respectively (Malendowicz et al., 1997). Subsequent investigations, carried out using adrenocortical cells cultured for 48 h in normal growth medium and then for 24 h in fetal calf serum-free medium, demonstrated that the 24 h exposure to 10 7M leptin lowered ACTH-stimulated, but not basal, corticosterone yield (Pralong et al., 1998). An acute inhibitory effect of leptin on ACTH-stimulated aldosterone and corticosterone production has also been shown in freshly dispersed adrenocortical cells of newborn rats (Salzmann et al., 2004) and on basal and ACTH-stimulated corticosterone secretion in adult rat adrenocortical slices (Tena-Sempere et al., 2000). Cherradi et al. (2001), although unable to observe any effect of leptin on basal and ACTH-stimulated pregnenolone production from primary cultures of rat adrenocortical cells, observed that leptin pretreatment significantly lowered the acute pregnenolone response to ACTH. These investigators suggested that the target for the inhibitory action of leptin resides upstream of pregnenolone synthesis and requires a chronic exposure to leptin. Accordingly, neither cAMP production nor CYP11A expression was affected by leptin, which, however, downregulated the steroidogenic acute regulatory protein (StAR) expression induced by an exogenous ACTH challenge (Cherradi et al., 2001; Salzmann et al., 2004). The biological effects of leptin on target tissues are mediated by several ObR isoforms (see Section 2.2), which could variously interact with native leptin and its fragments (see Fig. 2.1). This consideration prompted us to investigate the effects of leptin and several leptin fragments (10 8 and 10 6M) on corticosteroid-hormone secretion from dispersed or cultured rat adrenocortical cells (Malendowicz et al., 2003a, 2004b). In freshly dispersed cells, native murine leptin, its fragment 116–130, and human leptin fragments 138–167, 150–167, and [Tyr]26–39 acutely raised basal aldosterone and corticosterone secretion. Human leptin fragment 93–105 was ineffective, while fragment

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22–56 lowered corticosterone (but not aldosterone) output. In cultured ZF/R cells, the 96-h exposure to native leptin and leptin fragments 150–167 and 26– 39 enhanced corticosterone secretion, fragment 116–130 was ineffective, and fragments 138–167 and 22–56 exerted an inhibitory effect. Fragment 93–105 displayed a dose-dependent biphasic effect: stimulating and inhibitory actions at low and high concentrations, respectively. Collectively, these findings led to the conclusion that in rat adrenocortical cells leptin and its fragments may interact differently with Ob-R or interact with different isoforms. They also suggested that the direct adrenocortical secretagogue action of leptin depends on the C-terminal sequence 116–166 and that the N-terminal sequence is not needed for leptin to activate Ob-R positively coupled to steroidogenesis, but is possibly responsible for a direct inhibitory action on glucocorticoid secretion. Mice As expected, leptin was ineffective on steroid secretion of cultured adrenocortical cells obtained from db/db mice (Pralong et al., 1998) because this strain bears a spontaneous mutation of Ob-Rb, rendering it totally devoid of signal-transduction capability (Vaisse et al., 1996). Of interest, leptin was found to concentration-dependently raise 11b-hydroxysteroid dehydrogenase (11b-HSD) type I activity in primary cultures of ob/ob (but not db/db) mouse hepatocytes (Liu et al., 2003). Since 11b-HSD-I regenerates glucocorticoids from inactive 11-keto forms (Stewart and Krosowski, 1999), leptin could be an important metabolic signal activating intrahepatic corticosterone production. It is to be stressed that in vivo experiments should take into account this extra-HPA axis action of leptin. In vivo studies Humans The fluctuations of the 24-h pattern of circulating leptin were found to be inverse to those of ACTH and cortisol in human healthy volunteers (Licino et al., 1997), or leptin maxima followed cortisol maxima (Wagner et al., 2000). Dexamethasone or cortisol administration evoked an acute substained increase in blood leptin concentration (Miell et al., 1996; Newcomber et al., 1998), but rises in the plasma cortisol in the physiological range did not influence the level of circulating leptin (Nye et al., 2000).

Monkeys The infusion of recombinant human leptin (for 4 or 48 h) did not change basal and ACTH-stimulated cortisol blood levels in fed or fasted Rhesus monkeys (Lado-Abeal et al., 1999, 2000). However, the prolonged infusion with leptin blunted the CRH-induced increase in ACTH and cortisol plasma concentrations and lowered the morning (but not evening) surge in ACTH in ovariectomized animals (Wilson et al., 2005).

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Sheep Late fetal life is characterized in the sheep by an increased HPAaxis activity that prepares the fetus for extrauterine life and initiates the endocrine cascade leading to parturition (Challis and Brooks, 1989). The icv infusion for 5 days of leptin to the fetus in late gestation was found to inhibit a prepartum surge of ACTH and cortisol in the mother (Howe et al., 2002). The intravenous (iv) infusion of recombinant ovine leptin for 4 days of pregnant sheep at day 136 of gestation did not change the levels of ACTH and cortisol in the fetal blood, while the infusion at day 144 (i.e., near term; 145  2 days) suppressed cortisol without affecting ACTH (Yuen et al., 2004). Rats Earlier studies described an inhibitory action of leptin on the HPA axis and its involvement in the regulation of a diurnal pattern of corticosterone secretion (Ahima et al., 1996; Heiman et al., 1997). More recent findings confirmed this contention, showing that icv or ip administration of leptin lowered the blood level of corticosterone (Akirav et al., 2004; Clark et al., 2006). A negative feedback loop has been proposed between leptin and the HPA axis, where the increase in ACTH blood level raises leptin, which in turn inhibits corticosterone secretion (Spinedi and Gaillard, 1998). Accordingly, evidence has been provided that experimental conditions able to increase plasma leptin (as the monosodium L-glutamateinduced destruction of the hypothalamic arcuate nucleus) downregulated Ob-Rb mRNA expression in adrenals and enhanced their in vivo and in vitro corticosterone response to ACTH (Perello´ et al., 2003, 2004). Other studies obtained opposite findings. The icv injection of leptin was found to increase plasma corticosterone levels, the effect lasting 4 h and being especially intense at the onset of the dark phase (Van Dijk et al., 1996, 1997). Analogous observations have been described by Morimoto et al. (2000) and Jethwa et al. (2006), who reported a rise in both ACTH and corticosterone within 15–20 min after the leptin icv or ip injection. The bolus sc administration of recombinant murine leptin was found to raise the blood level of corticosterone within 60 min and the levels of both corticosterone and ACTH within 120 min (Hocho´l et al., 2000; Malendowicz et al., 1998). Further studies showed that the systemic administration not only of leptin, but also of leptin fragments 150–167, 138–167, 93–105, 22–56, and 26–39 was able to evoke a clearcut corticosterone response, suggesting that the in vivo stimulating action of leptin on glucocorticoid secretion is not connected, as occurred in vitro (see previously), to specific sequences of its molecule (Malendowicz et al., 2004a). The bolus sc injection of leptin also evoked within 120 min a net increase in the aldosterone plasma concentration (Malendowicz et al., 1998). In this connection, it is to be recalled that leptin icv injection increased the level of circulating AVP (see Section 4.1.2), which not only activates hypothalamic CRH neurons to

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drive ACTH secretion (Aguilera and Rabadan-Diehl, 2000; Engelmann et al., 2004), but also directly stimulates aldosterone production from ZG cells (Nussdorfer, 1996). Before concluding, it is necessary to mention the intriguing findings showing that leptin, although lowering corticosterone blood levels in normal rats (see previously), increased blood levels in streptozotocin-induced diabetic animals (Akirav et al., 2004). Mice The prolonged systemic administration of leptin (for up to 30 days) decreased corticosterone blood levels in ob/ob C57BL/6J, but not lean mice (Huang et al., 1998; Stephens et al., 1995). Accordingly, leptin partially blunted the fast-induced rise in the concentration of circulating corticosterone in C57BL/6J mice (Ziotopoulou et al., 2000). In summary, consistent findings show that leptin exerts a direct in vitro inhibitory effect on human and cow adrenocortical cells, connected with the downregulation of StAR and CYP17 expression. Contrasting results have been described in the rat, where leptin was found to either inhibit or stimulate steroid-hormone secretion. It is likely that the experimental model used (dispersed versus cultured cells) and the duration of exposure (short versus long term) may profoundly influence the in vitro leptin effects. In vivo studies indicate that leptin tends to inhibit the HPA axis in humans, monkeys, sheep, and mice. In the rat, contrasting findings have been again reported: Leptin appears to inhibit the HPA axis when centrally administered, but to stimulate it when given systemically. Probably, in this species, the in vivo effects of leptin are dependent on its route of administration and are the result of its combined action on the central and peripheral branches of the HPA axis. 5.1.2. Adrenocortical growth Adult and newborn adrenals The prolonged sc administration of leptin (six daily injections of 20 nmol/kg) resulted in a marked atrophy of adult rat adrenal cortex (Malendowicz et al., 2000b; Ziolkowska et al., 2001). The adrenal weight and the volume of ZF and its parenchymal cells were decreased. The proliferating cell nuclear antigen (PCNA) index was lowered in the ZG, and the apoptotic index (in situ TUNEL assay) was increased in the ZF and to a lesser extent in the ZR. Neither leptin nor leptin 116–130 altered the ZG mitotic index, as evaluated by the stachmokinetic method in adult rats, indicating that this technique is less sensible than the PCNA index assay (Malendowicz et al., 1999). In contrast, leptin fragment 116–130, but not native leptin, induced a 40% rise in the ZG mitotic index of immature (20-day–old) rats. Regenerating adrenals Adrenal regeneration after enucleation and contralateral adrenalectomy is a well-established model of adrenal growth, resembling that occurring during embryonal development and mainly involving cell proliferation (Dalmann, 1984–1985). The sc injection of

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leptin fragments 116–130, 138–167, and 150–167 (10 nmol/kg, 28, 16, and 4 h before the sacrifice) decreased the mitotic index of regenerating subcapsular tissue, while fragments 22–56 and [Tyr]26–39 and 93–105 were ineffective (Malendowicz et al., 2000a; Markowska et al., 2004). Adrenocortical cells cultured in vitro No effect of leptin on the proliferative activity of human adrenocortical cells in primary culture and cultured NCIH295 cells has been observed (Glasow and Bornstein, 2000; Glasow et al., 1999). In contrast, the 96-h exposure to leptin(1–147) lowered and fragment 26–39 enhanced proliferation of cultured rat adrenocortical cells, while fragments 22–56 and 138–167 were ineffective. Fragments 93–105 and 150– 167 exerted proliferogenic and antiproliferogenic effects at the concentrations of 10 8 and 10 6M, respectively (Malendowicz et al., 2004b). In summary, collectively, these reviewed findings indicate that in the rat, leptin suppresses the growth of the adrenal cortex. It is likely that the growth-promoting action of some leptin fragments (but not native leptin) on immature rat adrenals and adrenocortical cells cultured in vitro may be connected to their ability to bind and activate specific Ob-R isoforms specifically coupled to proliferogenic signaling pathways.

5.2. Other steroid-secreting cells Leptin plays a role in the functional regulation of steroid-secreting cells other than adrenocortical ones, namely those of endocrine gonads, and we will review this topic. 5.2.1. Testis Ob-Ra and Ob-Rb expression has been demonstrated in Leydig cells of the rat testis and Ob-Rb expression in a murine Leydig cell tumor line (Caprio et al., 1999). Leptin inhibited human chorionic gonadotropin (hCG)-stimulated testosterone secretion from rat Leydig cells (Caprio et al., 1999; Tena-Sempere et al., 2000), and the effect was associated with the downregulation of StAR and CYP11A (but not CYP17) expression (Tena-Sempere et al., 2001). As shown in adrenocortical cells, monosodioL-glutamate-induced hyperleptinemia lowered Ob-Rb mRNA in rat Leydig cells and counteracted the leptin-induced inhibition of their testosterone production (Giovanbattista et al., 2003). 5.2.2. Ovary Ob-Rb expression has been detected in human granulosa and cumulus cells (Cioffi et al., 1997) and in granulosa and corpus luteum cells of the rabbit ovary (Zerani et al., 2004). Leptin was reported to inhibit hCG-stimulated (but not basal) progesterone secretion from cultured human granulosa lutein cells in the presence of insulin (Brannian et al., 1999) and from a granulosa

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cell line (Barkan et al., 1999), as well as follicle-stimulating hormone (FSH)and IGF-I-stimulated progesterone yield from bovine granulosa lutein cells (Spicer et al., 2000), basal progesterone production from rabbit granulosa and corpus luteum cells (Zerani et al., 2004), and FSH-stimulated progesterone secretion from rat granulosa cells, this last effect being associated with downregulation of CYP11A expression (Barkan et al., 1999). A dosedependent biphasic effect of leptin has been observed in cultured porcine granulosa cells: At nanomole and micromole concentrations the cytokine increased and decreased progesterone synthesis and StAR mRNA expression, respectively (Ruiz-Corte´s et al., 2003). Earlier findings suggested a leptin-induced increase in estradiol (but not progesterone) production from human granulosa lutein cells, coupled to a rise in CYP aromatase expression (Kitawaki et al., 1999). However, subsequent investigations showed that leptin lowered basal (but not hCG- or IGF-I/II-stimulated) estradiol yield, although without inducing significant changes in StAR, CYP17, and CYP aromatase expression (Ghizzoni et al., 2001). Moreover, leptin was shown to inhibit FSH-stimulated estradiol secretion from rat granulosa cells (Barkan et al., 1999). Before concluding, we wish to recall that leptin, although inhibiting ovulation, was not found to alter in vivo progesterone and estradiol secretion from perfused rat ovary (Duggal et al., 2000). Taken together, the findings summarized herein provide firm evidence that leptin inhibits sex hormone secretion from endocrine gonads, its target point of action being the early step of steroid synthesis.

5.3. Adrenal medulla 5.3.1. In vitro studies Leptin was not found to affect catecholamine secretion from cultured human adrenomedullary cells (Glasow and Bornstein, 2000; Glasow et al., 1998). In contrast, leptin (3  10 9 and 3 10 8 M) enhanced catecholamine synthesis in cultured bovine adrenomedullary cells (Yanagihara et al., 2000). This last effect occurred via the activation of tyrosine hydroxylase (TH) through two mechanisms, one dependent on TH phosphorylation via the MAPK cascade and one independent of TH phosphorylation (Utsunomiya et al., 2001). In interest, according to Yanagihara et al. (2000), the leptininduced rise in catecholamine synthesis was not associated with the increase in their release. However, other findings indicate that leptin raises both catecholamine synthesis in and release from the adrenal medulla. Leptin (5 10 8 and 10 7M) enhanced catecholamine release via a mechanism mainly involving the activation of the voltage-gated Ca2þ L- and N-type channels (Takekoshi et al., 1999, 2001a) and the new synthesis of TH through the activation of the JAK/STAT, MAPK, and PKC-dependent Raf pathways (Takekoshi et al., 2001a,b).

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5.3.2. In vivo studies The icv injection of leptin was found to elicit a marked rise in the plasma catecholamine concentration in rabbits (Matsumura et al., 2000). Furthermore, evidence has been provided that leptin may increase catecholamine secretion from the adrenal medulla by stimulating the central sympathetic nervous system outflow (Satoh et al., 1999). Although rather scarce, these findings indicate that leptin stimulates catecholamine secretion from the adrenal medulla, at least of cows and pigs. Hence, leptin seems to exert opposite effects on the functions of the HPA and the hypothalamic sympathoadrenal axes.

6. Involvement of Leptin in the Pathophysiology of the Hypothalamic–Pituitary–Adrenal Axis 6.1. Response to stresses Leptin has been reported to dampen the HPA axis response (ACTH and/or glucocorticoid secretion) to an unpredictable situation in monkeys (Wilson et al., 2005) and starvation in mice (Ahima et al., 1996; Ziotopoulou et al., 2000). HPA axis activation by metabolic stress (glucose deprivation by means of 2-deoxyglucose administration), insulin-induced hypoglycemia, and restraint stress were also blunted by leptin in the rat (Giovanbattista et al., 2000; Heiman et al., 1997; Nagatani et al., 2001). However, in the rat only the corticosterone response was hampered; the plasma level of ACTH remained unchanged (Heiman et al., 1997). The sc bolus injection of leptin (5 nmol/kg) was found to induce a moderate magnification of ACTH response to ether stress at 2 h, followed by a net depression at 4 h. The corticosterone response was not affected (Hocho´l et al., 2000). It has been reported that newborn rodents exhibit adrenal hyporesponsiveness to stress during the first 2 weeks of life, probably induced by maternal leptin (Salzmann et al., 2004; Trottier et al., 1998). However, in 10-day–old rat pups daily leptin pretreatment from day 2 to day 9, although not inducing appreciable changes in basal HPA axis activity, was shown to lower the stress-evoked rise in CRH mRNA expression in PVN and to markedly shorten the duration of the ACTH response (Oates et al., 2000). In contrast, the sc bolus administration of leptin has been reported to enhance the ACTH response to cold stress, without altering the corticosterone response (Hocho´l et al., 2000). In conclusion, leptin prevents or attenuates the HPA response to various stresses, with the exception of cold stress. How leptin differentially regulates the activity of the HPA axis during stressful conditions is unclear (Nagatani et al., 2001). It is likely that the central mechanisms involved in the response

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to cold stress are different from those underlying the response to ether and other stresses.

6.2. Pituitary adenomas Leptin mRNA expression has been detected in three of four ACTHsecreting, one of four GH-secreting, one of four gonadotropin-secreting, and two of four nonsecreting pituitary adenomas ( Jin et al., 1999). Although, Knerr et al. (2001) found very low quantities of leptin mRNA in various pituitary tumors, leptin expression was demonstrated in two of three (Korbonits et al., 2001a,b) and four of five ACTH-secreting tumors (Isono et al., 2003). Electron microscopy showed leptin immunogold labeling only in ACTH-secreting adenomas (Vidal et al., 2000). Of interest, confocal microscopy ICC colocalized leptin-ir and ACTH-ir in the murine pituitary adenoma-derived cell line AtT20 (Chavez and Moore, 1997). Ob-R, almost exclusively of the b subtype, has been demonstrated in pituitary tumors (Dieterich and Lehnert, 1998; Jin et al., 1999, Knerr et al., 2001; Shimon et al., 1998). According to Korbonits et al. (2001a,b), Ob-Rb expression was present in five of nine ACTH-secreting, two of four GH-secreting, one of two prolactin-secreting, two of two gonadotropinsecreting, and 12 of 17 nonsecreting pituitary tumors. Both Ob-Ra and Ob-Rb isoforms are expressed in adult human pituitary, but only Ob-Rb is expressed in the fetal gland (see Section 3.2.2): hence, Shimon et al. (1998) suggested that human pituitary adenomas revert to a fetal type of Ob-R activity. The simultaneous expression of leptin and Ob-R makes it likely that the leptin system may be involved in the autocrine–paracrine regulation of secretion and differentiation of pituitary adenomas, and especially of the ACTH-secreting tumors. However, as pointed out by Garofalo and Surmacz (2006), its possible role in tumorigenesis is doubtful.

6.3. Adrenocortical tumors and pheochromocytomas Ob-Rb, but not leptin, mRNA, and protein expression, has been found in adrenocortical adenomas and carcinomas, but not pheochromocytomas (Glasow and Bornstein, 2000; Glasow et al., 1998, 1999). A 2-h exposure to leptin was reported to lower in vitro basal and ACTH-stimulated secretion from cortisol-secreting adenomas, without altering aldosterone production from aldosteronomas (Szucs et al., 2001). No studies have been carried out on the effects of leptin on catecholamine secretion from pheochromocytomas. High levels of circulating leptin have been measured in patients bearing cortisol-secreting adenomas causing Cushing’s syndrome (LealCerro et al., 1996; Masuzaki et al., 1997), but not in aldosteronoma patients

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(Haluzik et al., 2002; Torpy et al., 1999). Despite the reported inhibitory action of catecholamines on leptin secretion from human adipocytes (Scriba et al., 2000), no apparent changes in the leptin blood concentration were observed in patients bearing pheochromocytomas (Bo¨ttner et al., 1999). The surgical removal of cortisol-secreting adenomas or ACTH-secreting pituitary tumors causing Cushing’s disease, although normalizing the blood levels of ACTH and cortisol, did not alter the concentration of circulating leptin (Licino et al., 1997; Weise et al., 1999). Conversely, aldosteronoma removal caused a significant rise in the levels of circulating leptin (Haluzik et al., 2002; Torpy et al., 1999). Taken together, the findings now reviewed cast doubt on the possibility that leptin, acting as a circulating hormone, may be involved in adrenal tumorigenesis and/or may blunt the excessive hormone production from adrenal tumors.

6.4. Macronodular adrenal hyperplasia Macronodular adrenal hyperplasia (MAH) causes Cushing’s syndrome, which may be ACTH independent due to the presence in the hyperplastic tissue of ‘‘aberrant’’ secretagogue R for AVP, luteinizing hormone, angiotensin-II, and/or 5-hydroxytryptamine. In some instances, ACTHindependent MAH may be food dependent: Patients display marked cortisol surges after meals, which ensue from the presence in the MAH of ‘‘aberrant’’ R for gastric inhibitory polypeptide (GIP) (Antonini et al., 2006; Bertherat et al., 2005; Bourdeau et al., 2004; Lacroix et al., 1992, 2001). Of great interest, rather old studies showed that food-dependent, but not foodindependent, MAH displayed a clearcut in vitro cortisol secretory response to leptin (Pralong et al., 1999), whose molecular basis remains to be ascertained in light of the interrelationships among leptin, insulinotrophic GIP, and diabetes.

6.5. Hyperreninemic hypoaldosteronism Leptin via its sympathoexcitatory action (see Section 5.3.2.) induces a sizeable increase in blood pressure, which may account for the hypertension frequently associated with human obesity (Haynes et al., 1997; Shek et al., 1998). Evidence has been provided that leptin administration caused a moderate increase in plasma renin activity (PRA) and a significant decrease in aldosterone blood concentration in rats (Bornstein and Torpy, 1998; Shek et al., 1998). The rise in PRA could ensue from leptin-induced sympathetic activation and the lowering of plasma aldosterone from the direct inhibitory action of leptin on ZG cells (see Section 5.1). Critically ill patients (e.g., acute sepsis) frequently display hyperleptinemia (Bornstein et al., 1998), and these surveyed findings suggest that this may contribute to

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the hyperreninemic hypoaldosteronism observed in a substantial percentage of these patients (Zipser et al., 1981).

7. Concluding Remarks The preceding sections have shown that leptin plays a relevant role in the regulation of the HPA axis. Leptin and its R are both expressed in the central branch of the HPA axis, where they can modulate CRH and ACTH secretion acting in an autocrine–paracrine manner. In contrast, only leptin R is expressed in the adrenal gland, suggesting that leptin affects the peripheral branch of the HPA axis exclusively, acting as a circulating hormone. The levels of circulating leptin are in the nanomolar range in both lean and obese subjects (Baumgartner et al., 1999; Considine et al., 1996; Mann et al., 2003), which makes this possibility likely. However, despite extensive experimental work, many points remain unsettled, including the following, which are the most relevant. Although the bulk of evidence indicates that leptin exerts an overall inhibitory effect on the HPA axis, there is also proof that this cytokine in rodents may enhance HPA axis activity (see Sections 4 and 5). Hence, leptin could behave as an ‘‘antistress’’ or ‘‘emergency hormone,’’ depending on the species and probably the experimental conditions used. Parenthetically, the stimulating effect of leptin on catecholamine release and sympathetic outflow (see Section 5.3) appears to be in keeping with this latter action of leptin. At least five Ob-R isoforms are expressed in the HPA axis (see Section 3), but nothing is known about their functions. Leptin fragments frequently evoke effects other than those of the native molecule (see Section 5.1.1). Does this depend on their binding capacity to different Ob-R isoforms? This possibility could explain the rather conflicting results obtained in the rat on the effect of leptin on the peripheral branch of the HPA axis, but obviously it would be necessary to admit that in this species a proteasemediated posttranslational processing of leptin occurs, which may give rise to different levels of circulating leptin fragments. The signaling cascade coupled to Ob-R, and especially the long isoform b, has been extensively investigated (see Section 2.2). However, the signaling mechanisms mediating the effects of leptin on the HPA axis have not yet been examined. This is very surprising in view of the huge mass of studies devoted to ascertaining the signaling mechanisms of other regulatory peptides modulating HPA axis function and feeding (e.g., orexins, neuropeptides B and W, and cholecystokinin) (Mazzocchi et al., 2005; Nussdorfer et al., 2005; Spinazzi et al., 2006).

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Although leptin and Ob-R are expressed in pituitary adenomas and Ob-R in adrenal tumors, investigations on the possible involvement of leptin as a growth promoter are lacking. Likewise, the modulating action of leptin on tumor secretion has been poorly studied. For instance, despite findings that indicate that leptin enhances catecholamine release from adrenomedullary cells (see Section 5.3), no investigations are available on the effect of this cytokine on pheochromocytoma secretion. Resolving these and many other basic issues, along with the development of selective agonists and antagonists of Ob-R, will not only increase our knowledge of HPA axis physiology, but also, and more importantly, open novel perspectives for the treatment of diseases coupled with dysregulation of feeding and adrenal gland secretion.

ACKNOWLEDGMENTS We wish to thank Miss Alberta Coi for her secretarial support and invaluable help in the provision of bibliographic items.

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Focal Adhesion Kinase and p53 Signaling in Cancer Cells Vita M. Golubovskaya*,† and William G. Cance*,†,‡

Contents 104 105 105 111 114 122 122 128 129 130 132 132 133 133 134 136 136 136 137 137 138 138 138

1. Introduction 2. Structure and Function of Focal Adhesion Kinase Protein 2.1. FAK structure 2.2. FAK functioning in cells 2.3. FAK-protein binding partners 3. FAK in Tumorigenesis 3.1. Overexpression in tumors 3.2. FAK and stem cells 4. FAK and p53 Association 4.1. Structure and function of the p53 protein 4.2. p53 mutations in cancer cells 4.3. p53 binds the FAK promoter 4.4. Direct FAK and p53 protein binding 4.5. Cytoplasmic–nuclear protein shuttling 4.6. Feedback model of FAK–p53 protein interaction 5. FAK and p53 Targeted Therapy 5.1. Downregulation of FAK 5.2. FAK inhibitors 5.3. Targeting protein–protein interactions 5.4. p53 therapy 6. Summary Acknowledgments References

Abstract The progression of human cancer is characterized by a process of tumor cell motility, invasion, and metastasis to distant sites, requiring the cancer cells to be able to survive the apoptotic pressures of anchorage-independent conditions. * { {

Department of Surgery, University of Florida School of Medicine, University of Florida, Gainesville, Florida 32610 UF Shands Cancer Center, University of Florida, Gainesville, Florida 32610 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610

International Review of Cytology, Volume 263 ISSN 0074-7696, DOI: 10.1016/S0074-7696(07)63003-4

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One of the critical tyrosine kinases linked to these processes of tumor invasion and survival is the focal adhesion kinase (FAK). FAK was first isolated from human tumors, and FAK mRNA was found to be upregulated in invasive and metastatic human breast and colon cancer samples. Recently, the FAK promoter was cloned, and it has been found to contain p53-binding sites. p53 inhibits FAK transcription, and recent data show direct binding of FAK and p53 proteins in vitro and in vivo. The structure of FAK and p53, proteins interacting with FAK, and the role of FAK in tumorigenesis and FAK-p53-related therapy are reviewed. This review focuses on FAK signal transduction pathways, particularly on FAK and p53 signaling, revealing a new paradigm in cell biology, linking signaling from the extracellular matrix to the nucleus. Key words: Focal adhesion kinase, Cancer, p53, Apoptosis, Tumorigenesis. ß 2007 Elsevier Inc.

1. Introduction Focal adhesion kinase (FAK) was discovered about 15 years ago as a tyrosine phosphorylated protein kinase. Since then it has become clear that this protein plays a critical role in intracellular processes of cell adhesion, motility, survival, and cell cycle progression. Cancer is often characterized by defects of these processes. One of the critical tyrosine kinases that are linked to the processes of tumor invasion and survival is the FAK. The FAK gene encodes a nonreceptor tyrosine kinase that localizes at contact points of cells with extracellular matrix and is activated by integrin (cell surface receptor) signaling. The FAK gene was first isolated from chicken embryo fibroblasts transformed by v-src (Schaller et al., 1992). We were the first to isolate the FAK gene from human tumors, and in our initial report, we demonstrated that FAK mRNA was upregulated in invasive and metastatic human breast and colon cancer samples (Weiner et al., 1993). At the same time, matched samples of normal colon and breast tissue from the same patients had almost no detectable FAK expression. This was the first evidence that FAK might be regulated at the level of gene transcription, as well as by other mechanisms. Subsequently, we have demonstrated upregulation of FAK at the protein level in a wide variety of human tumors, including breast cancer, colon cancer, ovarian cancer, thyroid cancer, melanoma, and sarcoma (Cance et al., 2000; Judson et al., 1999; Owens et al., 1995, 1996). Recently, we cloned the regulatory promoter region of the FAK gene and confirmed transcriptional upregulation in cancer cell lines (Golubovskaya et al., 2004). We have found that the FAK promoter contains p53 binding sites and that p53 inhibits FAK transcription. In addition, our recent data show direct binding of FAK and p53 proteins in vitro and in vivo

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(Golubovskaya et al., 2005). Thus, this review will focus on FAK intracellular signaling in cancer, especially on the novel FAK and p53 signaling, linking signaling from the extracellular matrix to the nucleus. We will focus on the role of FAK expression, localization, transport, activity, protein interaction, and survival signaling in the development of cancer. We will discuss the structure, function, binding partners, and localization of FAK. Then we will discuss the novel cross-link of FAK and p53 signaling and its interaction, which opens a new paradigm in cell biology, and will pay attention to novel therapeutic approaches to target this interaction.

2. Structure and Function of Focal Adhesion Kinase Protein 2.1. FAK structure 2.1.1. FAK gene cDNA First, FAK cDNA encoding a 125-kDa protein was isolated from chicken embryo cells (Schaller et al., 1992). Then mouse FAK cDNA, encoding a 119-kDa FAK protein, was identified (Hanks et al., 1992). The human FAK (also known as PTK2a) gene has been mapped to chromosome 8 (Agochiya et al., 1999; Fiedorek and Kay, 1995), and there appears to be a high degree of homology between species. The human complete FAK mRNA sequence (NCBI Accession number: L13616) is a 3791-base long sequence that includes the 50 -untranslated 233-base pair region (Whitney et al., 1993). We were the first group to isolate human FAK cDNA from primary sarcoma tissue and found increased FAK mRNA in tumor samples compared with normal tissue samples (Weiner et al., 1993). Subsequently, Xenopus laevis FAK cDNA (Zhang et al., 1995) and rat FAK cDNA (Burgaya and Girault, 1996) were identified. Recently, Drosophila FAK cDNA (Dfak56) was isolated (Fujimoto et al., 1999). FAK cDNA is closely related to the homologous proline-rich calcium-dependent tyrosine kinase (45% amino acid identity) that is also located in humans at chromosome 8, locus p21.1, named PYK2 (RAFTK [related adhesion focal tyrosine kinase]), CADTK (calcium-dependent tyrosine kinase), CAK (cell adhesion kinase) b, and PTK 2b (protein tyrosine kinase 2b) (Avraham et al., 1995; Lev et al., 1995; Sasaki et al., 1995). Genomic structure Recently, the genomic structure of FAK has been characterized (Corsi et al., 2006). The gene coding sequence contains 34 exons (NCBI Gene ID: 5747), and the genomic sequence spans 230 kb (Corsi et al., 2006). The FAK gene contains four 50 noncoding exons and 34 coding exons, and it was shown to have multiple alternatively spliced forms. Comparison of the mouse and human FAK genes detected conservative

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and nonconservative 50 -untranslated exons, which suggests a complex regulation of FAK expression. Exons (13, 14, 16, and 31) are highly conserved among vertebrate species, suggesting their critical function in gene regulation (Corsi et al., 2006). It is known that alternative splicing often occurs and plays an important role in cancer (Caballero et al., 2001; Venables, 2006). Alternative splicing most often results from a different exon inclusion, but can also occur from intron retention or an alternative choice between two splice sites leading to changes in protein localization, structure, removal of phosphorylation sites, or proteasomal degradation (Venables, 2006). There were several cases of alternatively spliced genes that are involved in invasion and metastasis (Rac 1, b-catenin, Crk) or angiogenesis (VEGFR-2, VEGFR-3 [Flt-4]). Thus, a detailed study of alternatively spliced forms of FAK that are overexpressed in pre- and metastatic cancers will be critical for understanding mechanisms and regulation of FAK expression in carcinogenesis, either by changes in mRNA, by changes in the coding sequence (exon inclusion/exclusion), or by changes in protein levels (stability, etc.). FAK promoter We were the first group to clone the human FAK promoter regulating FAK expression (Golubovskaya et al., 2004). The core promoter contains 600 base pairs and includes many transcription binding sites, such as AP-1, AP-2, SP-1, PU.1, GCF, TCF-1, EGR-1, NF-kB, and p53 (Golubovskaya et al., 2004). We have demonstrated that NF-kB binds to the FAK promoter and upregulates its activity, as it was increased by an NFkB-inducing agent, tumor necrosis factor (TNF)-a, and blocked by an NFkB inhibitor, a superrepressor of NF-kB (Golubovskaya et al., 2004). Interestingly, we found two transcription binding sites for p53 in the FAK promoter and found that p53 can block FAK promoter activity (Golubovskaya et al., 2004). Recently, a mouse promoter has been cloned that was highly homologous to the human promoter and contained the same binding sites (Corsi et al., 2006). In addition, the FAK gene has an internal FRNK promoter or C-terminal, FAK-CD promoter that has been recently cloned by the Parsons group (Hayasaka et al., 2005), regulating expression of autonomously expressed FRNK protein.

2.1.2. FAK protein domains The FAK protein is a 125-kDa tyrosine kinase (p125FAK) with a large amino-N-terminal domain, exhibiting homology with a FERM (protein 4.1, ezrin, radixin, and moesin) domain with an autophosphorylation site (Y-397), a central catalytic domain, and a large carboxy-C-terminal domain that contains a number of potential protein interacting sites, including two proline-rich domains and an FAT domain (Hanks and Polte, 1997; Schaller and Parsons, 1994; Schaller et al., 1994) (Fig. 3.1).

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c-Met VEGFR-3

EGFR

Integrins

PDGFR

FAK

p53 N-terminus (1−415) Bmx/ Etk

FERM K152

Ezrin

p130Cas

Kinase domain (416−676) Pro

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GRAF

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K454 Y397 Y407 Y567 Y577

Y861

Y925

Paxillin

PIAS-1 ATP

JSAP1

Grb-7 RIP Shc

Src

S722 S732 S843 S910

Grb-2 PI-3K Nck-2

FIP200

RhoGEF

Hic-5

Figure 3.1 Focal adhesion kinase (FAK) structure. FAK has the N-terminal, kinase domain and the C-terminal domains.The N-terminal domain has theY-397-Y-autophosphorylation site. The kinase domain has the Y576/577 tyrosines, important for catalytic activity of FAK. The C-terminal part of FAK has Y861 and Y925 tyrosines. Different proteins bind to these domains and are involved in motility and survival signaling. The N-terminal domain (205^422 aa) of FAK is involved in interaction with Src, RIP, p53, PI3K, PIAS-1, Grb-7, EGFR/PDGFR, Ezrin, Bmx, Trio, and others. The kinase domain is involved in binding with FIP200 protein. ASAP, p130Cas, Grb-2, paxillin, talin, RhoGEFp190, and others bind the C-terminal domain of FAK. Interactions of FAK and other proteins demonstrated by group are shown in italics.

N-terminal domain The first function of the N-terminal domain, homologous to the FERM domain, was linked to the binding of integrins, via their b subunits (Schaller et al., 1995). The N-terminal domain of the FAK protein contains the major autophosphorylation site Y397-tyrosine, which in phosphorylated form becomes a binding site of the SH-2 domain of Src, leading to its conformational changes and activation (Hanks and Polte, 1997). Tyrosine phosphorylation of FAK and binding of Src lead to tyrosine phosphorylation of other tyrosine phosphorylation sites of FAK: Y407; Y576, Y577—the major phosphorylation sites in the catalytic domain of FAK; and Y861 and Y925 (Hanks and Polte, 1997; McLean et al., 2005)— to phosphorylation of FAK-binding proteins, such as paxillin and Cas (Schaller et al., 1999). That leads to subsequent cytoskeletal changes and activation of RAS-MAPK (mitogen-activated protein kinase) signaling pathways (Hanks et al., 2003; McLean et al., 2005). Thus, the FAK-Src signaling complex activates many signaling proteins involved in survival and

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Growth factors

Integrins

FAK VEGFR-3

RIP

p53

Paxillin

Talin N-terminus

C-terminus

FERM domain

Kinase domain

Y 397

Cytoplasm

F.A.T

Pro-1 Pro-2

P

Src-family PTKs

p130Cas

PI-3K

Angiogenesis ?

AKT

ERK/MAP Cell migration Lymphogenesis Cell invasion Survival signals

Cell proliferation

FAK

p53

p53 FAK Nucleus

Apoptosis P21, Bax

Figure 3.2 Focal adhesion kinase (FAK) functions in cells. Focal adhesion kinase integrates signals from growth factor receptors (EGFR,VEGFR) and integrins to control motility, survival, metastasis, lymphogenesis, and angiogenesis. Numerous binding partners of FAK mediate this signaling. The FAK and p53 complex is involved in apoptotic/survival signaling.

motility and the metastatic, invasive phenotype in cancer cells (Figs. 3.1 and 3.2). Phosphorylated Y397 FAK is able to recruit important signaling proteins: p85 phosphoinositide 3-kinase (PI3K), growth factor receptor bound protein Grb 7, phospholipase Cg (PLCg), and others. The crystal structure of the N-terminal domain of avian FAK, containing the FERM domain, has recently been reported (Ceccarelli et al., 2006). An interesting negative regulation of FAK function by the FERM domain was revealed by Cooper et al. (2003), where the N-terminal domain had an autoinhibitory effect through interaction with the kinase domain of FAK. Recently, our group discovered several novel binding partners of the N-terminal FAK domain in cancer cells, such as the epidermal growth factor receptor (EGFR) (Golubovskaya et al., 2002; Sieg et al., 2000), receptorinteracting protein (RIP) (Kurenova et al., 2004), and p53 (Golubovskaya et al., 2005) (Fig. 3.1, shown in italics). The N-terminal domain of FAK has been shown to cause apoptosis in breast cancer cells (Beviglia et al., 2003) and can be localized to the nucleus ( Jones and Stewart, 2004; Jones et al., 2001;

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Lobo and Zachary, 2000; Stewart et al., 2002). Thus, the N-terminal domain of FAK binds to the extracellular matrix receptors, integrins, growth factor receptors, or important signaling cytoplasmic, cytoskeletal, and nuclear proteins, mediating signaling from the extracellular matrix to the cytoplasm and nucleus and controlling cytoskeletal changes, survival, motility, and invasion. Kinase domain The central kinase (catalytic) domain of FAK (424–676 amino acids) is the most conserved domain in vertebrate and nonvertebrate organisms (Corsi et al., 2006). The central catalytic domain of FAK contains Y576 and Y577, major phosphorylation sites, and also K454, the ATPbinding site (Fig. 3.1). Phosphorylation of FAK by Src on Y576 and Y577 is an important step in the formation of an active signaling complex and is required for maximum enzymatic activity of FAK (Calalb et al., 1995). The crystal structure of the FAK kinase domain revealed an open conformation similar to the fibroblast growth factor receptor-1 (FGFR-1) and vascular endothelial growth factor receptor (VEGFR) (Nowakowski et al., 2002). The FAK kinase domain structure has an unusual bisulfite bond between the conserved cysteines 456 and C459, suggesting a possible role in protein– protein interactions and kinase function (Nowakowski et al., 2002). The ATP-binding site of protein kinases is the most common target for the small-molecule inhibitors design, although the design of these inhibitors can be complicated by structural similarities between kinase domains. Thus, finding small structural differences is a key in such a design. For example, the side chain of glutamic acid, E506, forms a bifurcated hydrogen bond to the 20 and 30 hydroxyl groups of the ribose (Nowakowski et al., 2002). The corresponding side chains in EphA2 and Aurora-A kinases are smaller and do not contact with sugar (Nowakowski et al., 2002). C-terminal domain Different proteins can bind to the C-terminal domain of p125FAK (677–1052 amino acids), including paxillin, p130cas, PI3K, and GTPase-activating protein Graf, leading to changes in the cytoskeleton and to activation of the Ras-MAPK pathway (Hanks et al., 2003; Parsons, 2003; Schaller and Parsons, 1994; Windham et al., 2002). The carboxy-terminal domain of FAK contains sequences responsible for its targeting to focal adhesions, also known as the FAT domain. Alternative splicing of FAK results in autonomous expression of the C-terminal part of FAK, FAKrelated nonkinase (FRNK) (Richardson and Parsons, 1995). The crystal structure of the C-terminal domain of FAK, the focal adhesion targeting domain (FAT), has been determined recently by several groups (Hayashi et al., 2002; Prutzman et al., 2004) and can exist as a dimer or monomer, allowing binding of several binding partners.

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2.1.3. Posttranslational modifications The main posttranslational modifications of FAK are phosphorylation of tyrosines and serines, and recently sumoylation of FAK has been reported (Kadare et al., 2003). Phosphorylation of tyrosines and serines FAK has numerous tyrosine phosphorylated sites: Y397, Y407, Y576/Y577, Y861, and Y925 (Fig. 3.1). Phosphorylation of Y397, as mentioned previously, created a binding site for Src, PI3K, PLC-g, Grb-7, and Grb-2-SOS. Phosphorylation of tyrosine 407, as well as Y397, correlated with differentiation and with the level of gastrin-releasing peptide and its receptor in colon cancer cells (Matkowskyj et al., 2003). Phosphorylation of Y576 and Y577 correlated with maximal activity of FAK (Calalb et al., 1995). Src-dependent phosphorylation of Y861 was induced by VEGFR in HUVEC endothelial cells (AbuGhazaleh et al., 2001). The FAT domain mediates signaling through Grb2 binding to the Y925 site of FAK (Arold et al., 2002). Inhibition of FAK, which resulted in decreased Y925 phosphorylation, resulted in decreased FAK-Grb2-MAPK signaling and VEGFR-induced tumor growth of 4T1 breast carcinoma cells (Mitra et al., 2006). In addition to tyrosine phosphorylation, several serine phosphorylation sites were reported to play a major role in FAK function, such as serines 722, 732, 843, and 910 (Fig. 3.1). The role of serine phosphorylation has been investigated less than the phosphorylation of tyrosines, but it was suggested that it plays a role in the binding/stability of proteins (Parsons, 2003). Some studies described serine phosphorylation of FAK. For example, phosphorylation of serine 722 was induced by Rho-dependent kinase in endothelial cells in response to VEGF (Le Boeuf et al., 2006). Serine 732 was reported to be phosphorylated by serine-threonine kinase Cdk5, which was important for microtubule organization, nuclear movement, and neuronal migration in cultured neuron cells (Xie et al., 2003). G-protein-coupled receptor activation induced phosphorylation of serine 843 (Fan et al., 2005). Recently, FAK phosphorylation at tyrosine 397 has been shown to be reverse correlated with phosphorylation at serine 843 ( Jacamo et al., 2007). While platelet-derived growth factor (PDGF) induced serine 910 phosphorylation of FAK in Swiss 3T3 cells, that differed in kinetics and in response to MAPK and PI3K inhibitors from Y397 tyrosine phosphorylation, suggesting activation of different signaling pathways (Hunger-Glaser et al., 2004). In addition, bombesin, lysophosphatic acid, and epidermal growth factor activated Ser-910 phosphorylation of FAK in Swiss 3T3 cells via an extracellular signalregulated kinase (ERK)-dependent pathway (Hunger-Glaser et al., 2003). In addition, recently mass spectrometry analysis of chicken FAK revealed 19 new sites of phosphorylation with some sites reported before: 15 serine, 5 threonine, and 5 tyrosine residues (Grigera et al., 2005). It was

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suggested that coordinated phosphorylation of FAK by tyrosine and serine/ threonine-specific kinases may be a critical step in regulation of FAK function (Grigera et al., 2005). Some of the sites were present only in chicken FAK, such as S386, T388, and T393, but several chicken phosphorylation sites were conservative in human, mouse, and frog species, such as S29, Y155, S390, S392, T394, Y397, T406, Y407, Y570, T700, S708, S722, S725, S726, S732, S766, S845 (S843 in human), S894, Y899, and S911 (S910 in human and mouse) (Grigera et al., 2005). Thus, now there are a total of 30 sites of phosphorylation of FAK, including those reported before, requiring detailed analysis of their biological functioning in vivo. Sumoylation Recently, the N-terminal domain of FAK was reported to contain lysine 152, which is sumoylated by a small ubiquitin-like modifier protein, SUMO, in the presence of PIAS1 ligase (Kadare et al., 2003). PIAS1 is a protein that initially was cloned as a protein inhibitor of activated STAT1 (signal transducer and activator of transcription) (Liu et al., 1998), which has been shown to interact with SUMO-1 and caused sumoylation of several proteins, such as p53 (Schmidt and Muller, 2002, 2003), regulating its transcriptional activity. Sumoylation of FAK at lysine 152 caused its nuclear localization and increased its autophosphorylation activity (Kadare et al., 2003). Since sumoylation occurs in the nucleus, and FAK was reported to be localized in the nucleus (Golubovskaya et al., 2005; Jones and Stewart, 2004; Lobo and Zachary, 2000), mechanisms of nucleocytoplasmic shuttling of FAK and its function between membrane, focal adhesions, and nucleus remain to be discovered.

2.2. FAK functioning in cells FAK has numerous functions in cell survival, motility, metastasis, invasion, and angiogenesis (Fig. 3.2). 2.2.1. Motility FAK has also been shown to be important for cell motility (Hanks et al., 2003; Hauck et al., 2001; Schaller, 2001; Schlaepfer and Mitra, 2004). FAKnull embryos exhibit decreased motility in vitro (Ilic et al., 1995). Furthermore, enforced expression of FAK stimulated cell migration (Hildebrand et al., 1993; Sieg et al., 1999). Cell migration is initiated by protrusion at the leading edge of the cell, by the formation of peripheral adhesions, exertion of force on these adhesions, and then the release of the adhesions at the rear of the cell (Tilghman et al., 2005). FAK is involved in the regulation of migration, although the precise mechanism of this FAK-regulated migration is unclear. FAK has been shown to be required for the organization of the leading edge in migrating cells by coordinating integrin signaling in

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order to direct the correct activation of membrane protrusion (Tilghman et al., 2005). The SH2 domain of Src, targeting Src to focal adhesions and Y397 activity, has been shown to be important for motility (Yeo et al., 2006). PI3K has also been shown to be critical for FAK-mediated motility in Chinese hamster ovary (CHO) cells (Reiske et al., 1999). The tumor suppressor gene PTEN, encoding phosphatase, has been shown to interact with FAK, causing its dephosphorylation and blocked motility (Tamura et al., 1998). Moreover, Y397FAK was important for PTEN interaction with FAK (Tamura et al., 1999a). Overexpression of FAK reversed the inhibitory effect of PTEN on cell migration (Tamura et al., 1998). 2.2.2. Invasion and metastasis Activation of FAK is linked to invasion and metastasis signaling pathways. FAK was important in Erb-2/Erb-3-induced oncogenic transformation and invasion (Benlimame et al., 2005). Inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis processes (Benlimame et al., 2005). In addition, FAK has been shown to be activated in invading fibrosarcoma and regulated metastasis (Hanada et al., 2005). Inhibition of FAK with dominant-negative FAK-CD disrupted invasion of cancer cells (Hauck et al., 2001). We have also shown high FAK expression in breast cancers associated with tumor aggressive phenotype (Lark et al., 2005). Subsequently, we analyzed FAK expression in preinvasive ductal carcinoma in situ (DCIS) tumors and detected protein overexpression in preinvasive tumors (Lightfoot et al., 2004), suggesting that the survival function of FAK occurs as an early event in breast tumorigenesis. 2.2.3. Survival FAK plays a major role in survival signaling and has been linked to detachment-induced apoptosis or anoikis (Frisch et al., 1996). It has been shown that constitutively activated forms of FAK rescued epithelial cells from anoikis, suggesting that FAK can regulate this process (Frisch, 1999; Frisch and Ruoslahti, 1997; Frisch and Screaton, 2001; Frisch et al., 1996; Windham et al., 2002). Similarly, both FAK antisense oligonucleotides (Smith et al., 2005; Xu et al., 1996), as well as dominant-negative FAK protein (FAK-CD), caused cell detachment and apoptosis in tumor cells (Beviglia et al., 2003; Gabarra-Niecko et al., 2003; Golubovskaya et al., 2002, 2003; Park et al., 2004; van De Water et al., 2001; Xu et al., 1996, 1998, 2000). The antiapoptotic role of FAK was also demonstrated in FAK-transfected FAK/ HL60 cells that were highly resistant to apoptosis induced with etoposide and hydrogen peroxide compared with the parental HL-60 cells or the vectortransfected cells (Kasahara et al., 2002; Sonoda et al., 2000). HL-60/FAK cells activated the AKT pathway and NF-kB survival pathways with the induction of inhibitor-of-apoptosis proteins (IAPs) (Sonoda et al., 2000). We have

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demonstrated that EGFR and Src signaling cooperate with FAK survival signaling in colon and breast cancer cells (Golubovskaya et al., 2002, 2003; Park et al., 1999, 2004). We have also demonstrated that simultaneous inhibition of Src and FAK or EGFR and FAK pathways was able to increase apoptosis in cancer cells (Golubovskaya et al., 2002, 2003). Thus, cancer cells use the cooperative function of kinases and growth factor receptor signaling to increase survival. 2.2.4. Angiogenesis VEGF is one of the known angiogenic growth factors, stimulating formation of new blood vessels or angiogenesis. FAK has been shown to play a major role in vasculogenesis. It has been shown that VEGF induced tyrosine phosphorylation of FAK in human umbilical vein endothelial cells (HUVEC) and other endothelial cell lines (Abedi and Zachary, 1997). VEGF-induced stimulation of FAK phosphorylation was also demonstrated in cultured rat cardiac myocytes, which was accompanied by subcellular translocation of FAK from perinuclear sites to the focal adhesions and increased association with the adaptor proteins Shc, Grb-2, and c-Src (Takahashi et al., 1999). VEGF-induced phosphorylation of FAK was inhibited by the tyrosine kinase inhibitors tyrphostin and genistein (Takahashi et al., 1999). VEGF-induced phosphorylation of FAK was induced in human brain microvascular endothelial cells (HBMEC) (Avraham et al., 2003). Furthermore, inhibition of FAK with the dominant-negative inhibitor FRNK or the C-terminal FAK (FAK-CD) significantly decreased HBMEC spreading and migration (Avraham et al., 2003, 2004). In addition, the angiogenic inhibitor endostatin blocked VEGF-induced activation of FAK (Kim et al., 2002). Recently, we have shown that FAK binds to the VEGFR-3 (Flt-4) protein in cancer cell lines (Garces et al., 2006), suggesting an important role of FAK in lymphogenesis in addition to angiogenesis. We have shown that the C-terminal domain of FAK binds to VEGFR-3. Disruption of this binding with VEGFR peptides caused apoptosis in breast cancer cells, allowing novel therapeutic approaches in breast tumors (Garces et al., 2006). The detailed interaction of FAK and VEGFR signaling and its mechanisms remain to be discovered. Overexpression of FAK in vascular endothelial cells promoted angiogenesis in transgenic mice (Peng et al., 2004). Overexpession of FAK induced human retinal endothelial cell (HREC) migration and in vivo angiogenesis (Kornberg et al., 2004). FAK activity and phosphorylation of the Y925 site of FAK promoted an angiogenic switch during tumor progression (Mitra et al., 2006). FAK-Grb2-MAPK signaling has been shown to be important for promoting angiogenesis. Furthermore, inhibition of FAK resulted in disruption of angiogenesis (Mitra et al., 2006). FAK and Src catalytic activities are important in promoting VEGF-dependent angiogenesis (Mitra and

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Schlaepfer, 2006). Thus, FAK is involved in angiogenesis and plays a major role in tumorigenesis. The link between FAK and VEGFR-3 signaling opens a new area of angionegensis/lymphogenesis linkage that seems to be critical in tumorigenesis. A correlation analysis between FAK and VEGFR on a population-based series of tumor samples is needed to define this mechanism.

2.3. FAK-protein binding partners FAK has numerous binding partners in the N-terminal, central, and C-terminal domains (Fig. 3.1). The N-terminal domain of FAK contains one proline-rich domain, and the C-terminal domain of FAK contains another two proline-rich domains that are sites of binding proteins, containing SH3 domains. The C-terminal part of the C-terminal domain of FAK (853–1012 aa) is called FAT, a domain that is necessary for the targeting of FAK to focal adhesion complexes through binding with different proteins (paxillin, talin, Rho, etc.). We will focus on more than 20 known protein-binding partners, including four recent novel ones, found recently by our group (EGFR, RIP, p53, and VEGFR), classified by the main binding domain. 2.3.1. Proteins binding to the N-terminal domain of FAK Src Src is a well-known binding partner of FAK (Schaller et al., 1994). C-Src is a member of related tyrosine kinases, including Fyn, Yes, Lck, Blk, Hck, Lyn, Fgr, and Yrk. Src proteins are located in the cytoplasm at cellular sites of integrin clustering. Following integrin binding, p125FAK becomes tyrosine phosphorylated at the Y-397 site (Hanks and Polte, 1997). This leads to efficient binding of Src via its SH2 domain to FAK, generating an FAK–Src complex (Calalb et al., 1995; Schaller and Parsons, 1994; Xing et al., 1994). The SH3 domain of Src can also bind the proline-rich domain of the N-terminal FAK, regulating downstream cell signaling (Thomas et al., 1998). Src can be autophosphorylated at tyrosine Y-416, while tyrosine Y-527 is critical for a negative regulation of Src activity ( Jones et al., 2000). Recently, Src has been shown to regulate anoikis in human colon tumors (Windham et al., 2002). Similarly, we have shown that increased Src activity led to additional survival signals in suppressing apoptosis in colon cancer cell lines, overexpressing FAK (Golubovskaya et al., 2003). The same result was obtained in a breast cancer model with stably expressing activated Src (Park et al., 2004). Shc The adapter protein Shc was identified as an SH2-containing protooncogene, involved in growth factor signaling (Ravichandran, 2001). Three Shc proteins are known: Shc A, Shc B, and Shc C. The Shc A protein has three isoforms that are expressed from the same messenger RNA in cells: 46-, 52-, and 66-kDa proteins (Ravichandran, 2001). The 66-kDa form of

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Shc A is expressed in all cells, while Shc B and C are mostly expressed in neuronal cells (Ravichandran, 2001). Shc contains two domains: one binds phosphotyrosine (SH2 and PTB), and another, central domain, contains phosphorylation sites. Importantly, knockout of Shc A caused embryonic lethality in mice, suggesting its important role in vivo (Ravichandran, 2001). Activation of Shc leads to mitogen-activated protein kinase (MAPK) activation, which has been well described (Ravichandran, 2001). Activation of FAK autophosphorylation at Y397 leads to binding with the Shc adaptor, its phosphorylation, and activation of downstream MAPK pathway signaling (Hanks and Polte, 1997). Also, Shc has been reported to be hyperphosphorylated in many types of tumors (Bonsi et al., 2005; Finlayson et al., 2003). JSAP1 The N-terminal domain of FAK binds to the c-Jun N-terminal kinase ( JNK)/stress-activated protein kinase-associated protein 1 ( JSAP1) (also known as JIP3, JNK-interacting protein 3), which was originally identified as a scaffolding protein for the JNK cascade by binding to JNK, SEK1, and MEKK1 proteins (Takino et al., 2002). The complex between JSAP and FAK was stimulated by c-Src, leading to JSAP tyrosine phosphorylation (Takino et al., 2002). JSAP mediated the association of FAK and JNK and is involved in cell migration (Takino et al., 2005). JSAP was suggested to play an important role in brain tumorigenesis (Takino et al., 2005). Cell spreading and adhesion in mouse embryonic fibroblasts with knockout of JSAP1 were slower than in the wild-type cells, suggesting a role of JSAP-1 in adhesion and cell spreading (Chae et al., 2006). BMX/Etk Etk/BMX is a member of the Btk family of tyrosine kinases, which is highly expressed in endothelial cells and metastatic carcinoma cells (Chen et al., 2001). The ETK interacts with the N-terminal domain of FAK, playing a critical role in motility (Chen et al., 2001). Importantly, physically Etk also associates with p53 through its Src homology 3 domain and the proline-rich domain of p53 ( Jiang et al., 2004). Thus, Etk interacts with p53, which has been shown to be a binding partner of FAK (see the following) in the cytoplasm, leading to bidirectional inhibition of the activities of both proteins ( Jiang et al., 2004). Ezrin Ezrin belongs to a ERM (ezrin/radixin/moesin) family of proteins, present in actin-rich cell structures, such as membrane ruffles, filopodia, and microvilli, linking the plasma membrane and the actin-based cytoskeleton (Poullet et al., 2001). ERM proteins belong to members of the 4.1 superfamily with a conserved N-terminal domain (FERM domain). ERM proteins are targeted to the plasma membrane by interacting with phosphoinositides and proteins, such as CD44, intercellular adhesion molecules (ICAMs), or EBP-50 (Poullet et al., 2001). The C-terminal end of

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the ERM proteins interacts with F-actin, and its N-terminal domain interacts with the N-terminal FERM domain of FAK (Poullet et al., 2001). Overexpression of ezrin increased phosphorylation of FAK Tyr-397, creating a docking site for FAK signaling partners (Poullet et al., 2001). c-Met Recently, it has been shown that the N-terminal domain of FAK interacts with the c-Met oncogene (Chen and Chen, 2006). The c-Met oncogene encodes a c-MET receptor protein that is activated by hepatocyte growth factor (HGF), which is a mesenchymally derived factor, transducing multiple cellular responses, including proliferation, motility, survival, and morphogenesis in various types of cells. Upon HGF/SF binding, c-Met autophosphorylation occurs on two tyrosine residues (Tyr-1234 and Tyr1235) within the activation loop of the tyrosine kinase domain, regulating kinase activity (Peruzzi and Bottaro, 2006). Phosphorylation of two tyrosine residues near the COOH terminus (Tyr-1349 and -1356) forms a multifunctional docking site for intracellular adapters (Grb2, Gab1, PI3K, phospholipase C-g, Shc, Src, Shp2, and Shp1) via Src homology-2 domains and other binding motifs, leading to downstream signaling (Peruzzi and Bottaro, 2006). Mainly tyrosines Tyr-1349 and, less, Tyr-1356 of c-Met are required for its interaction with the FERM domain of FAK. A patch of basic residues (216KAKTLRK222) in the FERM domain of FAK is critical for this interaction (Chen and Chen, 2006). Furthermore, the Met-FAK interaction activates FAK and plays a critical role in hepatocyte growth factor-induced cell motility and cell invasion (Chen and Chen, 2006). It has been suggested that the Met–FAK interaction plays an important role in tumorigenesis and serves as a target for therapeutic purposes (Chen and Chen, 2006). PI3 kinase FAK has been shown to directly interact with the p85 subunit of PI3K (Chen and Guan, 1994). PI3K is a known cytoplasmic kinase that is involved in different cellular functions, such as survival, cytoskeletal changes, and carcinogenesis (Bianco et al., 2006). The autophosphorylation of Y397FAK increased its interaction with PI3K (Chen and Guan, 1994). It was suggested that PI3K can be a substrate for FAK (Chen and Guan, 1994). The Y397 site of FAK is the site of binding with PI3K (Guan, 1997). The association of PI3K with FAK was mediated primarily through the association with the SH2 domain of p85, while the SH3 domain of PI3K was also able to bind FAK (Bachelot et al., 1996). FAK has recently been shown to activate the PI3K pathway to suppress doxorubicin-induced apoptosis (van Nimwegen et al., 2006). PIAS-1 Recently, the N-terminal domain of FAK has been shown to interact with FAK and to cause sumoylation in the presence of SUMO-1 (Kadare et al., 2003). The closely related protein Pyk-2 (CADTK/RAFTK)

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was not sumoylated by these proteins. This posttranslational modification of FAK increased its autophosphorylation activity. Interestingly, this sumoylation occurred in the nucleus, providing a basis for a novel functioning of the protein in the nucleus and for nucleocytoplasmic shuttling (Kadare et al., 2003). PIAS-1 also promoted sumoylation of p53 and inhibited its transcriptional activity (Schmidt and Muller, 2002). Trio Recently, a novel binding partner with FAK has been identified and named Trio, interacting with the N-terminal domain of FAK (Medley et al., 2003). The Trio guanine nucleotide exchange factor is essential for embryonic development and fetal skeletal muscle formation and organization of neural tissue (Medley et al., 2003). Trio is a large multidomain protein containing two functional DH GEF domains, which activate Rho-type GTPases, a protein serine/threonine kinase domain, two SH3-like domains, an Ig-like domain, and multiple spectrin-like repeats (Debant et al., 1996). Trio protein functions to activate Rho GTPases, such as RhoA, Rac1, and Cdc42, by promoting the exchange of GDP for GTP. Once activated, Rho GTPases function in several cellular processes, such as actin cytoskeleton organization, MAPK cascade signaling, and gene transcription (Medley et al., 2003). FAK phosphorylates 2737 tyrosine in the kinase domain of Trio. Trio can activate autophosphorylation and the kinase function of FAK. Thus, this interaction is a novel bidirectional signaling complex, playing an important role in cell motility and regulation of focal adhesion and cytoskeleton changes (Medley et al., 2003). Grb-7 Grb-7 is an Src homology (SH) 2-containing and pleckstrin homology domain-containing adaptor protein, which associates with the N-terminal domain of FAK through Y-397 tyrosine (Han and Guan, 1999). The Grb7 family members have similar structures, containing an amino terminal proline-rich region; a central part called the GM region, which includes a pleckstrin homology (PH) domain; and a carboxy-terminal SH2 domain. Like other adaptor molecules, Grb7 family members have been shown to interact with the different cell surface receptors and other signaling proteins (Shen et al., 2002).The FAK–Grb7 complex plays a critical role in cell migration stimulated by integrin signaling through FAK (Shen et al., 2002). Nck-2 A recently identified adaptor protein Nck-2, also known as Nckß or Grb4, contains three N-terminal SH3 domains and one C-terminal SH2 domain. The Nck-2 SH2 domain has been shown to bind the N-terminal FAK at Y397 and to decrease cell motility (Goicoechea et al., 2002).

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EGFR/PDGFR p125FAK interacts with other tyrosine kinases to affect survival functions. In breast cancer cell lines that overexpressed epidermal growth factor receptor (EGFR), we have found an association between EGFR and FAK, and demonstrated that FAK suppressed apoptosis by activating AKT/ERK1/2 pathways (Golubovskaya et al., 2002). Dual inhibition of FAK and EGFR induced death-receptor-mediated apoptosis, involving activation of caspases –8 and –3 and cleavage of poly(ADP-ribose) polymerase (PARP) (Golubovskaya et al., 2002). The N-terminal domain of FAK interacts with EGFR (180 kDa) and PDGFR protein complexes, linking growth factor receptors and integrin signaling pathways (Sieg et al., 2000). RIP To study death-induced apoptosis, we performed an analysis of death receptor proteins that bind FAK and found a physical association between FAK and a 66-kDa apoptosis-related death domain RIP (Kurenova et al., 2004). We have shown that RIP provides proapoptotic signals that are suppressed by its binding to FAK (Kurenova et al., 2004). The RIP protein has dual survival and apoptotic functions: it can recruit CRADD (apoptosis) and NF-kB (survival/proliferation) (Thakar et al., 2006). p53 The first indirect link between FAK and p53 was provided by Ilic et al. (1998). It was shown that extracellular matrix survival signals mediated by FAK suppressed p53-directed apoptosis (Ilic et al., 1998). We have shown direct binding of FAK and p53 in different cancer cells (Golubovskaya et al., 2005). The N-terminal domain of p53 (1–92 aa) interacts with the 205–422 aa of the N-terminal domain of FAK (Golubovskaya et al., 2005). We have shown previously that p53 can bind the FAK promoter and inhibit its luciferase activity (Golubovskaya et al., 2005). Moreover, FAK can block p53 transcriptional activity of p21, BAX, and Mdm-2. Thus, there is a feedback loop mechanism of regulation for these two proteins.

2.3.2. Proteins binding the kinase domain of FAK: FIP200 FIP200 (focal adhesion interaction) protein is a novel 200-kDa binding partner of FAK. FIP200 binds to the kinase domain of FAK and inhibits its kinase activity (Abbi and Guan, 2002; Gan et al., 2005). This protein plays an important role in various cellular functions, such as cell adhesion, motility, and survival. FIP has been shown to inhibit the G1–S phase transition of the cell cycle by increasing p21 and decreasing cyclin D1 protein expression (Melkoumian et al., 2005). FIP200 has been shown to interact with p53, stabilizing the protein half-life (Melkoumian et al., 2005). Targeted deletion of FIP200 in the mouse led to embryonic death associated

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with heart failure and liver degeneration (Gan et al., 2006). FIP200 knockout embryos had increased apoptosis and reduced phosphorylation of c-Jun N-terminal kinase in response to TNF-a treatment (Gan et al., 2006). A competition model of association between FIP200-p53 and FIP200-FAK has recently been proposed by Mitra and Schlaepfer (2006). 2.3.3. Proteins binding with the C-terminal domain of FAK Paxillin Paxillin is a 68-kDa cytoplasmic multidomain protein that localizes to focal adhesions and is known to interact with FAK, PYK2, Src, vinculin, and Crk (Turner, 1998). It is a phosphorylation substrate of FAK that binds the C-terminal domain of FAK, FAT (Schlaepfer et al., 2004; Turner, 2000). Paxillin is localized to focal adhesions through its LIM domains. Its primary function is as a scaffold or adapter protein that provides multiple binding sites at the plasma membrane for a variety of signaling and structural proteins, causing changes in the organization of the actin cytoskeleton that are necessary for cell motility and tumor metastasis (Schaller, 2004; Turner, 2000). Paxillin contains the N-terminal leucine-rich LD motifs, binding directly to FAK, and three LIM (Lin-11, Isl-1, and Mec-3) domains in the C-terminus (Wade and Vande Pol, 2006). It was demonstrated that paxillin LIM domains 1, 2, and 3, but not the LIM 4 domain, were required for FAK tyrosine phosphorylation (Wade and Vande Pol, 2006). Cas Cas (Crc-associated substrate) is a 130-kDa protein that binds to and is phosphorylated by FAK (Hanks and Polte, 1997). Phosphorylation of FAK caused Src- and FAK-dependent phosphorylation of the Cas protein, leading to the recruitment of a Crk adaptor protein and activation of a small GTPase and JNK, promoting membrane protrusion and cell migration (Cox et al., 2006). The Cas proteins have numerous protein–protein interaction domains, including: (1) an SH3 domain, mediating interaction with proline-rich PxxxP proteins; (2) the substrate domain with tyrosine phosphorylation sites, mediating interaction with SH2 domain proteins, a proline-rich motif; (3) the serine-rich region; and (4) a C-terminal dimerization domain, mediating homo- and heterodimerization (O’Neill et al., 2000). The FAK-binding domain is the N-terminal SH3 domain of Cas that associates with the proline-rich domain of the C-terminal domain of FAK. Cas proteins serve as docking molecules for numerous interacting proteins (O’Neill et al., 2000). The extracellular matrix and growth factor receptor signals are transduced through the interaction of FAK, Src, and Cas proteins, stimulating cells to migration and actin rearrangements. In transformed cells, Cas proteins can be constitutively tyrosine phosphorylated and might play a role in the development of cancer (O’Neill et al., 2000).

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Talin The cytoskeletal protein talin plays a key role in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton (Critchley, 2005). Talin contains the N-terminal 47-kDa globular head domain and a 190-kDa C-terminal domain. Talin binds to integrins via the 4.1 band of the FERM domain (Ratnikov et al., 2005). Talin binds to the FAT domain in the C-terminal domain of FAK (Schlaepfer et al., 2004), linking FAK indirectly to integrins (Chen et al., 1995) (Fig. 3.1). It has been shown that phosphorylation of type Ig phosphatidylinositol phosphate kinase (PIPKIg661) on tyrosine 644 (Y644) is critical for the interaction of PIPKIg with talin and its localization to focal adhesions (Ling et al., 2003). PIPKIg661 is specifically phosphorylated on Y644 by Src, which is regulated by FAK (Ling et al., 2003). Grb-2 Grb-2 is an adaptor protein (24 kDa) that binds to the Y925 site of the C-terminal domain of FAK through the Grb-2 SH-2 domain (Schlaepfer and Hunter, 1996; Schlaepfer et al., 1994). FN-stimulated Grb2 binding to FAK can activate ERK2-MAPK intracellular signaling (Schlaepfer et al., 1997). It has been shown that the Src-dependent association of FAK with both Grb2 and p130(Cas) is required for the regulation of cell cycle progression by FAK (Reiske et al., 2000). RhoGEF RhoGEF (guanine nucleotide exchange factor; 190 kDa) is a RhoA-specific guanosine diphosphate/guanosine triphosphate (GDP/GTP) exchange factor protein that directly associates with the C-terminal domain of FAK, FAT (Zhai et al., 2003). Rho GTPases are members of the Ras GTPase superfamily that play an important role in a number of biological processes, such as actin cytoskeleton reorganization, cell motility, gene expression, and cell cycle progression. RhoGTPases are inactive when bound to GDP and active once bound to GTP (Zhai et al., 2003). Several regulatory proteins bind to RhoGTPases, such as RhoGEF, to promote conformation from the inactive to the active form. Binding of RhoGEF to FAK supports the role of FAK in the activation of the Rho pathway and plays an important role in different cellular functions. GRAF Another Rho pathway protein GAP, GTPase-activating protein termed GRAF (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42 (Hildebrand et al., 1996). GRAF colocalizes with actin stress fibers, cortical actin structures, and focal adhesions in Graf-transfected chicken embryo cells (Hildebrand et al., 1996). GRAF is expressed in embryonic brain and liver tissues (Hildebrand et al., 1996) and may function in mediating cross talk between FAK and the Rho family GTPase, controlling integrin-initiated signaling events. GRAF has been shown to be GAP for Rho in vivo in Swiss

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3T3 cells, and microinjection of GRAF cDNA changed actin cytoskeletal localization and cell shape (Taylor et al., 1999). The human GRAF gene is 90% homologous to the chicken GRAF gene, is located on the 5q31 chrosomosome, and functions as a GAP for RhoA in vivo (Borkhardt et al., 2000). This gene might be associated with hematological malignancies containing deletions of 5q (Borkhardt et al., 2000). ASAP1 ASAP1 (ADP ribosylation factor [ARF]-GTPase-activating protein [GAP] containing SH3, ANK repeats, and the PH domain) has been shown to bind Src and has been phosphorylated by Src (Brown et al., 1998). The second proline-rich domain of the C-terminal domain of FAK has been shown to bind the SH3 domain of ASAP-1 (Liu et al., 2002). Overexpression of wildtype ASAP1 prevented the organization of paxillin and FAK in focal adhesions during cell spreading, while it did not change vinculin localization and organization (Liu et al., 2002). Hic-5 Hic-5 (hydrogen peroxide-inducible clone), a senescence-related protein, binds the C-terminal domain of FAK (Fujita et al., 1998). hic-5 was initially identified as a gene induced by transforming growth factor b1 or by H2O2 (Fujita et al., 1998). The exogenous overexpression of Hic-5 induced growth arrest, senescence-like morphology, and the increased expression of p21/WAF1/Cip1/sdi1 and extracellular matrix-related proteins such as fibronectin (FN), collagen, and collagenase (Fujita et al., 1998). Structurally, Hic-5 contains four LIM domains with 62% homology to paxillin in the C-terminal half and LD domains in the N-terminal half. Hic-5 is localized to focal adhesions and is involved in integrin-mediated signal transduction. Hic-5 expression has been shown to be decreased in immortalized mouse fibroblasts (Ishino et al., 2000). Overexpression of Hic-5 decreased the colony-forming ability of MEF from FAK(þ/þ) mice but not of FAK (–/–) cells. These observations suggested the involvement of Hic-5 in the regulation of cellular proliferation (Ishino et al., 2000). VEGFR-3 We have performed a phage display assay to map protein– protein interactions with the C-terminal domain of FAK and demonstrated that VEGFR-3 binds to the C-terminal and FAT domains of FAK (Garces et al., 2006). We synthesized TAT-conjugated peptide, containing 12 amino acid binding sites of VEGFR-3, introduced it into the cancer cells, and caused cell detachment and displacement of FAK from focal adhesions (Garces et al., 2006). Thus, this approach can be used for specific targeting of protein–protein interactions and for future cancer therapy. A detailed study of FAK–VEGFR-3 association and downstream signaling is required for development of in vivo tumorigenesis models.

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3. FAK in Tumorigenesis 3.1. Overexpression in tumors Our laboratory was the first to isolate FAK from a primary human tissue and to link FAK to human tumorigenesis (Cance et al., 2000; Owens et al., 1995, 2001). FAK is elevated in a variety of human tumors, including colorectal cancer (Han et al., 1997), breast (Cance et al., 2000; Owens et al., 1995), sarcomas (Weiner et al., 1993), cervical carcinomas (McCormack et al., 1997), and prostatic carcinoma tumors and cancer cell lines (Tremblay et al., 1996). We identified and cloned tyrosine kinase fragments from primary tumors (Weiner et al., 1993, 1994) and identified FAK from a primary high-grade human sarcoma (Weiner et al., 1993). We analyzed FAK mRNA expression in normal, invasive, and metastatic human tissues. Northern blot analysis demonstrated low levels of FAK mRNA in normal tissues, while primary and metastatic tumors significantly overexpressed FAK (Fig. 3.3) (Weiner et al., 1993). Subsequently, we cloned the human FAK cDNA and generated anti-FAK antibodies that were used to demonstrate that FAK is overexpressed in different types of tumors (Owen et al., 1999; Owens et al., 1995, 1996; Weiner et al., 1993). In a recent study, we performed real-time PCR analysis on colorectal carcinoma and liver metastasis samples with matching normal tissues and demonstrated increased FAK mRNA levels in tumor and metastatic tissues versus normal tissues (Lark et al., 2003). To confirm this hypothesis, we first cloned the regulatory promoter region of FAK with potential transcription binding sites, identified the start of transcription of the FAK gene, and found that FAK mRNA was higher in cancer cells compared to normal fibroblast and monocyte cell lines (Golubovskaya et al., 2004). Although FAK gene amplification was demonstrated in tumors (Agochiya et al., 1999; Okamoto et al., 2003), it is not the only mechanism of FAK increased expression. Moreover, recently we analyzed breast and liver metastatic tumors with increased FAK mRNA from liver metastatic samples with increased FAK mRNA (Lark et al., 2003) by FISH and did not find gene amplification in these samples. Moreover, analysis of FAK expression in head and neck squamous cell carcinomaderived cells demonstrated that FAK protein expression correlated with mRNA levels, but not with FAK copy DNA levels (Canel et al., 2006). It was suggested the increase in FAK protein levels in head and neck tumors can be due to its upregulated transcription (Canel et al., 2006). Thus, defining the mechanisms of FAK upregulation in different types of tumors will be important in understanding the role of FAK in tumorigenesis. Next, we will describe FAK expression and signaling in different types of tumors.

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Breast cancer Normal breast tissue

B

Matched breast cancer tissue

Colon cancer Normal colon tissue with adjacent dysplasia

Invasive colon adenocarcinoma

Figure 3.3 Overexpression of focal adhesion kinase (FAK) in different types of tumors. Immunohistochemical staining of FAK in tumor samples. Breast cancer (A), colon cancer (B). FAK is overexpressed in these tumors.The arrow in colon normal tissues indicates it is adjacent to normal tissue dysplasia with overexpressed FAK.

3.1.1. Ovarian cancer We have shown that FAK expression was increased in ovarian cancers (26 cancer samples from patients with Stage I–IV ovarian carcinoma and two ovarian carcinoma cell lines) compared to normal samples ( Judson et al., 1999). It was suggested that FAK may be a potential target for therapeutic disruption of ovarian carcinoma progression ( Judson et al., 1999). Recently, FAK overexpression in ovarian cancer and its absence in normal ovarian epithelial cells were also demonstrated by several other groups (Gabriel et al., 2004; Grisaru-Granovsky et al., 2005; Sood et al., 2004). 3.1.2. Colon cancer We have shown that FAK was overexpressed in invasive and metastatic colon tumors on both protein and mRNA levels (Fig. 3.3) (Lark et al., 2003; Weiner et al., 1993). FAK protein overexpression was seen in 32 out of 80 cases of colon adenocarcinoma (Theocharis et al., 2003). An increase of tyrosine phosphorylation of FAK (Y-397) was detected in colorectal cancer

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cells (Yu et al., 2004). Phospho-FAK correlated with invasiveness and lymph node metastasis in colon cancers (Yu et al., 2006). We have demonstrated that simultaneous inhibition of FAK and Src pathways caused increased apoptosis in colon cancer cell lines, suggesting that multiple signaling is involved in tumorigenesis (Golubovskaya et al., 2003). 3.1.3. Prostate cancer Increased FAK protein and mRNA levels and phosphorylation were observed in metastatic prostate cancer cells that correlated with the progression and metastasis of tumors (Tremblay et al., 1996). It has been suggested that FAK signaling plays a critical role in the invasiveness and motility of prostate cancers (Zeng et al., 2006; Zheng et al., 1999). 3.1.4. Breast cancer We found that p125FAK was significantly elevated in 17 (100%) of 17 invasive and metastatic colonic lesions and in 22 (88%) of 25 invasive and metastatic breast tumors, suggesting that FAK can be a marker of invasive tumors (Owens et al., 1995). We have shown that dual inhibition of FAK and EGFR (which are both overexpressed in breast tumors) signaling pathways cooperatively enhanced apoptosis in breast cancers (Golubovskaya et al., 2002). We have also shown that overexpression of an activated form of the Src tyrosine kinase in breast cancer cells (BT474 and MCF-7) suppressed the loss of adhesion and apoptosis induced by dominant-negative adenoviral FAK-CD, indicating cooperative FAK and Src signaling in breast tumorigenesis (Madan et al., 2006; Mitra and Schlaepfer, 2006; Park et al., 2004). Overexpressed HER2 has been shown to be involved in the tumor malignancy and metastatic ability of breast cancer through FAK and Src signaling pathways (Schmitz et al., 2005). We analyzed FAK expression in DCIS breast tumors and demonstrated upregulation of FAK expression in DCIS is an early event in breast tumorigenesis (Lightfoot et al., 2004). Subsequently, we analyzed FAK expression in 629 formalin-fixed, paraffin-embedded tissue sections (Lark et al., 2005). High FAK expression was associated with poor prognostic indicators, including high mitotic index, nuclear grade 3, architectural grade 3, estrogen and progesterone receptor negative, and overexpression of HER-2/neu (Lark et al., 2005). The association of FAK and Her-2 in breast tumors indicated its important cooperative signaling to promote breast tumorigenesis (Lark et al., 2005). 3.1.5. Melanoma cancer Increased FAK constitutive phosphorylation was observed in human metastatic melanoma cells (Scott and Liang, 1995). Focal adhesion increased expression correlated with motility of human melanoma cells (Akasaka et al., 1995). FAK expression appeared to be important for tumor cell adhesion in melanoma (Maung et al., 1999). Constitutive activation of FAK was

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regulated by the cytoskeleton in melanoma cells important for aggressive tumor growth (Kahana et al., 2002). We treated melanoma cell lines with antisense FAK oligonucleotides, inhibiting FAK expression, and demonstrated increased cell apoptosis and sensitivity to the chemotherapy drug 5-fluorouracil (Smith et al., 2005). Treatment of melanoma cells with dominant-negative FAK-related FRNK increased the aggressive phenotype of the cells, demonstrated by decreased invasion and motility, and inhibited the Erk1/2-mediated signaling pathway (Hess and Hendrix, 2006). 3.1.6. Thyroid cancer We analyzed p125FAK expression in 30 human thyroid tissue samples that included paired normal and malignant specimens (Owens et al., 1996). The highest levels of p125FAK were seen in follicular carcinomas and tumors associated with distant metastatic foci (Owens et al., 1996). FAK was not expressed in normal tissue and nodular hyperplasia but was expressed in all of the follicular, papillary, medullary, and anaplastic thyroid carcinomas (Kim et al., 2004). This result indicates that the upregulation of FAK may play a role in the development of thyroid carcinogenesis. 3.1.7. Oral cancer FAK was found to be overexpressed in oral cancers (Kornberg, 1998). Overexpression of FAK in low-invading cells resulted in a 4.5-fold increase in the rate of invasion compared with control cell lines, suggesting that enhanced expression of FAK in oral carcinoma cells may lead to a selective growth advantage and increased invasive potential of the primary oral tumor (Schneider et al., 2002). In another study, it was suggested that activation of FAK by phosphorylation of c-Met could mediate the HGF/SF-induced motility of human oral squamous cell carcinoma cells and that Rho protein could regulate the tyrosine phosphorylation of FAK through translocation from the nucleus to the membrane (Kitajo et al., 2003). Increased FAK phosphorylation and expression were observed in oral carcinoma of the larynx (Aronsohn et al., 2003). 3.1.8. Head and neck cancer In a recent study, hepatocyte growth factor/scatter factor HFG/SF induced phosphorylation of FAK and Erk in cultured squamous cell carcinoma of the head and neck, indicating its role in tumorigenesis (Fleigel et al., 2002). Inhibition of FAK with a dominant-negative C-terminal FAK, Ad-FRNK, caused increased apoptosis and decreased cell motility in epithelial cells from head and neck carcinomas (SCCHN cells) (Kornberg, 2005). Analysis of FAK expression by immunohistochemistry in 211 head and neck squamous cell carcinoma (HNSCC) tissue samples, including 147 primary tumors, 56 lymph node metastases, 3 benign hyperplasias, and 5 dysplasias, demonstrated elevated FAK expression in 62% of tumor samples (Canel et al., 2006).

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Positive immunostaining was also detected in benign hyperplasias and preinvasive dysplastic lesions, indicating that FAK overexpression is an early event during tumorigenesis. FAK DNA copy number ratios were higher in 39% of the tumors compared with normal matched samples. However, not all cases of FAK overexpression contained gene amplification, indicating additional mechanisms of FAK expression regulation (Canel et al., 2006). Inhibiting of FAK by adenoviral Ad-FRNK and introduction of exogenous p53 with Ad-p53 cause increased apoptosis of the carcinoma cells and an improved cytotoxic effect of chemotherapy drugs (Kornberg, 2006). Photodynamic therapy, effective in the treatment of head and neck invasive cancers, decreased cell motility and FAK phosphorylation and downstream survival signaling (Yang et al., 2006). FAK overexpression correlated with the invasiveness and lymph node metastasis of 91 thoratic esophageal squamous cell carcinoma tumor samples (Miyazaki et al., 2003). Patient survival with FAK-overexpressing tumors was significantly lower than survival without FAK-overexpression tumor ( p ¼ 0.006). The 5-year survival rate of patients without FAK overexpression was 69%, whereas that of patients with FAK overexpression was 38% (Miyazaki et al., 2003). This suggests that FAK can be a prognostic factor in these tumors. 3.1.9. Hepatocellular carcinoma Analysis of FAK expression in 60 cases of hepatocellular carcinoma demonstrated overexpression of FAK mRNA and protein in tumor samples compared to matched nontumor liver samples (Fujii et al., 2004). The fact that FAK overexpression correlated significantly with tumor size ( p ¼ 0.034) and serum AFP level ( p ¼ 0.030) led to the suggestion that FAK can be a prognostic therapeutic factor in these tumors (Fujii et al., 2004). Downregulation of FAK with the dominant-negative FRNK caused metastatic adhesion of carcinoma cells within liver sinusoids (von Sengbusch et al., 2005). The hepatitis B virus (HBV) involved in hepatic cell transformation activated FAK, suggesting the importance of FAK signaling in HBV-associated hepatocellular carcinogenesis (Bouchard et al., 2006). 3.1.10. Brain tumors Anaplastic astrocytoma brain tumors expressed higher levels of FAK and autophosphorylation compared to nonneoplastic adult normal brain tissues (Hecker et al., 2002). Overexpression of FAK promoted Ras activity through the formation of the FAK/p120RasGAP complex in malignant astrocytoma cells cultured in aggregate suspension or as monolayers adherent to vitronectin (Hecker et al., 2004). Overexpression of FAK in serumstarved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in enhancement of basal migration and in a more significant increase of PDGF-BB-stimulated migration. Both expression of mutant FAK(397F) and FAK downregulation with small interfering siRNA inhibited basal and

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PDGF-stimulated migration (Natarajan et al., 2006). Recently, a novel kinase inhibitor of FAK (TAE226) has been shown to increase apoptosis in brain tumors (Shi et al., 2007). 3.1.11. Lung cancer In lung surgical specimens, phosphorylated FAK has been shown to be the major component among 100- to 130-kDa phosphorylated proteins correlating with poor patient prognosis (Nishimura et al., 1996). In addition, increased phosphorylation of FAK has been demonstrated in lung cancer samples and its absence in normal tissues (Imaizumi et al., 1997). The increased phosphorylation of FAK was closely correlated with the nodal involvement of cancer and disease-free survival time (Imaizumi et al., 1997). Expression of laminin 5, regulating cell adhesion, and anoikis and increased phosphorylation of FAK in lung adenocarcinoma cells suggested the importance of the laminin–integrin–FAK pathway in tumorigenesis (Kodama et al., 2005). Stimulation of small-cell lung cancer cells with hepatocyte growth factor (HGF) activated c-Met and increased phosphorylation of Y397 FAK, suggesting that this pathway is a therapeutic target in lung tumorigenesis (Maulik et al., 2002). FAK signaling has been demonstrated to be important in the early stages of mammary adenocarcinoma lung metastasis (van Nimwegen et al., 2005). In an experimental model of mammary metastasis, formation of lung metastasis was prevented when the dominant-negative FAK inhibitor, FRNK, was expressed 1 day before tumor cell injection, whereas at 11 days after injection expression of FRNK had no effect (van Nimwegen et al., 2005). Immunohistochemical staining of FAK in 60 formalin-fixed and paraffin-embedded non-small-cell lung cancer (NSCLC) tumors demonstrated increased FAK levels compared with nonneoplastic tissues (Carelli et al., 2006). Moreover, Western blotting and real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed a statistically significant correlation between FAK upregulation and higher disease stages (I þ II versus III þ IV, p ¼ 0.019 and 0.028, respectively), indicating FAK involvement in tumorigenesis (Carelli et al., 2006). 3.1.12. Kidney cancer Comparative analysis of FAK expression in metastatic and nonmetastatic renal carcinoma cells revealed that FAK and paxillin mRNA expression were upregulated 2.0- to 2.5-fold in the metastatic Caki-1 cells over normal renal cortex epithelial cells (RCEC), suggesting its potential role as a marker of metastasis ( Jenq et al., 1996). Expression of FAK and HGF synergistically increased transformation of Madin–Darby canine kidney epithelial cells (Chan et al., 2002).

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3.1.13. Neuroblastoma Treatment of neuroblastoma cells with okadaic acid (OA), a serine phosphatase inhibitor, increasing serine/threonine phosphorylation and inhibiting tyrosine phosphorylation, induced focal adhesion loss, actin cytoskeleton disorganization, and cellular detachment, which corresponded to a loss of FAK Tyr-397 (Kim et al., 2003). It was suggested that drugs causing FAK dephosphorylation may be potentially therapeutic in neuroblastoma cells. A detailed study of FAK expression and phosphorylation in neuroblastoma will be critical in understanding FAK regulation in these cells. 3.1.14. Pancreatic cancer Inhibition of FAK with FAK siRNA potentiated gemcitabine-induced cytotoxicity in pancreatic cells. FAK siRNA treatment downregulated AKT activity (Duxbury et al., 2003). In addition, downregulation of FAK with siRNA caused cell increased anoikis in pancreatic adenocarcinoma PANC1, BxPC3, and MiaPaCa2 cell lines (Duxbury et al., 2004). FAK siRNA also inhibited metastasis in a nude mouse model (Duxbury et al., 2004). Furthermore, downregulating FAK with siRNA decreased cell motility and invasiveness in pancreatic cells (Huang et al., 2005). Analysis of FAK expression in 50 patients with pancreatic invasive ductal carcinoma by immunohistochemistry showed its detection in 24 samples (48%) (Furuyama et al., 2006). There was a statistically significant correlation between FAK expression and tumor size ( p ¼ 0.004), although FAK expression did not significantly correlate with other factors such as tumor histological grade, lymph node metastasis, distant metastasis, histological stage, and overall survival. It was concluded that FAK expression may not be a prognostic marker for pancreatic cancer patients (Furuyama et al., 2006). The more extended study of pancreatic cancer needed to be analyzed for FAK expression, as it correlated with tumor size. 3.1.15. Osteosarcoma Expression analysis of 16 osteosarcoma tumors, 5 osteosarcoma cell lines, and 6 normal tissues demonstrated FAK overexpression in high-grade osteosarcomas (Schroder et al., 2002). FAK expression analysis performed in our group in 13 high-grade sarcomas showed high levels in all tumor samples compared to benign, noninvasive mesenchymal specimens (Owens et al., 1995). This analysis shows that FAK can be a potential target in these tumors, although a more detailed study with correlation analysis of other tumor markers will be critical.

3.2. FAK and stem cells We would like to review several studies on FAK functioning in stem cells, as these cells are now considered precursors of cancer cells. Stem cell studies generate an important and promising field in cancer research. FAK has been

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shown to be upregulated differently by various cytokines in primary murine bone marrow (BM) cells (Kume et al., 1997). In unstimulated BM cells, FAK mRNA was detected in myeloid and lymphoid cells, but not in erythroid cells. FAK mRNA and protein levels have been shown to be upregulated by granulocyte macrophage colony-stimulating factor (GM-CSF), but not by interleukin-3 ( IL-3) (Kume et al., 1997). FAK increased levels correlated with motility and morphology cell polarization, suggesting the involvement of FAK in the maturity of GM-CSF-stimulated monocyte–macrophage lineages. In the developing brain, precursor cells give origin to cells that mature into microglia and neurons (astrocytes and oligodendrocytes). Oligodendrocytic progenitors are motile cells and are stimulated for proliferation by a number of growth factors: neurotrophin-3, fibroblast growth factor-2, and PDGF form isoforms. By RT-PCR and immunohistochemical analysis of 17 different protein kinases, FAK and c-Met have been shown to be expressed by cultured rat oligodendrocytes, suggesting their role in cell motility (Kilpatrick et al., 2000). In embryonic mouse stem cells, a decrease of mobility has been observed in FAK-deficient endodermal cells (Ilic et al., 1996). FAK has been shown to play a role in laminin-5 and extracellular matrix contact-induced differentiation of human mesenchymal stem cells into osteoblasts (Salasznyk et al., 2007a,b). Thus, these reports suggest that FAK functioning is important during stem cell maturation and motility. Future studies will reveal the role of FAK and stem cell signaling in tumorigenesis.

4. FAK and p53 Association FAK activity and expression can be regulated not only by cooperation with oncogens, but also by association with tumor suppressor gene proteins. One of the known proteins encoded by a tumor suppressor gene that can regulate FAK activity is PTEN, which is able to bind and dephosphorylate FAK and thus negatively regulate motility and invasion (Tamura et al., 1999b). Another protein is the neurofibromatosis type 2 (NF2) gene product, Merlin, encoded by a tumor suppressor gene frequently inactivated in malignant mesothelioma that has been shown to inactivate FAK and also inhibit invasion and motility (Poulikakos et al., 2006). A recent report demonstrated that tumor suppressor neurobifromin, NF1, modulated AKT, RAS, and FAK signaling pathways and affected cytoskeletal organization (Boyanapalli et al., 2006). We have recently demonstrated a novel direct interaction of FAK and p53, where FAK blocked tumor suppressor

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apoptotic activity (Golubovskaya et al., 2005). These data link FAK with tumor suppressors, especially important in association with p53 signaling. It is known that the p53 tumor suppressor is the most frequent target for genetic alterations in human cancers and is mutated in almost 50% of all tumors (Baker et al., 1990; Hollstein et al., 1999; Sidransky et al., 1992; Wang et al., 2003; Willis and Chen, 2002). Inactivation of the p53 gene is a critical step in tumorigenesis (Fearon and Vogelstein, 1990). Following induction by a variety of cell stresses such as DNA damage, hypoxia, or the presence of activated oncogenes, p53 upregulates a set of genes that can promote cell death and growth arrest, such as p21, GADD45, cyclin G, and Bax, reviewed in Giaccia and Kastan (1998). Recently, it was shown that p53 can repress promoter activities of a number of antiapoptotic genes and cell-cycle genes: survivin (Hoffman et al., 2002), cyclin B1, cdc2, (Taylor and Stark, 2001; Wu et al., 2001), cdc25 c (Krause et al., 2000), stathmin ( Johnsen et al., 2000), Map4 (Murphy et al., 1996), and bcl-2 (Wu et al., 2001). Overexpression of p53 induced apoptosis (Oren, 2003; Yonish-Rouach et al., 1991) and p53 inactivation caused a decrease in radiation-induced apoptosis (Fridman and Lowe, 2003; Lowe et al., 1993, 1994). The first report that analyzed a role of FAK and p53 genes in apoptosis was performed by Ilic et al. (1998). They reported that FAK suppressed a p53-regulated apoptotic pathway in anchorage-dependent cells (fibroblasts and endothelial cells) (Ilic et al., 1998). They also demonstrated that in the absence of FAK function, p53-regulated apoptosis was activated by protein kinase C and phospholipase A2, and this process was suppressible by dominantnegative p53 and Bcl-2. (Ilic et al., 1998). They analyzed apoptosis in anchorage-dependent human fibroblasts and endothelial cells but did not study these processes in tumors and cancer cell lines (Ilic et al., 1998; Zhang et al., 2004). In addition, immunohistochemical analysis of 115 endometrial carcinoma samples by our group demonstrated a correlation between FAK and p53 overexpression (Livasy et al., 2004). Our group showed a direct association between FAK and p53. Thus, we will focus briefly on the structure and biology of p53 to understand its novel interaction.

4.1. Structure and function of the p53 protein 4.1.1. p53 gene structure p53 is a tumor suppressor gene that is located at the chromosome 17p13 region, spans 20 kb, and contains 11 exons (Levine, 1997). The p53 protein is a phosphoprotein transcription factor that binds to the 50 Pu-Pu-PuC-A/T-T/A-G-Py-Py-Py30 (Pu-purine; Py-pyrimidine) consensus DNA sequence in the promoters of the genes and activates their transcription (Farmer et al., 1992). The p53 gene encodes the 393 amino acid protein. The promoter of p53 lacks a TATA box and contains various binding sites

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for known transcription factors, such as NF-kB, Sp1, or c-Jun (Bouchet et al., 2006). 4.1.2. p53 protein structure The p53 protein contains three domains: an acidic N-terminal transcriptionalactivating domain (1– 92 aa), a central DNA-binding domain (102–292 aa), and a C-terminal (102–292 aa) tetramerization domain (325–393 aa) (Fig. 3.4). The p53 protein contains many sites for phosphorylation by different kinases: ATM, Chk2, ATR, JNK, MAPK, CKI, and CKII. N-terminal transactivation domain A DNA-binding domain crystal structure shows that this domain contains a scaffold of b-sheets, supporting flexible helixes and loops, involved in interaction with DNA. Mutations in this domain alter this structure and disrupt DNA–protein interactions. Codons 17–28 interact with the double minute-2 homologue (Mdm-2), which plays a major role in p53 inhibition and degradation via the ubiquitin–proteasome pathway (Levine, 1997). Mdm-2 regulates p53 via an autoregulatory feedback loop in which both proteins control its cellular expression. p53 activates Mdm-2 transcription and expression. In turn, Mdm-2 binds to p53 and negatively regulates its stability and activity. Codons 22–26 are involved in binding of histone acetyltransferase P300 (Lacroix et al., 2006). A proline-rich domain (codons 63–97) that contains five PXXP motifs is required for interaction with various proteins and regulates apoptosis (Lacroix et al., 2006). PXXP motifs have been shown to create a binding site for Src homology SH3 domains (Yu et al., 1994). A mutant deleted from the proline-rich domain in human p53 (D62–91) is not able to cause apoptosis, but maintains the ability for cell cycle arrest (Edwards et al., 2003). Interestingly, a polymorphism at codon 72 has been demonstrated with either proline or arginine that can lead to differences in binding of transcription factors, apoptosis/survival signaling, and chemotherapy responses (Bergamaschi et al., 2003).

FAK

Transactivation domain TAD 1−92 a.a.

p53 DNA-binding central domain DBD

102−292 a.a.

Tetramerization domain TD 325−393 a.a.

Figure 3.4 p53 structure. p53 contains three domains: the N-terminal, transactivation domain, the central DNA-binding domain, and the C-terminal tetramerization domain. Focal adhesion kinase (FAK) interacts with the N-terminal domain of p53 (1^92 aa). The FAK protein binds directly to the N-terminal transactivation domain of p53, which is marked by an arrow.

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Central domain, DNA-binding domain This region is highly conserved, homologous to other p53 family member (p63 and p73) regions. The DNA-binding domain binds to promoter binding sites 50 Pu-Pu-Pu-C-A/ T-T/A-G-Py-Py-Py30 (Levine, 1997). C-terminal tetramerization domain This domain is involved in protein tetramerization and regulation of p53 activity. It contains three nuclear localization signals that allow a p53 nuclear import tetramerization region (323–356 aa) and a negative regulatory region (363–393 aa) (Lacroix et al., 2006).

4.2. p53 mutations in cancer cells p53 is the most commonly mutated gene in human malignancies, with up to 75% mutations in invasive cancers. The majority of mutations are missense mutations with four out of five mutations in the DNA-binding domain; only 1% is found in the N-terminal domain and 4% in the tetramerization domain (Stoklsa and Golab, 2005). The latest version of the International Agency for Research on Cancer p53 mutation database (http://www-p53.iarc.fr/) from July 2005 reported 21,512 somatic mutations. The most frequent ‘‘hot-spot’’ mutations are in codons: 175 Arg!His (breaks the bond between the L2 and L3 loop), Arg!Gln (Trp) (breaks contact with DNA in the minor groove), 273 Arg!His (Cys) (breaks contact with DNA in the major groove), and 282 Arg!Trp (destabilizes the H2 helix and DNA binding in the major groove and breaks contact on the b-hairpin) (Stoklsa and Golab, 2005). Among reported mutations, 75% are missense mutations; 80% are located in the DNA-binding domain of p53 (Bouchet et al., 2006), and 30% are located in five hotspot codons: 175, 245, 248, 273, and 282. Arginine residues (248 and 273) involved in interaction of p53 with DNA and arginines (175 and 282) stabilize the DNA-binding sequence (Bouchet et al., 2006). Wild-type p53 binds to promoters differently; for example, p53 activates the p21 promoter with higher affinity than the Bax promoter (Bouchet et al., 2006). Some p53 mutants are able to transactivate different genes, such as EGFR, MDR1, c-Myc, PCNA, IGF-2, or VEGF, providing growth-promoting phenotypes and drug resistance (Bouchet et al., 2006).

4.3. p53 binds the FAK promoter Our group was the first to clone the human FAK promoter and to find two p53-binding sites in it (Golubovskaya et al., 2004). We have shown that p53 can bind the FAK promoter and inhibit its transcriptional activity (Golubovskaya et al., 2004). In addition, several other transcription factors, such as SP-1, AP-2, TCF-1, and NF-kB, were shown to be present in the

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FAK promoter. The NF-kB protein has been shown to be linked to the p53 pathway (Benoit et al., 2006). For example, activation of Cox-2 transcription required the cooperation of NF-kB and p53 (Benoit et al., 2006). Thus, regulation of the FAK promoter can also include the association of these two transcription factors, thus providing an additional indirect p53-regulated FAK expression mechanism. Moreover, while the wild-type inhibited FAK promoter activity, the mutant p53 did not. The recent global analysis of the p53 transcription factor-binding sites demonstrated that induction of HCT116 colon cancer cells with 5-fluorouracil transcriptionally downregulated FAK (Wei et al., 2006). Thus, it was suggested that p53 can suppress metastasis through downregulation of metastasis-related genes, such as FAK. Since p53 is often mutated in cancer, a population-based study is needed to identify which mutations of p53 will correlate with upregulation of FAK.

4.4. Direct FAK and p53 protein binding In a recent report, we demonstrated that the N-terminal transactivation domain (1–92 aa) of p53 is physically directly associated with the N-terminal part (205–422 amino-acids) of FAK (Golubovskaya et al., 2005). In addition, there have been several reports on the localization of the N-terminal part of FAK in the nucleus (Beviglia et al., 2003; Jones and Stewart, 2004; Lobo and Zachary, 2000; Stewart et al., 2002). Furthermore, the N-terminus of FAK was shown to cause apoptosis in breast cancer cell lines (Beviglia et al., 2003), and its nuclear localization was regulated by caspase inhibitors in endothelial cells (Lobo and Zachary, 2000). In addition, p53 has been reported to be localized in the cytoplasm (Chipuk and Green, 2004). p53 directly activated Bax and released proapoptotic molecules, activating multidomain proteins in the cytoplasm. This mechanism required 62–91 residues in the proline-rich N-terminal domain of p53 (Chipuk and Green, 2004). Thus, understanding of the mechanism and functions of FAK/p53 interaction may ultimately have important implications for targeted cancer therapy.

4.5. Cytoplasmic–nuclear protein shuttling Moving proteins between cellular localizations is a tightly regulated process and provides an important mechanism for controlling protein functioning in the cell. FAK and p53 can both be localized under different conditions in either cytoplasm or nucleus. Although p53 was known mainly as a nuclear protein, recently evidence appeared to demonstrate that an extranuclear function of p53 in the cytoplasm caused apoptosis (Chipuk et al., 2004). p53 has been shown to directly bind the proapoptotic BAX protein, allowing for mitochondrial membrane permeabilization, cytochrome c release, and apoptosis (Chipuk et al., 2004). Thus, evidence was provided for the apoptotic

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transcription-independent function of p53 in the cytoplasm. Importantly, the proline-rich subdomain of the N-terminal p53 domain has been found to be critical for this p53 functioning (Chipuk et al., 2004). Cytoplasmic sequestration of p53 by the E1B 55-kDa protein played an important role in inhibiting p53 activities (Zhao et al., 2003). Different mechanisms of p53 nuclear export have been proposed: either through Mdm-2 binding in the nucleus and shuttling it to the cytoplasm, through p53 nuclear export signals (NES), or through Mdm-2-directed ubiquitination of p53 in the nucleus to promote its nuclear export (Zhang et al., 2001). It was found that a novel nuclear export signal in the N-terminal domain of p53 is required for the nuclear export of p53 (Zhang et al., 2001). p53 Arg-72 was critical for Bax binding with the p53 N-terminal proline-rich domain (Chipuk et al., 2004). The polymorphism with arginine instead of proline expressed more apoptotic functioning in mitochondria (Chipuk et al., 2004). These data suggest that the p53 N-terminal domain interaction with FAK can be critical for protein localization, transport, and shuttling from the nucleus to the cytoplasm. It was shown that p53 uses microtubules for nuclear localization (Vousden and Woude, 2000). FAK is mainly a cytoplasmic protein, but it was also recently reported to be localized in the nucleus ( Jones and Stewart, 2004; Jones et al., 2001; Lobo and Zachary, 2000). The N-terminal domain of FAK has been shown to be localized in the nucleus of endothelial cells (Lobo and Zachary, 2000). Loss of FAK from focal adhesions induced aggregation of the N-terminal domain of FAK in the nuclei in apoptotic glioblastoma cells, demonstrating FAK functioning not only in the cytoplasm and in the nucleus ( Jones et al., 2001). Treatment of cells with an inhibitor of nuclear transport, leptomycin B, resulted in the accumulation of the nuclear localization of FAK ( Jones and Stewart, 2004). Thus, FAK can shuttle between the nuclear and cytoplasmic compartments ( Jones and Stewart, 2004). In addition, recently a novel functioning of FAK was demonstrated in the nucleus, such as the PIAS-1-mediated sumoylation of FAK that occurred at lysine 152 in the N-terminal domain (Kadare et al., 2003). Thus, it is important to understand the mechanisms of protein shuttling between the cytoplasm and the nucleus that are critical for the regulation of their activity (Vousden and Woude, 2000).

4.6. Feedback model of FAK–p53 protein interaction We have shown that p53 can suppress FAK transcription (Golubovskaya et al., 2004). Recently, global characterization of 65,572 p53 ChIP DNA fragments was done in an HCT116 colorectal cancer cell line treated with 5-fluorouracil for 6 h, which activated p53 (Wei et al., 2006). Novel targets of p53 were identified that are involved in cell adhesion, migration, and metastasis, and PTK2 or FAK was one of these kinases (Wei et al., 2006).

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Interestingly, in HCT116 cells treated with 5-fluorouracil, which increases the p53 level, PTK2 (FAK) expression was also inhibited (Wei et al., 2006), which is consistent with our data (Golubovskaya et al., 2004). We have also shown that FAK can suppress the transcriptional activity of p53 through its interaction, as p53-mediated activation of p53 targets: p21, Mdm-2, and Bax were blocked by overexpression of FAK (Golubovskaya et al., 2005). Thus, p53 can regulate FAK, and in turn, FAK can regulate p53. Thus FAK and p53 can be regulated by comprising a feedback mechanism (Fig. 3.5). Mutations of p53 that are frequently found in cancers can lead to upregulation and overexpression of FAK (Fig. 3.3). This mechanism was recently discussed in Mitra and Schlaepfer (2006). Interestingly, the novel link between FAK and p53 was supported by a recent report on the novel FAK inhibitor FIP200, which binds to FAK directly and inhibits cellular functions, such as cell adhesion, spreading, and motility (Melkoumian et al., 2005). FIP200 overexpression resulted in increased levels of p21 and caused growth arrest (Melkoumian et al., 2005). It was found that FIP200 interacted with FAK and p53 (Melkoumian et al., 2005). An interesting model of the FAK–p53–FIP200 interaction has been proposed (Mitra and Schlaepfer, 2006). It was suggested that FIP200/FAK and FIP200/p53 bindings can compete, and when FAK has low expression, FIP200 can bind p53 and

p21

Bax Mdm-2 Apoptosis Wild-type p53 protein

p53

p53

FAK protein

Low expression of FAK protein FAK protein

FAK promoter

Mutant p53 protein

p53

Other factors (NF-kappa B) High expression of FAK protein p53

FAK FAK FAK protein protein protein

FAK promoter

Figure 3.5 The feedback model of focal adhesion kinase (FAK) and p53 interaction. The wild-type p53 binds to the FAK promoter and blocks its transcriptional activity, resulting in low FAK expression. Overexpressed FAK protein binds to p53 protein and blocks p53 transcription by repressing p53-induced apoptotic targets: p21, Mdm-2, and Bax, thus generating a feedback loop mechanism.

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increase its expression (Mitra and Schlaepfer, 2006). For the reverse, it was also suggested that FAK overexpression can limit binding of FIP200 to p53, enhancing cellular survival. Thus, novel mechanisms of FAK survival function remain to be discovered during carcinogenesis.

5. FAK and p53 Targeted Therapy 5.1. Downregulation of FAK It was recently proposed that FAK is a new therapeutic target (McLean et al., 2005). Several in vitro approaches have been used to downregulate FAKadenoviral FAK-CD (dominant-negative FAK) (Xu et al., 2000), antisense oligonucleotides (Smith et al., 2005), and siRNA for FAK (Han et al., 2004). We linked FAK expression to apoptosis by treating FAK-positive tumor cell lines with different antisense oligonucleotides to FAK that specifically inhibited p125 FAK expression ( Judson et al., 1999; Xu et al., 1996). The melanoma cells treated with antisense oligonucleotides lost their attachment and underwent apoptosis (Smith et al., 2005; Xu et al., 1996). The same effect was observed with Ad-FAK-CD in different cancer cells. While breast cancer cells underwent apoptosis by downregulation of FAK with FAKCD, normal MCF-10A and HMEM cells did not (Xu et al., 2000). These inhibitor applications are limited due to in vivo cell toxicity. Thus, developing small-molecule drugs is critical for the future of FAK-targeting therapy, involving kinase inhibitors and drugs, targeting FAK–protein interactions.

5.2. FAK inhibitors There were no pharmacological inhibitors reducing FAK kinase activity. Recently, Novartis Inc. developed novel FAK inhibitors, causing downregulation of its kinase activity (Choi et al., 2006). A series of 2-amino-9aryl-7H-pyrrolo[2,3-d]pyrimidines was designed and synthesized to inhibit FAK using molecular modeling in conjunction with a cocrystal structure. Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. The novel Novartis FAK inhibitor TAE226 was recently employed in brain cancer and effectively inhibited FAK signaling and caused apoptosis in these cells (Shi et al., 2007). We also used this inhibitor in breast, neuroblastoma, and pancreatic cancer cells and found that this inhibitor can effectively cause apoptosis, although it can also inhibit other signaling pathways in addition to FAK. Thus, the specificity of this drug remains to be discovered. Future detailed studies will be needed to address the specificity of these drugs.

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5.3. Targeting protein–protein interactions One of the approaches to inhibit FAK function can be targeting its protein– protein interaction with its binding partners such as p53. Small-molecule drugs can be found either through high-throughput screening or through database searches using protein crystal structures.

5.4. p53 therapy Experimental approaches that target p53 for cancer treatment include therapies to activate p53 as well as to inactivate p53. Anticancer therapies include: (1) a gene therapy approach to replace wild-type p53 with p53expressing retroviruses or adenoviruses, (2) killing of p53-deficient cells with modified adenoviruses, and (3) pharmacological activation by inhibition of Mdm-2 binding with nutlins or by direct activation with polyamines or reactivation of wild-type activity in mutant cells with PRIMA-1 (Bouchet et al., 2006). Retroviruses are attractive agents for cancer gene therapy, as in the stable form they integrate into the genome and require cell division for transduction. The second strategy is adenoviruses that are double-stranded DNA viruses. In contrast to retrovirus, their effect is not limited to high-proliferating cells, and there is no risk of insertional mutagenesis. One such drug is Ad5CMV-p53 (Advexin INTROGEN Therapeutics Inc.; Gendicine Shenzden SiBiono Gene Tch. Co. Ltd.), which is a recombinant E1-deleted serotype 5 adenoviral vector encoding p53. In vitro and in vivo studies confirmed the therapeutic affect of Ad5CMV-p53. This therapy approach was used effectively in clinical trials, Phase I and II, although some cases of tumor resistance were also demonstrated (Bouchet et al., 2006). The second major approach to kill p53-defective cells is the use of E1Bdefective adenoviruses. The delta 1520 virus (dl1520; ONYX-015) is an adenovirus that contains a deletion in the E1B region. ONYX-015 can be used as a selective drug against tumors with mutant p53. The same problems as with adenoviruses can occur in some types of tumors. Small molecule drug inhibitors are effectively used to target p53 protein–protein interactions, particularly with Mdm-2 protein (Vassilev, 2005). The first potent inhibitors targeting p53–Mdm-2 interaction have been identified by high-throughput screening followed by structure-based optimization (Vassilev, 2005). The screening identified nutlins that represent a class of cis-imidazole analogues that bind to the p53 pocket interacting with Mdm-2. The same strategy can be used to target interaction with FAK. Recently, we used the crystal structure of the N-terminus of FAK and screened 20,000 small molecules from the NCI bank for their ability to target this binding site and for their oral bioavailability using Lipinski rules. We identified 10 potential lead compounds and tested them on human

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breast, colon, and melanoma cell lines for their ability to disrupt p53 FAK binding and to induce cancer cell death. We found several potential compounds that were able to decrease FAK phosphorylation, decrease cell viability, and activate PARP, suggesting their potential role in future therapy.

6. Summary Thus, an understanding of FAK biology during tumorigenesis, the mechanisms of its upregulation in different tumors, its role in stem cell biology, angiogenesis, and motility, and especially the mechanisms of its direct physical interaction with the p53 protein and downstream signaling pathways will be critical in developing targeted therapeutics. Studies with peptide inhibitors already have indicated that blockade of specific protein–protein interactions has therapeutic promise for treating a variety of diseases, including cancer (Aarts et al., 2002; Akhter et al., 1998; Aramburu et al., 1999; Chen et al., 1999; May et al., 2000; van Rooij et al., 2002). Small-molecule drugs are particularly attractive as inhibitors of intracellular protein–protein interactions due to their ability to modify their structures to achieve optimal target binding. As we further define the mechanisms of FAK signaling in cancer cells, we will identify the optimal sites for targeting this protein and disrupting its signaling to cause apoptosis in human tumors. Thus, the interaction of FAK and p53, as well as complexes with other binding partners, can be important therapeutic targets in cancer treatment programs.

ACKNOWLEDGMENTS We apologize to those whose papers we have not cited because of space limitations. The research is supported by NIH RO1-CA065910 (W.G.C.) and by Susan G. Komen for the Cure grant (V.M.G.).

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C H A P T E R

F O U R

Cell and Molecular Biology of the Spindle Matrix Kristen M. Johansen and Jrgen Johansen

Contents 1. Introduction 2. Microtubule Spindle Dynamics and Force Production 3. Evidence for a Spindle Matrix 3.1. Early indications of abundant nonmicrotubule components of the spindle and spindle remnants 3.2. Chromosome movement after UV microbeam severing of microtubules 3.3. Molecular identification of a multiprotein spindle matrix complex in Drosophila 3.4. Spindle length and the spindle matrix 3.5. Membranes and the spindle matrix 3.6. Other molecular candidates for an internal spindle matrix structural element 3.7. Spindle assembly factors (SAFs) and the spindle matrix 4. Concluding Remarks Acknowledgments References

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Abstract The concept of a spindle matrix has long been proposed to account for incompletely understood features of microtubule spindle dynamics and force production during mitosis. In its simplest formulation, the spindle matrix is hypothesized to provide a stationary or elastic molecular matrix that can provide a substrate for motor molecules to interact with during microtubule sliding and which can stabilize the spindle during force production. Although this is an attractive concept with the potential to greatly simplify current models of microtubule spindle behavior, definitive evidence for the molecular nature of a spindle matrix or for its direct role in microtubule spindle function has been lagging. However, as reviewed here multiple studies spanning the evolutionary

Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011 International Review of Cytology, Volume 263 ISSN 0074-7696, DOI: 10.1016/S0074-7696(07)63004-6

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2007 Elsevier Inc. All rights reserved.

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spectrum from lower eukaryotes to vertebrates have provided new and intriguing evidence that a spindle matrix may be a general feature of mitosis. Key Words: Spindle matrix, Spindle formation, Microtubules, Mitosis, Chromosome segregation. ß 2007 Elsevier Inc.

1. Introduction A mitotic spindle is present in all known eukaryotic cells and its function is essential for chromosomal segregation and cell division to occur (Mitchison and Salmon, 2001). The spindle apparatus is a complex molecular machine known to be made up of polymerized tubulin and various associated motor proteins (Gadde and Heald, 2004; Karsenti and Vernos, 2001; Sharp et al., 2000a). Although much work has been directed toward understanding mitotic spindle apparatus structure and function, it is still unclear what directs and stabilizes the assembly of the spindle. Furthermore, although numerous models have been proposed for how the spindle apparatus may transmit forces, none of these models have been able to account for all the experimentally observed properties of spindle behavior (Bloom, 2002; Gadde and Heald, 2004; Kapoor and Compton, 2002; Mitchison and Salmon, 2001; Scholey et al., 2001; Wittmann et al., 2001); especially, the discovery of microtubule flux and the constant treadmilling of tubulin dimers toward the poles (Cassimeris et al., 1988; Mitchison, 1989; Mitchison and Salmon, 2001; Rogers et al., 2005; Sawin and Mitchison, 1991, 1994) have made it difficult to model how forces are generated to actually move chromosomes on the basis of a metastable structure not anchored in place. For these reasons and based on theoretical considerations of the requirement for force production at the spindle, the concept of a spindle matrix has long been proposed (Johansen and Johansen, 2002; Pickett-Heaps et al., 1982, 1997; Wells, 2001). The spindle matrix is hypothesized to provide a stationary or elastic molecular matrix that can provide a substrate for motor molecules to interact with during microtubule sliding and which can stabilize the spindle during force production (PickettHeaps et al., 1997) (Fig. 4.1). Molecules forming a spindle matrix complex would be expected to exhibit several characteristics: (1) they should associate together to form a true fusiform structure coaligned with the microtubule spindle; (2) they should remain associated, forming a polymerized complex in the absence of microtubules; (3) perturbation of one or more of the components should affect spindle assembly and/or function; and (4) one or more members of the complex should interact with microtubules or microtubule-associated molecules such as motor proteins. Such a matrix could also be envisioned to have the added properties of helping to organize

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A

B

Depolymerized tubulin

Figure 4.1 Properties of the spindle matrix.The spindle matrix hypothesis proposes the existence ofa stationaryorelastic molecular matrix thatcontributesto microtubule spindle function and/or assembly. Molecules forming a spindle matrix would be expected to exhibit several characteristics: they should associate together to form a true fusiform structure coaligned with the microtubule spindle, and they should remain associated forming a polymerized complex in the absence of microtubules as illustrated in the micrographs. (A) and (B) Confocal images of Drosophila syncytial embryo nuclei at metaphase double labeled with antibodies to tubulin (red) and the spindle matrix protein Megator (green). After depolymerization of microtubules by cold treatment, the Megator antibody-labeled matrix is still intact maintaining a fusiform structure (B).

and stabilize the microtubule spindle as well as the midbody, an electron dense body implicated in cytokinesis. Whereas the spindle matrix is an attractive concept, for many years there has been little direct experimental evidence for such a structure, and its molecular nature has remained enigmatic. However, a number of studies in Drosophila (Qi et al., 2004, 2005; Rath et al., 2004; Walker et al., 2000) as well as in vertebrates (Chang et al., 2004; Mitchison et al., 2005; Tsai et al., 2006) have revived interest in the spindle matrix. Here we review evidence for the existence of a spindle matrix and its possible molecular composition in the context of microtubule spindle dynamics and force production.

2. Microtubule Spindle Dynamics and Force Production The mitotic spindle is a dynamic, complex macromolecular machine constantly being remodeled during the progression through the stages of mitosis (prophase, prometaphase, metaphase, anaphase, and telophase). Most research to date has focused on two classes of molecules that play

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critical roles in transducing the forces necessary to align and separate chromosomes precisely to two daughter nuclei, namely microtubules and motor proteins (Kapoor and Compton, 2002; Karsenti and Vernos, 2001; Mitchison and Salmon, 2001; Sharp et al., 2000a,b; Wittmann et al., 2001). Microtubules contribute to these forces in two ways: they are a rigid, polarized cytoskeletal element on which dynein- and kinesin-family motor proteins can move cargo in a directional manner, and they also can directly promote movement within the spindle via ‘‘treadmilling’’ in which tubulin subunits are dissembled from the minus end while being added to the plus end (Margolis and Wilson, 1978; Margolis et al., 1978; Mitchison et al., 1986). Thus the biophysical properties of microtubules make them an ideal cytoskeletal structural element to confer many of the essential functional properties required of a mitotic apparatus. Microtubules are hollow cylindrical tubes of 13 parallel protofilaments, each composed of ab-tubulin heterodimers arranged end-on-end with the a-tubulin subunit exposed at the minus end and the b-tubulin subunit exposed at the plus end (Fan et al., 1996; Hirose et al., 1995; Nogales et al., 1999). This tubular organization of protofilaments is stabilized by lateral interactions forming a ‘‘B lattice’’ in which contacts are made between homologous subunits, a a and b b (Song and Mandelkow, 1993), but closing of the cylinder leaves a ‘‘seam’’ in which these lateral contacts are slightly altered (Chre´tien et al., 1996; Kikkawa et al., 1994; Sosa and Milligan, 1996; Wade and Hyman, 1997). Studies suggest this seam may be an important site of action for the regulation of microtubule dynamics (Sandblad et al., 2006). However, better understood is how MT dynamics are affected by the status of the guanine nucleotide bound to the b-tubulin subunit. Each tubulin subunit binds a GTP nucleotide with hydrolysis of GTP to GDP occurring only on the b-tubulin subunit (David-Pfeuty et al., 1977; Spiegelman et al., 1977). When the b-tubulin subunits at the end of a MT contain GTP (a ‘‘GTP-cap’’; Carlier and Pantaloni, 1981), the MT tends to be stable and growing, but loss of this GTP cap by hydrolysis to GDP favors ‘‘catastrophe’’ (Davis et al., 1994), a situation in which the MT rapidly depolymerizes until a ‘‘rescue’’ event may reverse the process (Hyman et al., 1992; Mitchison, 1993; Nogales et al., 1999). The rapid switching between a depolymerizing state and a growing state is the hallmark of ‘‘dynamic instability’’ for which MTs are known and is a key feature allowing for remodeling of MTs (Cassimeris et al., 1987; Kristofferson et al., 1986; Mitchison and Kirschner, 1984). The metaphase spindle is a fusiform-shaped structure anchored by two poles with microtubules organized with their plus-ends pointing away from the pole (Euteneuer and McIntosh, 1981; Haimo, 1985; Telzer and Haimo, 1981). Within the mitotic spindle there are different classes of MTs: (1) astral fibers, which radiate toward the cortex of the cell and are believed to assist in spindle orientation and cleavage plane specification; (2) kinetochore

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microtubules (kMTs), which typically are bundled into k-fibers, extend from the pole and attach to the chromosomes at specialized attachment sites called kinetochores; and (3) interpolar microtubules (ipMTs), which extend across the spindle midzone, interdigitating with microtubules originating from the opposite half of the spindle without interacting with chromosomes (Compton, 2000). This latter class of MTs can be of diverse lengths, since their minus-ends do not always originate at a pole but can be spread out over half of the spindle length (Mastronarde et al., 1993). This observation was unexpected because previously it had been thought that all MTs initiate from a ‘‘microtubule organizing center’’ (MTOC) that for most spindles is provided by one of the duplicated and separated centrosomes found at each half-spindle pole. However, experiments have shown that microtubule spindles can form and function in the complete absence of centrioles and centrosomes (Basto et al., 2006; Bonaccorsi et al., 1998; Hinchcliffe et al., 2001; Khodjakov et al., 2000; Megraw et al., 2001; Szo¨llo¨si et al., 1986). Furthermore, g-tubulin, which is involved in MT nucleation, has been observed to be distributed throughout the spindle fibers and not strictly localized to the centrosomes (O’Brien et al., 2005; Raynaud-Messina and Merdes, 2007; Wilde and Zheng, 1999). Thus, based on these findings several different models have been developed to account for the multiple mechanisms that combine to generate the microtubule spindle (Gadde and Heald, 2004; Wadsworth and Khodjakov, 2004). In the ‘‘search and capture’’ model, MTs that emanate from the poles are highly dynamic, undergoing multiple rounds of growth and shrinkage until they become stabilized by ‘‘capture’’ of a chromosome kinetochore (Kirschner and Mitchison, 1986; Mitchison and Kirschner, 1985; Nicklas and Kubai, 1985). Once the bivalent kinetochore has been captured by MTs emanating from opposite poles, the chromosome will congress to the metaphase plate to form a bipolar metaphase spindle (Nicklas and Kubai, 1985). But a different mechanism for spindle assembly must occur in acentrosomal cells such as certain oocytes and higher plant cells. It has been proposed that such cells build their spindles by a ‘‘self-organization’’ mechanism in which MTs are nucleated from the chromosomes and from within the spindle itself instead of from a MTOC; once formed the MTs are progressively focused by the actions of motor proteins and other scaffolding proteins to form a bipolar spindle (Albertson and Thomson, 1993; Matthies et al., 1996; McKim and Hawley, 1995; Smirnova and Bajer, 1992; Steffen et al., 1986). However, the observation that cells that would normally utilize a centrosomalmediated mechanism could build spindles by a chromosome-mediated pathway if an essential centrosome component was mutant (Bonaccorsi et al., 1998; Megraw et al., 2001) or if the centrosome was removed or inactivated by chromophore-assisted laser inactivation (Hayden et al., 1990; Khodjakov et al., 2000) suggested that cells could be induced to use alternate pathways. High-resolution imaging studies revealed that although the

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centrosome-mediated pathway dominates in Drosophila S2 cells, both pathways are operational (Maiato et al., 2004). Thus, the evidence points toward a ‘‘combined model’’ in which multiple redundant mechanisms such as centrosome-mediated nucleation of MTs, chromosome-based assembly of MTs, and recruitment, sliding, and bundling of MTs created at other sites interact to give rise to spindle assembly (Gadde and Heald, 2004; Janson et al., 2007; Mahoney et al., 2006; Wadsworth and Khodjakov, 2004). Whereas microtubule dynamics of assembly and disassembly during spindle formation is regulated through a complex balance between different MTstabilizing and MT-destabilizing activities (Andersen, 2000; Kline-Smith and Walczak, 2004), less well understood is how spindle MT fluxes are generated or how flux may be linked to actual force generation capable of moving chromosomes during anaphase (Kwok and Kapoor, 2007; Rogers et al., 2005; Sharp et al., 2000a). In addition to microtubules, a large number of MT-based motors participate in spindle action via a number of different mechanisms, including cross-bridging and sliding adjacent MTs, transporting chromosomes and other mitotic cargoes along the MTs, and by influencing MT growth and shrinkage (Goshima and Vale, 2003; Goshima et al., 2005a; Kwon et al., 2004; Rogers et al., 2004; Tao et al., 2006). Furthermore, a balance of plus-end–directed and minus-end–directed motors regulate many aspects of spindle morphogenesis and dynamic function (Fuller and Wilson, 1992; Heald and Walczak, 1999; Sharp et al., 2000a,b). Thus the mitotic apparatus is under constant tension, with both microtubule dynamics and opposing motor proteins generating inward and outward forces. Because the metaphase spindle does not collapse, these forces must balance each other out or alternatively the motor proteins must be anchored to a stabilizing structural element that acts as a scaffold or strut ( Johansen and Johansen, 2002; Sharp et al., 2000a). Theoretical calculations have been derived in support of the hypothesis that observed spindle dynamics (i.e., centrosome separation, spindle assembly, spindle elongation, and spindle disassembly) can be satisfactorily accounted for based on a structure comprised solely of microtubules and motors (Cytrynbaum et al., 2003; Nedelec, 2002; Scholey et al., 2001). However, even if the forces in the model can be ‘‘balanced’’ such that it is not necessary to invoke an additional scaffolding element to assist in the dynamics of spindle assembly or mitotic motility, certain biophysical constraints argue for the existence of a matrix to strengthen the spindle apparatus. Force calculations predict 1- to 10-pN force is generated per motor (Block, 1995; Howard, 1995; Scholey et al., 2003; Svoboda and Block, 1994), and with the cooperation of hundreds to thousands of force generators (motors), the actual spindle force measured is in the range of a 1000 pN (Nicklas, 1983). The force to buckle a microtubule is only in the pN range and although bundling of microtubules increases the buckling threshold to an 100 pN range (Elbaum et al., 1996; Freitas, Jr., 1999; Fygenson et al., 1997), this is still well below the measured forces exerted on the spindle (Nicklas, 1983). It is also difficult to

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account for why most spindle microtubules are curved. Whether they are envisioned to exert pulling forces or pushing forces on the chromosomes, microtubules would be expected to be ‘‘straight’’ without some kind of tensile element to act against. Thus, current models to explain spindle structural dynamics based solely on the activities of microtubules and motors are likely to be incomplete. Furthermore, there may be many missing elements because the molecular composition of the spindle is still not fully defined: a proteomic analysis of the human mitotic spindle identified 795 proteins, only 151 of which had been previously known to associate with the spindle apparatus (Sauer et al., 2005). Consequently, our understanding of the molecular composition of the mitotic spindle apparatus and how it generates forces to segregate the chromosomes is likely to be only rudimentary. The demonstration of the existence of a spindle matrix would have the potential to clarify many of the outstanding issues and to greatly simplify current models for force generation.

3. Evidence for a Spindle Matrix Some of the first experimental observations that hinted at the existence of a spindle matrix were from the early experiments of Goldman and Rebhun (1969) and Forer (1969), who found that the volume of the nonmicrotubule portion of the spindle was much greater than that of microtubules. That this could correspond to a ‘‘spindle matrix’’ was suggested by data indicating that birefringence in the spindle originates from nonmicrotubule, as well as from microtubule, components. Similar conclusions were made from EM evidence demonstrating linear arrays of particles in the absence of microtubules (Behnke and Forer, 1966; Goldman and Rebhun, 1969). When spindles are treated with nocodazole, a ‘‘spindle remnant’’ can be isolated that retains spindle-like morphology despite the absence of microtubules (Leslie et al., 1987; Pickett-Heaps et al., 1984; Wein et al., 1998). Tektin-like antigens associate with spindles as well as spindle remnants generated by cold treatment, implying other structural elements exist in the spindle (Steffen and Linck, 1992). Chromosomes are still pulled to spindle poles in ultraviolet (UV)-microbeam experiments during mitosis, despite the microtubules having been severed (Sillers and Forer, 1983; Spurck et al., 1997) (Fig. 4.2). At least four proteins from two different nuclear compartments that interact with each other and that redistribute during prophase forming a fusiform spindle structure that persists in the absence of polymerized tubulin have been identified in Drosophila ( Johansen et al., 1996; Qi et al., 2004, 2005; Rath et al., 2004; Walker et al., 2000). The coiled-coil protein NuMA forms a pericentriolar matrix that has been shown to be necessary for proper spindle formation and function

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Figure 4.2 Chromosome movement after k-fibers are severed in an irradiated cranefly spermatocyte spindle. 0.00: Cell before treatment. Chromosomes are aligned at the metaphase plate (arrowheads). 17:52: UV irradiation creates a k-stub (arrow) and an ARB (area of reduced birefringence), indicating loss of microtubules. 21:03: Both k-stub and chromosome move toward the pole in anaphase, despite lack of k-fiber connection to the pole. 22:09: Chromosomal movement during anaphase continues. Note the increased separation between partners (arrowheads) and continued presence of a k-stub (arrow). 24:53: Cell after lysis reveals k-stub (arrow) and separated chromosomes (arrowheads). Final panel: confocal of cell in previous panel stained for tubulin shows clearly that the UV-severed k-fiber remained a stub and was not in contact with the pole. Asterisk denotes a k-stub from a previously irradiated region. Note that both chromosome partners moved toward poles despite severed k-fibers. (Micrographs were generously provided by Drs. A. Forer,T. Spurck, and J. D. Pickett-Heaps).

(Dionne et al., 1999; Merdes et al., 1996). In addition, a nonmicrotubule filamentous structure that colocalizes with spindle microtubules has been observed in yeast nuclei (van Hemert et al., 2002). Studies indicating a nonmicrotubule matrix contributes to force generation in the spindle have also been reported, for example, severing all of the microtubules in a metaphase half-spindle resulted in spindle shortening, suggesting the existence of compressive forces on the central spindle at metaphase (Spurck et al., 1990). Similarly, based on responses to microtubule destabilization experiments in Xenopus egg extracts, Mitchison et al. (2005) suggested an unidentified tensile element acts in parallel with conventional microtubule lattice factors to generate spindle-shortening forces. One molecular candidate for such an internal matrix in this system is poly(ADP-ribose), a nonprotein macromolecule required for bipolar organization of Xenopus extract spindles (Chang et al., 2004). Another candidate molecule is lamin B, a component of the interphase nuclear lamina shown to be required for spindle assembly (Tsai et al., 2006).

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Thus a wide range of experimental observations in different organisms has been consistent with the existence of a spindle matrix.

3.1. Early indications of abundant nonmicrotubule components of the spindle and spindle remnants Porter (1955) reported the presence of fibrillar elements in the spindle that were later shown to be composed of microtubules (Ledbetter and Porter, 1963, 1964; Slautterback, 1963) and which became the focus of most studies of spindle function. However, even at that time a number of different lines of investigation were raising questions about whether additional components in the spindle remained to be identified, Mazia and Dan (1952) first took advantage of the synchronized mitotic populations of sea urchin eggs to develop procedures to obtain substantial quantities of isolated mitotic spindles for such analysis, and multiple versions of this original spindle isolation protocol have since been developed (e.g., Kane, 1965, 1967; Mazia et al., 1961; Sauer et al., 2005; Zieve and Solomon, 1982). In independent studies by Goldman and Rebhun (1969) and by Forer (1969), it was found that the volume of the nonmicrotubule portion of the spindle was greater than that of microtubules. That this could correspond to a ‘‘spindle matrix’’ was suggested by data indicating that birefringence in the spindle originates from nonmicrotubule as well as from microtubule components (Forer, 1966; Goldman and Rebhun, 1969). Similar conclusions were made from EM evidence demonstrating linear arrays of particles in the absence of microtubules (Behnke and Forer, 1966; Goldman and Rebhun, 1969). Thus, very early on it was clear the mitotic apparatus was a complex structure composed of significantly more material than simply microtubules. However, different isolation procedures could give rise to very different results, as shown in the studies of Forer and Goldman (1969). In these studies isolated spindles included protein, RNA, and carbohydrate-containing material, but the actual composition varied depending on pH and isolation times. This has prompted some to question whether mitotic spindles act as ‘‘a sponge’’ during the isolation procedure and has led to concerns about the physiological relevance of many components that copurify with the mitotic apparatus (Wells, 2001). Nonetheless, when isolated spindles are extracted with calcium and/or shifted to low temperatures, a ‘‘spindle remnant’’ can be isolated that retains spindle-like morphology despite the absence of microtubules (Leslie et al., 1987; Pickett-Heaps, 1986; Rebhun and Palazzo, 1988; Wein et al., 1998). Rebhun and Palazzo (1988) reported that Ca2þ-extracted spindles contained a 55 kDa polypeptide with an amino acid composition similar to intermediate filament proteins. Leslie et al. (1987) used antibodies to study the distribution of kinesin in isolated sea urchin spindles. Whereas the kinesin colocalized with MT spindles including asters in unperturbed spindle preparations, following

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MT disassembly it remained associated with the spindle remnant in amorphous, spindle-shaped structures with no astral extensions. This localization was not affected by exogenous ATP addition, supporting the interpretation that in addition to its association with microtubules, kinesin likely also associates with a spindle matrix component. These conclusions were strengthened by studies in Cylindrotheca fusiformis in which the kinesin-related protein DSK1 was shown to be part of a matrix in the extracted diatom mitotic apparatus that exists independent of the microtubule spindle (Wein et al., 1998). Other molecules besides motor proteins show this behavior. Affinity purified polyclonal antibodies against tektin C label tubulin-depleted spindle remnants as well as spindles (Steffen and Linck, 1992). Because tektin itself does not appear to localize to the mitotic apparatus but under renaturing transblot conditions this antibody could recognize tektins A and B due to their high degree of structural similarity, it was proposed that tektinrelated protein(s) might be present in the spindle and potentially provide additional structural support (Steffen and Linck, 1992). Other potential cytoskeletal proteins have also been described in spindles, including the keratin-related proteins cytocentrin (Paul and Quaroni, 1993) and astrin (Gruber et al., 2002; Mack and Compton, 2001), titin or titin-related proteins (Wernyj et al., 2001; Zastrow et al., 2006), protein 4.1 (Huang et al., 2004; Krauss et al., 1997, 2004) and lamin B (Beaudouin et al., 2002; Georgatos et al., 1997; Harel et al., 1989; Maison et al., 1997; Paddy et al., 1996; Tsai et al., 2006).

3.2. Chromosome movement after UV microbeam severing of microtubules An early indication of a functional spindle matrix was reported by Forer (1965), who used UV-microbeam irradiation to create an ‘‘area of reduced birefringence’’ (ARB) in the spindle fiber and found that it moved to the pole with constant velocity and shape until it disappeared. The fiber simultaneously recovered birefringence from the kinetochore end, suggesting a poleward flux of spindle fiber material. In addition, EM analysis showed that lesions associated with ARBs are devoid of microtubules (Snyder et al., 1991; Wilson and Forer, 1988). Thus this technique was ideally suited to test the prevailing ‘‘Pac-man’’ model that proposed chromosome segregation to the poles was powered by disassembly of kMTs at the kinetochore. If this were the sole mechanism generating the separation forces severing k-fibers in the middle of the half-spindle, UV-microbeam irradiation should have halted chromosome segregation during anaphase, but remarkably, this was not the case. Even after UV-mediated kMT disassembly, the chromosomes continued to be pulled to the spindle poles (Pickett-Heaps et al., 1997; Sillers and Forer, 1983; Spurck et al., 1997) (see Fig. 4.2).

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These results suggested poleward forces for chromosome motion are not produced by the k-fibers but rather act on them and that an intact kMT is not required for force production. In addition, the observation that although some chromosomes moved with unchanged speed, other chromosomes showed a transient acceleration, returning to the normal segregation speed only after the kMT-stub contacted the centrosome (Spurck et al., 1997), led to the proposal that the rate of MT disassembly in the spindle might serve to limit the rate of chromosome motion rather than power it (Forer, 1974; Nicklas, 1975; Pickett-Heaps et al., 1982). Taking into consideration an external source of power generating chromosome movement as well as that MTs adjacent to the ARB showed a tendency to buckle, Spurck et al. (1997) favored a model in which the force, for chromosome segregation is generated by a component associated with a spindle matrix and where the role of spindle MTs is to resist this force, thus setting up the spindle as a kind of tensegrity structure (Ingber, 1993, 2003). Although it has become clear there are multiple and perhaps redundant mechanisms at work generating segregation forces in the spindle, these were critical experiments in advancing the idea that forces external from the kMTs are also involved. However, the debate about the existence of a ‘‘spindle matrix’’ continued, with some favoring its role in contributing to these external forces (Pickett-Heaps and Forer, 2001; Pickett-Heaps et al., 1984, 1996, 1997; Spurck et al., 1997), whereas others envisioned that motor proteins could move chromosomes on intact ipMTs outside of the ARB region after cross-bridging with the kMT-stub (Scholey et al., 2001; see also Maiato et al., 2004 regarding k-stub regrowth and reincorporation into the spindle), an issue that has still not been definitively resolved.

3.3. Molecular identification of a multiprotein spindle matrix complex in Drosophila 3.3.1. Molecular components A longstanding reservation regarding the potential existence of a spindle matrix has been a lack of direct molecular information on its biochemical composition. However, some of the most promising molecular candidates, Skeletor, Chromator, Megator, and EAST (Qi et al., 2004, 2005; Rath et al., 2004; Walker et al., 2000), for constituting a bona fide spindle matrix complex presently fulfilling at least three of its defining criteria have been identified in Drosophila. Skeletor, the founding member of this complex, is an 81-kDa protein originally identified by use of a mAb with an intriguing dynamic staining pattern during mitosis in Drosophila embryos (Walker et al., 2000) (Fig. 4.3). However, Skeletor encodes a low-complexity protein with no obvious motifs, so it was difficult to predict Skeletor’s potential role in spindle matrix function. Consequently, in a search for

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Figure 4.3 Drosophila embryo nuclei labeled by the Skeletor mAb1A1 from various stages of the cell cycle. (A) interphase; (B) prophase; (C) late prophase; (D) prometaphase; (E) metaphase; (F) late anaphase; (G) early telophase; (H) late telophase. At prophase (B), the chromosome localization of the spindle matrix component Skeletor undergoes a redistribution to a structure forming a spindle-like scaffold from late prophase (C) through late anaphase (F). During interphase (A) and prophase (B, C), mAb1A1 labeling appears to be also associated with the nuclear envelope. The micrographs are Nomarski images of mAb1A1 labelings visualized using HRP-conjugated secondary antibody. (Modified from J. Cell Biol. [2000]. 151, 1401^1411. Copyright 2000 Rockefeller University Press.)

other members of a spindle matrix molecular complex, Rath et al. (2004) used a yeast two-hybrid screen to identify a novel protein, Chromator, which directly interacts with Skeletor. Chromator is a 101-kDa protein that contains a chromodomain, and analysis of P-element mutations has demonstrated that Chromator is an essential protein (Rath et al., 2004). However, for a spindle matrix to form independently or to form a structural scaffold aligned with the microtubule spindle, one or more of its molecular components would be predicted to have the ability to form polymers, and neither Skeletor nor Chromator appear to contain molecular motifs with such properties. Qi et al. (2004, 2005), therefore, used immunocytochemistry and cross-immunoprecipitation experiments to show that two additional proteins, Megator and EAST, also interact with Skeletor and Chromator during mitosis. Megator is a 260-kDa protein with a large NH2-terminal coiled-coil domain and a shorter COOH-terminal acidic region that originally was referred to as the Bx34 antigen (Zimowska et al., 1997). EAST is another large protein of 253 kDa, which, apart from seven potential nuclear

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localization sequences and 12 potential PEST sites, does not have any previously characterized motifs or functional domains (Wasser and Chia, 2000). Although Skeletor, Chromator, and EAST appear to have no obvious mammalian homologs, the coiled-coil protein Megator has a striking overall structural and sequence similarity to the mammalian nuclear pore complex Tpr protein (Zimowska et al., 1997) and to the yeast nuclear pore complexassociated proteins Mlp1p and Mlp2p (Kosova et al., 2000; Strambio-deCastillia et al., 1999). Mlp2p binds directly to core components of the spindle pole body (SPB) and is required for proper SPB function and normal cell division (Niepel et al., 2005). Interestingly, several other nuclear pore complex-associated proteins have also been linked to proper microtubule spindle assembly and function (Orjalo et al., 2006; Schetter et al., 2006; Scott et al., 2005). Furthermore, the presence of a large coiled-coil domain in Megator raises the intriguing possibility that Megator could comprise the structural element of the spindle matrix complex. Interestingly, Megator deletion construct analysis in S2 cells indicates that the NH2-terminal coiledcoil containing domain has the ability to self assemble into spherical structures in the cytoplasm (Qi et al., 2004). This is in contrast to the acidic COOH-terminal domain, which is targeted to the nucleus, implying the presence of a functional nuclear localization signal. Furthermore, the COOH-terminal domain is sufficient for localization to the nuclear rim as well as for spindle localization. Thus, an attractive hypothesis is that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain may be responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex. Supporting this notion is the finding that Megator spindles persist in the absence of microtubules depolymerized by cold or nocodazole treatment (Qi et al., 2004) (see Fig. 4.1). 3.3.2. Dynamic redistribution of spindle matrix proteins during mitosis Coimmunoprecipitation studies, yeast-two hybrid interaction assays as well as immunocytochemistry show that all four proteins interact to form a true fusiform spindle complex during mitosis (Qi et al., 2004, 2005; Rath et al., 2004; Walker et al., 2000). Two of the proteins, Skeletor and Chromator, are localized to chromosomes during interphase (Rath et al., 2004; Walker et al., 2000), whereas the other two, Megator and EAST, occupy the intranuclear space surrounding the chromosomes, with Megator additionally being localized to the nuclear rim (Qi et al., 2004; Zimowska et al., 1997) (Fig. 4.4). Thus, the four proteins are derived from two different nuclear compartments. As illustrated in Fig. 4.5A, the establishment of the spindle matrix (as labeled using the Skeletor antibody mAb1A1) appears to precede microtubule spindle formation at prophase. During metaphase the spindle matrix and the microtubule spindles are coaligned (Fig. 4.5B).

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Spindle matrix proteins in Drosophila Name

Kind

Size

Interphase localization

Skeletor

Novel protein

81 kD

Chromosomes

Chromator

Chromodomain protein

101 kD

Chromosomes

Megator

Coiled-coil protein

260 kD

Intranuclear space

EAST

Novel protein

253 kD

Intranuclear space

Chromator

EAST

Megator

Metaphase

Interphase

Skeletor

Figure 4.4 Spindle matrix proteins in Drosophila. The four nuclear spindle matrix proteins, Skeletor, Chromator, Megator, and EAST, redistribute during prophase and interact with each other to form a fusiform spindle structure at metaphase.Two of these proteins, Skeletor and Chromator, are localized to chromosomes during interphase, whereas the other two, Megator and EAST, occupy the interchromosomal space surrounding the chromosomes.The micrographs in the upper panel are confocal images from third instar larval salivary gland nuclei double-labeled with antibodies specific to each of the four spindle matrix proteins (red or green) and Hoechst (blue).The lower panel shows confocal images of metaphase nuclei from syncytial embryos labeled with antibodies specific to each of the four spindle matrix proteins (red or green).

Importantly, the mAb1A1-labeled spindle matrix maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate (Fig. 4.5D). At telophase when the chromosomes start to decondense, mAb1A1 labeling still defines a spindle in the midregion (Fig. 4.5C). When embryos are treated with nocodazole to disassemble the microtubules, the mAb1A1-labeled spindle structure persists (Fig. 4.5E). Thus, the mAb1A1-defined spindle exhibits all the properties predicted for the spindle matrix (Walker et al., 2000). It should be emphasized that antibodies to Chromator, EAST, and Megator label the metaphase spindle matrix structure in an identical way to that of the Skeletor antibody (Qi et al., 2004, 2005; Rath et al., 2004). Especially, the finding that the spindle matrix maintains its fusiform spindle structure

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during chromosome segregation makes it an ideal candidate for being a scaffold that provides structural support for motor proteins and counterbalancing force production. That this spindle may provide a substrate for the alignment of microtubules is further supported by the finding that microtubules are colocalized with the spindle matrix that remains in the central region during midbody formation at telophase. This alignment of microtubules at the midbody region is largely unaccounted for by most other models but could be explained if the spindle matrix as hypothesized by Walker et al. (2000) helps in organizing microtubule fibers. Based on these observations Walker et al. (2000) proposed a model in which the spindle matrix constituted by these proteins is assembled during late prophase (Fig. 4.5F) at a time prior to or coinciding with microtubule extension into the nucleus. During mitosis the nuclear lamina in Drosophila first breaks down at the poles, thus allowing access of the microtubules into the nuclear interior where they coalign with the spindle matrix (Fig. 4.5G). The spindle matrix remains intact during metaphase and anaphase, thereby providing a stable scaffold to balance the forces and counterforces generated by motor proteins while microtubules shorten and chromosomes are moved to the poles (Fig. 4.5H). A prediction of this model is that mutations in components of the spindle matrix compromising this scaffold or resulting in its loss will lead to abnormal microtubule spindles and chromosome segregation defects (Fig. 4.5I). 3.3.3. Functional analysis Unfortunately, a definitive analysis of the role of these proteins in spindle matrix function by mutant and RNAi approaches has been challenging. One of the reasons is that components of the spindle matrix may have essential functions at interphase as well as during mitosis. For example, it is likely Megator plays several important functional roles as a component of multiple subcellular structures that include the nuclear pore complex, the interphase interchromosomal domain, and the spindle matrix (Qi et al., 2004). Thus, although a null allele of the Megator gene has been identified, Megator function in early homozygous embryos could not be tested due to the presence of maternally derived Megator protein that masks any potential phenotypes (Qi et al., 2004). Furthermore, these animals die before hatching, precluding larval neuroblast analysis. For these reasons Qi et al. (2004) used RNAi methods in S2 cells to deplete Megator protein levels. When Megator levels were knocked down, the number of S2 cells undergoing mitosis was greatly reduced. However, cells with obvious defects in tubulin spindle morphology or chromosome segregation defects were not observed, suggesting that depletion of Megator prevents cells from entering metaphase. This could be due to an essential function of Megator in maintaining nuclear structure and/or in maintaining the integrity of the nuclear rim and pore complexes during interphase or a necessary function for nuclear reorganization during prophase. Thus, if Megator plays multiple functional roles as its

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A

B

C

Skeletor

Comp

DNA

Anaphase

D

E

Skeletor

Tubulin

DNA

Metaphase

Comp

Nocodazole treated F

G Late prophase

Metaphase

H Anaphase

I Spindle matrix mutant

Figure 4.5 Spindle matrix properties in Drosophila. (A^C) Spindle matrix formation begins before microtubule entry into the nucleus or nuclear lamina breakdown. Double labelings with mAb1A1 to visualize the spindle matrix protein Skeletor (red) and anti^ a-tubulin to visualize the microtubules (green) show that in late prophase, when the microtubules have not yet entered the nuclear space, the Skeletor antibody-labeled spindle is already aligned within the nucleus (A). During metaphase the two spindles are coaligned, although the Skeletor antibody-labeled spindle appears broader than the microtubule spindle (B). During telophase the Skeletor antibody-labeled spindle persists in the central region where midbody formation of the microtubules is found to take place (C). QuickTime movies of 3D reconstructions of Skeletor and tubulin labelings

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dynamic localization pattern suggests (Zimowska and Paddy, 2002), it would prevent the analysis of a mitotic function using RNAi approaches. Therefore, the further study of Megator function will require additional methods such as genetic approaches to generate new alleles that behave as conditional lethals or knockout-specific functions of the Megator protein or cell biological/biochemical approaches such as isolation of the matrix. For the analysis of Chromator function using a mutant approach, a similar situation exists. A null Chromator allele has been isolated, but homozygous animals die as embryos or first instar larvae, preventing the study of Chromator function during mitosis (Rath et al., 2004). However, with the report of the generation of hypomorphic alleles of Chromator (Rath et al., 2006), it may soon be possible to study Chromator’s effect on cell division in larval neuroblasts. So far the best evidence for a functional role of a spindle matrix protein in mitosis was provided by Rath et al. (2004) using RNAi assays in S2 cells, demonstrating that depletion of Chromator protein leads to abnormal spindle morphology and that chromosomes are scattered in the spindle, indicating defective spindle function in the absence of Chromator (Fig. 4.6A and C). These types of defects would be expected if Chromator functions as a spindle matrix-associated protein that promotes interactions between motor proteins and a stationary scaffold and if these interactions were necessary for chromosome mobility. Interestingly, this phenotype resembles the mitotic chromosome segregation defects observed after RNAi knockdown of some kinesin motor proteins in S2 cells including KLP67A by Goshima and Vale (2003) and KLP59C by Rogers et al. (2004) (Fig. 4.6A and B). Thus, these data provide evidence that Chromator is a nuclear-derived protein that plays a role in proper spindle dynamics leading to chromosome separation during mitosis and are compatible with the hypothesis that Chromator may constitute a functional component of a spindle matrix molecular complex. Although no Skeletor mutants have yet been characterized, studies of chromosome behavior in east loss-of-function mutations during mitosis and can be accessed at http://www.jcb.org/cgi/content/full/151/7/1401/DC1. (D) The Skeletor antibody-labeled spindle (red) persists as an intact spindle extending across the metaphase plate as the chromosomes (blue) segregate to the poles. (E) Nocodazole-treated Drosophila embryo at metaphase triple-labeled with mAb1A1 (red), a-tubulin antibody (green), and Hoechst (DNA in blue).The microtubule spindles have completely depolymerized as indicated by the absence of microtubule labeling (green).The mAb1A1-labeled spindle (red) is still intact albeit slightly deformed, demonstrating that this structure persists independently of the microtubule spindle. All panels represent confocal images. (F^H) Diagram of spindle matrix protein redistribution during mitosis.The spindle matrix is indicated in red, condensed chromosomes in blue, centrosomes and microtubules in green, and nuclear lamina in yellow. (I) The spindle matrix hypothesis predicts impaired function of one or more spindle matrix proteins (gray) would lead to abnormal chromosome segregation and/or microtubule spindle defects. (Modified from J. Cell Biol. [2000]. 151, 1401^1411. Copyright 2000 Rockefeller University Press.)

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A

B

Chromator RNAi

Number of segregation defects per field

C

200

KLP59C RNAi

p 80%) in the cell appears to undergo continuous exchange between soluble- and spindle-associated pools as determined by fluorescence recovery after photobleaching (FRAP) analysis (Kisurina-Evgenieva et al., 2004). Because NuMA has been shown to directly bind and bundle microtubules (Haren and Merdes, 2002) and immunodepletion of NuMA from in vitro mitotic assembly extracts causes spindles to develop into irregular, unfocused MT arrays, it was proposed that a NuMA-based matrix structure acts to stabilize the mitotic spindle poles (Merdes et al., 1996). In this same study, Merdes et al. (1996) observed that NuMA is found in a complex with dynein and dynactin that they proposed localizes NuMA to the poles where it then forms a matrix that promotes and stabilizes the fusiform spindle. This notion is supported by experiments showing that cell free assembly systems require the presence of NuMA to organize MTs into mitotic asters (Gaglio et al., 1995). By immunogold EM techniques, NuMA was found to localize to an electron-dense matrix at the spindle pole, and in cell fractionation experiments, it was retained predominantly in the insoluble fraction, even after nocodazole treatments had shifted tubulin

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and conventional MAPs into the soluble fraction (Dionne et al., 1999). Thus, NuMA was proposed to comprise part of the mitotic spindle matrix, and it was envisioned that this matrix functioned to organize MT minus ends and counterbalance the forces exerted by microtubule motors at the spindle pole (Dionne et al., 1999). However, NuMA shows a pericentrosomal localization to the spindle at metaphase and does not form a complete spindle. Thus NuMA may contribute to the spindle matrix, but it is likely there are additional components essential for providing a complete framework. 3.6.2. Fin1p, Ase1p, and the midzone matrix One candidate for a filamentous molecule that extends throughout the mitotic spindle was reported in a study by van Hemert et al. (2002), where they identified a novel Saccharomyces cerevisiae 14-3-3-interacting protein that they named Fin1p (for filaments in between nuclei). This protein contains two putative coiled-coil domains suggesting a potential for self-assembly. To test this possibility, van Hemert et al. (2002) found that 6xHis-tagged Fin1p purified from yeast extract could self-assemble in vitro into 10-nm filamentous structures independently of microtubules or other proteins. When a GFP-tagged Fin1p was introduced into yeast cells, the distribution of Fin1p dynamically reorganized during the cell cycle from a nonfilamentous nuclear form in nondividing cells to a filamentous structure that colocalized with spindle microtubules extending between the two nuclei of dividing cells during mitosis. More recently, using a 13xmyc-tagged Fin1p construct expressed at endogenous levels Woodbury and Morgan (2007) observed that Fin1p’s targeting to spindles occurs specifically at anaphase and only after it is dephosphorylated by the Cdc14 phosphatase. Mutation of Fin1p’s consensus phosphorylation sites to alanine (Fin15A) results in a dominantlethal phenotype, but Fin15A-GFP can be expressed in cycling cells using an inducible GALS promoter (Woodbury and Morgan, 2007). Although these cells showed a reduced growth rate and premature association of Fin15AGFP with the spindle prior to anaphase, there were no obvious spindle defects. In contrast, when Fin15A-GFP was overexpressed in metaphasearrested cells, the spindles collapsed, yielding unseparated spindle poles and unusually long astral microtubules. Thus, in cycling cells mislocalization of Fin15A may cause microscopically undetectable spindle defects that impair chromosome segregation, ultimately leading to cell lethality, a notion that was supported by finding a sevenfold increase in chromosome loss after a transient, sublethal pulse of Fin15A was delivered. Although expression of Fin15A caused metaphase spindle collapse, it appears to play a role in stabilizing anaphase spindles, as shown using an artificial anaphase system in which metaphase-arrested cells can be triggered to enter anaphase by induction of the TEV protease (Higuchi and Uhlmann, 2005; Uhlmann et al., 2000). In this system sister chromatids move to the poles, but because cyclins are

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stable, spindle elongation at anaphase is abnormal, and spindles eventually break. The presence of wild-type Fin1, which due to cyclin stabilization remains phosphorylated and hence does not associate with the spindle, has no effect. However, nonphosphorylatable Fin15A does target to the spindle and greatly reduces spindle breakage frequency (Woodbury and Morgan, 2007). Interestingly, this requirement for a ‘‘Fin1p matrix’’ coincides with the transition of microtubules from a state of high dynamic instability at metaphase to suddenly becoming stable at anaphase (Higuchi and Uhlmann, 2005) and would support previous hypotheses for a role of the spindle matrix in stabilizing the microtubule spindle. One potential inconsistency with the idea that Fin1p determines a spindle matrix scaffold required for stabilizing microtubules is the observation that deletion of Fin1 does not affect cell viability or cause any obvious spindle defects (van Hemert et al., 2002; Woodbury and Morgan, 2007). However, just as establishment of a proper microtubule spindle is so critical to the cell that redundant pathways have evolved to ensure its successful formation, it is likely redundant pathways to build a spindle matrix have evolved as well. This idea is supported by the finding that a second coiledcoil protein, Ase1p (anaphase spindle elongation), has been identified that localizes to the spindle midzone, binds and bundles MTs, and is necessary to maintain anaphase spindle integrity (Loı¨odice et al., 2005; Pellman et al., 1995; Schuyler et al., 2003). Similarly with FIN1, the null allele of ASE1 is viable (Pellman et al., 1995). However, cells lacking both Fin1p and Ase1p are inviable (Woodbury and Morgan, 2007), consistent with the idea that they may comprise redundant molecular components of a spindle matrix. 3.6.3. Poly(ADP-ribose): A regulatory switch or a spindle matrix ‘‘gel’’? The addition of negatively charged poly(ADP-ribose) (PAR) moieties is an unusual and versatile posttranslational modification originally linked to DNA damage detection and repair but now associated with an increasing number of biological functions, including chromatin modification, transcription, and cell survival/cell death pathways (Kim et al., 2005; Schreiber et al., 2006). The surprising discovery of several different poly(ADP-ribose) polymerases (PARPs) on the mitotic spindle hinted at a potential role for ADP-ribosylation in spindle function as well (Earle et al., 2000; Kickhoefer et al., 1999; Smith, 2001; Smith and de Lange, 1999). This idea received experimental support when Chang et al. (2004) discovered that mitotic spindles contain approximately tenfold higher levels of PAR than the surrounding cytoplasm and PAR levels and/or function are necessary for proper spindle assembly and structure. When PAR polymer levels were decreased enzymatically in preassembled spindles by poly(ADP-ribose) glycohydrolase (PARG), a highly specific processive endoglycosidase and exoglycosidase (Hatakeyama et al., 1986), or functionally blocked with purified anti-PAR antibodies, Chang

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et al. (2004) observed rapid breakdown of spindle structure with microtubules splaying outward and the two half-spindles becoming disconnected. In spindle assembly assays, these treatments resulted in the formation of monopolar microtubule asters, suggesting PAR is not required for microtubule nucleation or dynamics but instead has a specific role in organizing the bipolar spindle, a role consistent with that proposed for a spindle matrix molecule. The mechanism by which PAR regulates bipolar spindle formation is not known. Given that proteins can be reversibly poly(ADP-ribosyl)ated by the opposing activities of PARPs and PARGs and the significant biochemical consequences that result due to addition or removal of such large, negatively charged groups, one model postulates that PARsylation of proteins acts as a molecular switch regulating protein activity, much in the way phosphorylation does. In this case, the challenge is to identify the potential targets of PARsylation. Using RNAi approaches, Chang et al. (2005a) identified tankyrase-1 as the PARP responsible for the spindle-associated PAR, and one relevant target that has already been identified is NuMA (Chang et al., 2005a,b). However, because PAR was found to extend across the entire spindle (Chang et al., 2004) and is not restricted to the pericentrosomal region where NuMA resides, presumably there are other targets yet to be identified. PARsylation of spindle proteins might regulate their function or binding activities in an analogous manner to how tankyrase-1mediated PARsylation regulates telomere length by inducing telomeric repeat binding factor 1 (TRF1) displacement to allow telomerase access (Chong et al., 1995; Smith et al., 1998). An alternative model posits that a PARsylated spindle matrix might be composed of a cross-linked poly(ADP-ribose) gel that stretches and stores elastic energy due to its attachment to spindle poles and plus end-directed motors (Mitchison et al., 2005). The stored elastic energy would provide an explanation for the tensile forces such as were observed in Mitchison et al. (2005) and in earlier experiments (Forer, 1966; Forer and Wilson, 1994; Pickett-Heaps et al., 1984, 1996, 1997; Sillers and Forer, 1983) that could not be ascribed to microtubules. Although the actual molecular composition of such a matrix is not yet defined, its regulation by PARsylation would provide a convenient tag to assist in its identification. 3.6.4. Lamin B and a membranous spindle matrix Early studies in Drosophila that had noted a mitotic ‘‘spindle envelope’’ also found that a fraction of the lamin T40 antigen (now known to be the lamin B homolog) remains associated with the mitotic apparatus (Harel et al., 1989). The dynamics of mitotic spindle formation and nuclear lamina breakdown inferred a functional role for the lamina in mitotic spindle formation (Paddy et al., 1996), but because in Drosophila a large fraction of the nuclear envelope remains localized to a rim in the nuclear periphery until well into metaphase (Paddy et al., 1996), it was not clear whether this was a consequence of the

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so-called ‘‘semi-closed’’ mitosis in Drosophila. However, the subsequent observation that lamin B also associates with mitotic spindles in mammalian cells (Beaudouin et al., 2002; Georgatos et al., 1997; Maison et al., 1997) argues for a more widely conserved function for lamin B in the spindle. In Caenorhabditis elegans reduction of lamin B in early embryos resulted in a range of both nuclear and mitotic defects (Liu et al., 2000). Nonetheless, it was difficult to differentiate cause and effect in these studies and a functional requirement for lamin in spindle assembly was not explored until more recently. To assess the potential requirement in spindle assembly and/or function for LB3, the major lamin B isoform in Xenopus eggs (Lourim et al., 1996), Tsai et al. (2006) took advantage of a powerful spindle assembly system in which spindles can be assembled in vitro from Xenopus M-phase extracts with or without depleting factors of interest (Lohka and Maller, 1985; Sawin and Mitchison, 1991; Sawin et al., 1992). Immunostaining of spindles assembled in complete M-phase egg extract revealed the presence of LB3 in the spindle and peripheral region surrounding the spindle. Depleting LB3 from the extract severely disrupted the spindle assembly process with the vast majority of mitotic figures appearing as half spindles or asters, indicating a requirement for the presence of lamin B for proper bipolar spindle assembly. To address whether the lamin B spindle localization defined a ‘‘spindle matrix’’ independent of microtubules, the authors treated normally assembled spindles with nocodazole to depolymerize the MTs. Despite the complete absence of spindle MTs, lamin B3 remained spindle-associated, although it adopted a more granular and vesicular appearance suggesting the presence of membranes. To test whether membranes were associated with the spindle as well as the lamin B matrix, Tsai et al. (2006) used CM-diI, a membrane dye that is well retained throughout fixation and permeabilization. Both the complete assembled spindle and the nocodazole-generated lamin B spindle matrix preparations were stained with CM-diI, indicating the presence of membranes. But when exposed to Triton X-100 prior to fixation, the lamin B matrices were completely disrupted, suggesting this matrix is dependent on a membrane component. Although not examined in this study, it would be interesting to know whether, once assembled, the microtubule spindle is stable through Triton X-100 treatment or whether there is a functional requirement for continued presence of the lamin B matrix. In vivo data also supports the indication from the in vitro studies that lamin B plays a role in spindle assembly. Tsai et al. (2006) analyzed lamin B distribution in HeLa cells and found by immunostaining that a fraction of both lamin B isoforms (LB1 and LB2) was associated with mitotic spindles. Reducing expression of either lamin B isoform by siRNA resulted in spindle abnormalities including unfocused spindle poles, loss of chromosome congression, and defects in spindle morphology. However, because the lamin B appears to remain associated with membranes, it is not clear

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how this mitotic spindle matrix may be organized or how it may relate to force production of the microtubule spindle as originally envisioned for a spindle matrix. It has therefore been suggested the assembly of this lamin B-containing membranous matrix in mitosis may provide a connection between microtubule spindle function and the partitioning of membrane systems during cell division (Zheng and Tsai, 2006). 3.6.5. Actin and myosin A number of immunolocalization studies in a variety of systems have reported on the presence of actin and myosin in the mitotic spindle (Cande et al., 1977; Czaban and Forer, 1992; Espreafico et al., 1998; Forer and Jackson, 1976, 1979; Fujiwara and Pollard, 1976; Robinson and Snyder, 2005; Sampson, 2004; Sanger, 1975; Schmit and Lambert, 1987; Seagull et al., 1987; Silverman-Gavrila and Forer, 2000a, 2003; Traas et al., 1987; Yasuda et al., 2005). However, other studies have failed to identify significant F-actin in the spindle proper (Clayton and Lloyd, 1985; Derksen et al., 1986; Palevitz and Liu, 1992), while yet others suggested that lack of antibody specificity or fixation artifacts may be responsible for apparent actin fibers in the spindle (Aubin et al., 1979; Barak et al., 1981; Schroeder, 1973). It is also possible that actin structures can be lost due to fixation artifacts (Traas et al., 1987). In some cases few actin filaments were observed within the spindle, but an actin network instead appeared as a stretched, ‘‘elastic cage’’ encasing the microtubule spindle (Schmit and Lambert, 1987). In an EM study of HeLa cell mitotic spindles, Pollard et al. (1984) found no examples of long actin fibers but did detect a large number of short, actin-like filaments. Thus, the presence of actin or myosin in the spindle has historically been controversial. Nevertheless, a variety of functional studies indicating a requirement for actin and myosin for proper spindle function have been reported and thus raise the prospect that an actin cytoskeletal system may be involved in spindle matrix function in at least some cell types. Inhibition of actin polymerization with cytochalasin D or latrunculin B induces abnormal chromosome alignment and segregation (Forer and Pickett-Heaps, 1998; Sampson et al., 1996). Treatment of cells with the myosin inhibitor 2,3-butadione monoxine (BDM) has been associated with aberrant chromosome movements on the spindle and a loss of microtubule poleward flux (Sampson, 2001; Silverman-Gavrila and Forer, 2000a,b, 2001, 2003). Although a study has called into question BDM’s target specificity (Ostap, 2002), the observation that these same defects are also observed with actin inhibitors (Silverman-Gavrila and Forer, 2000a,b) suggests the relevant target in these studies was myosin. Analysis of the severed ‘‘k-stubs’’ created when kinetochore fibers are severed by UV microbeam revealed that in the presence of either actin or myosin inhibitors, subsequent fiber elongation is blocked (Forer et al., 2007). Based on these results, it was suggested that actin

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and myosin are involved in generating microtubule flux that results in elongation of kinetochore microtubules (Forer et al., 2007; SilvermanGavrila and Forer, 2000a). Because microtubule flux has also been attributed to motor activity at the poles (Goshima et al., 2005a; Miyamoto et al., 2004; Rogers et al., 2005; Shirasu-Hiza et al., 2004), there may be multiple mechanisms underlying flux generation or the motors that drive microtubule flux may require interaction with an actinomyosin-based spindle matrix. In this model actin and myosin are a functional component of a spindle matrix, generating a tensegrity structure that can provide the energy necessary to propel kinetochore fibers poleward (Pickett-Heaps and Forer, 2001; Pickett-Heaps et al., 1984, 1996, 1997; Spurck et al., 1997). This force is resisted by the microtubules which in this model are acting as struts (Forer, 1974) and whose rate of disassembly thus determines the rate of chromosome segregation (Forer and Wilson, 1994; Pickett-Heaps et al., 1986, 1997). One issue that may underlie the difficulty in firmly establishing a connection between an actin spindle matrix component and microtubule function in the spindle is that due to a variety of technical reasons it has been difficult to document interactions between the two systems. These reasons include difficulties fractionating two filamentous, polymeric entities and different fixation conditions required for each to optimize preservation for fluorescent imaging studies, as well as masking of less abundant F-actin structures by the high local concentration of cortical F-actin (discussed in Sider et al., 1999 and references therein). Sider et al. (1999) exploited the Xenopus oocyte extract system to examine whether microtubules and F-actin could interact and observed a consistent lengthwise colocalization of F-actin with astral microtubules that was correlated with a bending or kinking of microtubules similar to that previously described in vivo in lamellipodia by Waterman-Storer and Salmon (1997). Interestingly, the association between microtubules and F-actin was dependent on the presence of one or more microtubule-associated proteins (MAPs) because it was not observed to occur in assays with purified brain tubulin and muscle F-actin unless oocyte microtubule-binding proteins were included in the assay (Sider et al., 1999). Examples of microtubule-actin interactions have now been observed to play an important role in directed cell migration, neuronal growth cone guidance, wound healing, cytokinesis, and cortical flow (Rodriguez et al., 2003). In addition, the MyTH4-FERM domain cassette present in several unconventional myosins has, in the case of myosin-10, been demonstrated to bind microtubules directly (Weber et al., 2004). Thus, there is precedence for functional interactions between the two cytoskeletal systems. Furthermore, some cells appear to be dependent on the actinomyosin cytoskeletal system for proper mitotic spindle assembly. Rosenblatt et al. (2004) found that perturbation of myosin II by either depleting protein by

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RNAi or by inhibiting myosin activity with blebbistatin or inhibiting actin polymerization with latrunculin B led to defects in centrosomal separation and abnormalities in spindle formation. However, in this case they identified cortical myosin as the necessary player and proposed that astral microtubule interaction with the cortex plays a role in triggering centrosomal separation (Rosenblatt et al., 2004). They did not find a requirement for actin or myosin after nuclear envelope breakdown had occurred. In another study in starfish oocytes, nuclear envelope breakdown was found to trigger actin polymerization required for efficient chromosome capture by the spindle during the first meiotic division (Le´na´rt et al., 2005). Chromosomes or DNA-coated beads were observed to trigger the formation of actin patches connected to each other by a network of actin filaments. Contraction of this network delivered the embedded chromosomes to the animal pole where they could then be captured by spindle microtubules (Le´na´rt et al., 2005). When the actin cytoskeleton was disrupted by latrunculin B, chromosome congression failed in 75% of the cells, resulting in chromosome loss and aneuploid eggs (Le´na´rt et al., 2005). Defects in spindle formation have also been observed in Xenopus oocytes when the actin cytoskeleton was disrupted with cytochalasin B (Gard et al., 1995; Ryabova et al., 1986). Thus, cells with large nuclear volumes such as oocytes appear to utilize an actinbased system to propel chromosomes toward the centrosomes to increase the efficiency of the microtubule-based ‘‘search and capture’’ mechanism for spindle assembly (Le´na´rt et al., 2005). Thus, although the role of actin and myosin in cytokinesis is well established (Glotzer, 2005), their requirement for mitotic spindle assembly or function has been debated over the years. Mutations in myosin II in Schizosaccharomyces pombe and Dictyostelium did not lead to apparent mitotic defects (Bezanilla et al., 1997; de Hostos et al., 1993), and RNAi depletion of myosin II appeared to affect only cytokinesis (Somma et al., 2002). Perturbing myosin function by injection of anti-myosin II antibodies or myosin II fragments into amphibian eggs did not have any apparent effect on spindle formation or function, though after one cell division subsequent cell cycles were blocked (Kiehart et al., 1982; Meeusen et al., 1980). Furthermore, actin depolymerizing drugs do not prevent spindle formation or function in Xenopus egg extracts (Desai et al., 1998, 1999; Sawin and Mitchison, 1991). Interestingly, in some studies where inhibition of actin or myosin blocked anaphase chromosome movements, such blockage was only temporary with chromosomes resuming movement, albeit more slowly, after a delay (Fabian and Forer, 2005; Forer and Pickett-Heaps, 1998; Silverman-Gavrila and Forer, 2001). Thus, contributions of the actinomyosin cytoskeletal system to force production in the spindle may reflect the evolution of multiple, independent mechanisms to create an operational spindle to reliably power chromosome movement despite the

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diverse array of physiological demands placed upon different cell types in the functioning organism (Fabian and Forer, 2005).

3.7. Spindle assembly factors (SAFs) and the spindle matrix The development of an in vitro spindle assembly system from Xenopus egg extracts (Lohka and Maller, 1985; Sawin and Mitchison, 1991) has provided a very powerful system in which to dissect out some of the molecular requirements for spindle assembly. Whereas the first models of spindle assembly had been almost exclusively based on the ‘‘search and capture’’ principle in which two opposing centrosomes nucleated microtubules that were stabilized after capturing a kinetochore, thus generating a bipolar spindle (Compton, 2000), subsequent studies employing the Xenopus in vitro assembly system revealed that chromatin-coated beads were also highly efficient at promoting formation of bipolar spindles (Heald et al., 1996), and further analysis with this system identified that it was the gradient of RanGTP generated by the chromosomes that was responsible for this activity (Carazo-Salas et al., 2001). Both the spindle localization and MT stabilizing effects of RanGTP were subsequently confirmed in vivo in the Drosophila embryo system (Trieselmann and Wilde, 2002) and in HeLa cells using a fluorescent biosensor that shows increased fluorescence resonance energy transfer (FRET) signal when liberated from importin-b by RanGTP (Kala´b et al., 2006). The discovery that RanGTP regulates spindle assembly was somewhat unexpected as the focus on Ran had previously uncovered its critical role in regulating transport of RNA and proteins between the nucleus and cytoplasm (Moroianu, 1999). Ran is an abundant, small G-protein that when in the nucleus primarily exists in its GTP-bound form due to the activity of the chromosomally localized GEF (guanine nucleotide exchange factor) RCC1 (the regulator of chromosome condensation) and the high nuclear GTP concentration. RanGTP promotes the dissociation of importin b-like nuclear transport receptors from their cargoes upon reaching the nuclear interior, a critical part of the nuclear import pathway. Among the cargoes imported into the nucleus in this pathway are a number of spindle assembly factors (Goodman and Zheng, 2006). Nuclear localization of these factors sequesters them from the microtubules during interphase. However, upon nuclear envelope breakdown the mixing of nuclear and cytoplasmic compartments results in nuclear SAFs becoming bound and thus inactivated by available nuclear import receptors. RanGTP is able to stimulate MT assembly in the vicinity of the chromosomes as a consequence of the chromosomal RCC1 RanGEF activity that produces a gradient of RanGTP (Carazo-Salas et al., 1999). Just as nuclear RanGTP acts to release cargo from the receptor during the import process, the binding of RanGTP to the nuclear import receptor in the proximity of chromosomes results in release of the SAFs, which are then free to stabilize

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MTs and thereby promote spindle assembly (Dasso, 2001; Goodman and Zheng, 2006; Walczak, 2001). One such SAF is NuMA, a protein that had already been proposed to comprise part of the spindle matrix. This raises the question of whether other spindle matrix-associated components may be similarly regulated by RanGTP. A number of RanGTP targets have been identified, although it is not yet known if they mediate their activities in conjunction with the spindle matrix. 3.7.1. TPX2 TPX2 (targeting protein for Xklp2) was identified as a microtubuleassociated protein that directly links the motor protein Xklp2 to microtubules (Wittmann et al., 1998). TPX2 is a basic 82.4-kDa protein with two coiled-coil domains (Wittmann et al., 2000) required for bipolar spindle formation in in vitro assembly assays using chromatin beads (Gruss et al., 2001) and in in vivo studies in HeLa cells (Gruss et al., 2002). TPX2 is inactivated by importin-b via the adaptor protein importin-a. Release of TPX2 from importin-a/b by Ran-GTP frees it to promote spindle assembly (Gruss et al., 2001) Although TPX2 was originally defined as a microtubule associated protein (MAP) (Wittmann et al., 1998) and its colocalization with the lamin B spindle matrix was dependent on the presence of microtubules (Tsai et al., 2006), the report that addition of dominant negative lamin constructs in the assay prevented association of any of the SAFs studied (Tsai et al., 2006) suggests TPX2 predominantly interacts with the microtubule spindle but its association may also be affected by a spindle matrix. 3.7.2. TACC (D-TACC and maskin) TACC (transforming acidic coiled coil) proteins were first identified in humans where genomic rearrangements involving the genes encoding these proteins were associated with different types of cancers (Chen et al., 2000; Lauffart et al., 2002; Pu et al., 2001; Still et al., 1999a,b). In addition to being highly acidic, the common feature of these proteins was a highly conserved 200-amino acid coiled-coil domain termed the TACC-domain, but the biological function(s) of these proteins was not known. Using a ‘‘reverse genetics’’ approach in Drosophila in which microtubule-binding proteins were isolated from microtubule affinity columns for subsequent molecular, cellular, and genetic characterization (Kellogg et al., 1989), a protein containing a TACC domain was identified and named D-TACC (Gergely et al., 2000b). Further analysis showed D-TACC is a 220 kDa centrosomal and mitotic spindle protein required for mitotic spindle function. A hypomorphic mutation in the d-tacc gene resulted in many embryos showing mitotic defects with many failing to develop beyond the first mitotic division (Gergely et al., 2000b). Although a D-TACC-GFP fusion protein primarily concentrated to the spindle poles, it also oscillated to and from the centrosomes consistent with an association with microtubule

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plus-ends (Lee et al., 2001). In this same study, Lee et al. (2001) found D-TACC associates with Mini-spindles (Msps), the Drosophila homolog of XMAP215/ch-TOG (Cullen et al., 1999). XMAP215 plays a critical role in the regulation of MT dynamics by modulating plus-end behavior, both by promoting microtubule assembly (Charrasse et al., 1998; Gard and Kirschner, 1987; Vasquez et al., 1994) and disassembly (Shirasu-Hiza et al., 2004), and its activity is essential for centrosome integrity, spindle pole organization, and bipolar spindle assembly (Cassimeris and Morabito, 2004; Gergely et al., 2003). Thus a model was proposed that centrosomal D-TACC was responsible for loading Msps (Drosophila XMAP215) onto the ends of growing microtubules to regulate the microtubule dynamics required for spindle assembly, in particular enhancing MT growth from the centrosome (Lee et al., 2001). D-TACC appears to comprise part of the pericentriolar matrix and maintains its centrosomal localization even after the microtubules have been depolymerized by colchicine (Gergely et al., 2000b), a finding that was also confirmed for the human TACC proteins (Gergely et al., 2000a). A single TACC homolog had also been previously identified in Xenopus oocytes where, due to its activity as an mRNA-binding protein that represses polyadenylation and translation of certain maternally provided stores of mRNA, it had been named Maskin (Stebbins-Boaz et al., 1999). Maskin had been found at the mitotic spindle where it was proposed to regulate localized cyclin B1 mRNA translation during the cell cycle (Groisman et al., 2000), but more recently, using the Xenopus spindle assembly assay system, Maskin has been shown by two independent groups to also play a direct role in mitotic spindle assembly (O’Brien et al., 2005; Peset et al., 2005). Depletion of maskin resulted in small asters, poorly organized spindles with reduced numbers of microtubules, and misaligned chromosomes (O’Brien et al., 2005; Peset et al., 2005). Maskin associates with XMAP215, and its activation and localization to the centrosome is regulated by phosphorylation by the Aurora A kinase (Kinoshita et al., 2005; O’Brien et al., 2005; Peset et al., 2005). Although Maskin lacks a conventional nuclear localization signal, it is still able to bind importin-b, maintaining it in an inactive state until the presence of RanGTP promotes its release (Albee et al., 2006), placing it in the family of Ran-regulated SAFs. 3.7.3. NuSAP Raemaekers et al. (2003) identified NuSAP (nucleolar spindle-associated protein) as a 55 kDa, basic protein found at increased levels in dividing cells that contains an N-terminal SAP domain, a helix-extension-helix motif that is implicated in organizing nuclear architecture by binding MARs (AT-rich nuclear Matrix Attachment Regions of the DNA) and/or RNA (Aravind and Koonin, 2000) and a C-terminal–charged helical domain. Immunolocalization revealed NuSAP is primarily nucleolar at interphase but

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relocalizes to the spindle at metaphase. An in vitro sedimentation assay showed purified, recombinant NuSAP is able to directly bind to microtubules via its C-terminal domain, suggesting NuSAP may play a role in regulating microtubule dynamics. Indeed, overexpression of NuSAP in COS cells resulted in the appearance of long, curved, highly bundled microtubules that were extremely stable even in the presence of nocodazole, whereas reduction of NuSAP delayed mitotic entry, resulting in defects in chromosome condensation, chromosome alignment, and spindle organization (Raemaekers et al., 2003). NuSAP is regulated by RanGTP in a complex manner because it must release the blocking of NuSAP’s microtubule-stabilizing activity mediated by importin-a and importin-7, as well as the blocking of NuSAP’s MT cross-linking activity mediated by importin-b (Ribbeck et al., 2006). The cross-linking activity of NuSAP was observed to be especially dramatic, resulting in the formation of large networks of bundled microtubules that included not only intact microtubules, but also other polymerization intermediates including protofilament sheets (Ribbeck et al., 2006). In biochemical reconstitution experiments, NuSAP efficiently adsorbs to chromatin or DNA, where it can efficiently promote microtubule formation and retention (Ribbeck et al., 2007). This ability to stabilize such a meshwork of microtubules around the chromosomes may serve a critical role in stabilizing the nascent spindle structure as it encounters forces and counterforces during assembly. It is not currently known whether, in addition to binding microtubules, NuSAP interacts with a spindle matrix. However, the presence of a SAP domain implicated in interacting with nuclear matrix attachment sites during interphase raises the tantalizing prospect that NuSAP may serve as a bridge between the spindle matrix and the microtubule spindle. 3.7.4. HURP HURP (hepatoma up-regulated protein) had previously been identified as a cancer-related marker for detecting transitional cell carcinoma (Chiu et al., 2002), but its cellular function was not known. Using proteomic, biochemical fractionation, and microarray expression approaches, three different labs independently identified HURP’s involvement in mitosis and further characterized it as playing an essential role in spindle organization, including mediating k-fiber stabilization and chromosome congression (Koffa et al., 2006; Sillje´ et al., 2006; Wong and Fang, 2006). HURP was found to be a direct cargo of importin-b released by high concentrations of Ran-GTP, whereupon it localizes to kinetochore MTs near the chromosomes. Western blot and mass spectroscopy analysis revealed that HURP migrated at several different sizes, including a high molecular weight form that appeared to represent a covalently linked dimeric or oligomeric species, raising the possibility of it forming a meshwork (Koffa et al., 2006). In this latter study, HURP was isolated as part of a complex that includes TPX2, Aurora A,

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XMAP215, and Eg5, four Ran-GTP–regulated SAFs that have been suggested to interact with the lamin B spindle matrix (Tsai et al., 2006). The immobility of Eg5 on bipolar spindles treated with a moderate dose of monastrol (an Eg5 inhibitor) relative to the vigorous continued flux of tubulin at this monastrol concentration has been interpreted to indicate a static spindle matrix (Kapoor and Mitchison, 2001). Thus, although HURP’s potential association with a spindle matrix has not yet been examined, it appears to show many characteristics consistent with such a role. The demonstration that HURP can polymerize free tubulin in vitro into a new configuration composed of antiparallel protofilaments wrapped around the growing end of a normal microtubule could suggest a novel mechanism for regulating MTs, although it is not yet known whether this activity is biologically relevant (Santarella et al., 2007). 3.7.5. Rae1 Rae1 is another direct cargo of importin-b released (activated) by high concentrations of Ran-GTP. A role for the Rae1 RNP complex in regulation of MT dynamics was uncovered by Blower et al. (2005) using in vitro assembly assays to identify factors necessary to promote Ran-GTP–induced aster formation. Rae1, which was previously known for its role as an mRNA export factor (Pritchard et al., 1999), was found to associate with spindle microtubules, with the highest levels at the poles, as well as with the aligned chromosomes. Rae1 apparently does not compose a MT-independent structure, as its spindle—but not chromosomal—localization was lost after nocodazole treatment (Blower et al., 2005). Rae1 is found in an RNP complex with at least 10 other polypeptides, and Rae1’s aster- and spindlepromoting activity requires the presence of other cofactors including RNA, because RNase treatment blocked assembly. Propidium iodide staining of in vitro-assembled spindles revealed the presence of RNA on and adjacent to the mitotic microtubules. Given that Rae1 contains four b-propeller WDrepeats, a domain proposed to serve as a scaffolding platform (Li and Roberts, 2001), Blower et al. (2005) speculate that Rae1 may function as a scaffold that tethers functionally important factors to the mitotic microtubule network.

4. Concluding Remarks As reviewed here, multiple studies spanning the evolutionary spectrum from lower eukaryotes to vertebrates have provided new and intriguing evidence that a spindle matrix may be a general feature of mitosis. Nonetheless, definitive evidence for its molecular nature and for its role in microtubule spindle function is still lacking. Considering the diversity of potential and unrelated spindle matrix molecules so far described, the

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possibility exists that different molecules may have attained the same function in different organisms. Alternatively, this could reflect the presence of multiple redundant systems or that different spindle matrices are operational at different stages of the cell cycle or in different cell types. Thus, the challenge remains to directly demonstrate a stationary or elastic molecular matrix independent of microtubule polymerization contributes to microtubule spindle function and/or assembly. However, with the identification of several potential spindle matrix molecules in experimentally tractable systems, such experiments may soon be forthcoming. Especially the characterization of a multiprotein spindle matrix complex in Drosophila where a wide range of genetic and biochemical approaches are available promises to provide an avenue to directly test the spindle matrix hypothesis and to give insight into its functional dynamics. For example, mutagenesis strategies can be applied to generate a range of mutant alleles that will allow a genetic dissection of the functional requirements of the spindle matrix proteins in different cellular contexts. Such alleles can also be used to explore the potential interaction of spindle matrix proteins with microtubule spindle assembly factors and microtubule-based motors. If spindle matrix proteins form a structural component essential for microtubule spindle function, the expectation is that microtubule spindle assembly and alignment will be impaired with abnormal congregation and segregation of chromosomes in dividing cells with spindle matrix protein mutations. Another interesting issue that can be addressed in live preparations in Drosophila by creating transgenic animals with fluorescently tagged spindle matrix proteins is whether nuclear proteins reorganize and begin to form a fusiform spindle structure prior to nuclear envelope breakdown and microtubule invasion of the nuclear space. Immunocytochemical studies of fixed preparations and analysis of cell division in east mutants carried out thus far suggest that the spindle matrix in Drosophila begins to form independent of microtubule assembly and additionally may play an important role in guiding chromosome congression. However, it is also possible that the microtubule spindle apparatus forms independently and the alignment with the spindle matrix complex is a secondary process. If that is the case, the spindle matrix may only serve to provide structural support. Although the studies described here support the spindle matrix hypothesis and indicate it plays an important role in microtubule assembly and function, an alternative hypothesis for spindle matrix function is that it serves as a means to distribute important nuclear components to the forming daughter nuclei. One major strategy to accomplish this goal is exemplified by structural components of the nucleus such as the nucleolus and the nuclear lamina that are completely dismantled and reassembled in the forming daughter nuclei only after chromosome segregation. Many of the proteins making up these structures are either degraded or are recycled through incorporation into vesicles (Moir et al., 2000; Olson et al., 2000). Other proteins known as ‘‘the

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chromosomal passengers’’ become associated with the condensing chromosomes during prophase, accumulate at the inner centromeres in prometaphase, and then at the onset of anaphase leave the chromosomes and transfer to the central spindle before concentrating at the midbody at cytokinesis (Vagnarelli and Earnshaw, 2004). The ‘‘chromosomal passenger protein complex’’ (Adams et al., 2001; Terada, 2001) has been functionally implicated in chromosome condensation and segregation as well as in completion of cytokinesis. The spindle matrix complex through interactions with the microtubule spindle may play a similar role in assuring equal distribution to the daughter nuclei of essential proteins that for structural reasons are difficult to degrade or resynthesize and reassemble on a rapid time scale. However, it should be noted that this hypothesis is not mutually exclusive with the ‘‘spindle matrix hypothesis’’ in which the spindle matrix proteins play an important role in chromosome congression and segregation as well as in microtubule spindle function. Thus, the further study of the cellular and molecular biology of the spindle matrix promises to provide important new information on the highly choreographed process for how chromosomes and/or nuclear proteins are segregated during mitosis.

ACKNOWLEDGMENTS We thank Drs. A. Forer, T. Spurck, J. D. Pickett-Heaps, and Dr. D. Sharp for generously providing micrographs. We also thank Dr. A. Forer, H. Maiato, and the members of our laboratory for critical reading of the manuscript and for helpful comments. The authors’ work on the spindle matrix is supported by NSF grant MCB0445182.

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C H A P T E R

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Mitochondrial Biology and Disease in Dictyostelium Christian Barth, Phuong Le, and Paul R. Fisher

Contents 1. Introduction 2. Mitochondrial Biology 2.1. Mitochondrial genetics 2.2. Protein import into mitochondria 2.3. Mitochondrial morphology and division 2.4. Mitochondria and programmed cell death in Dictyostelium 3. Mitochondrial Disease 3.1. Mitochondrial disease in humans 3.2. Genetic methods for creating mitochondrial disease in Dictyostelium 3.3. Mitochondrial disease phenotypes and associated signaling pathways 3.4. Phenotypic thresholds in mitochondrial disease in Dictyostelium 3.5. Phenotypes associated with mitochondrial defects not known to affect respiration 3.6. AMPK—the missing link between phenotype and genotype in mitochondrial disease 3.7. Implications 4. Concluding Remarks References

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Abstract The cellular slime mold Dictyostelium discoideum has become an increasingly useful model for the study of mitochondrial biology and disease. Dictyostelium is an amoebazoan, a sister clade to the animal and fungal lineages. The mitochondrial biology of Dictyostelium exhibits some features which are unique, others which are common to all eukaryotes, and still others that are otherwise found only in the plant or the animal lineages. The AT-rich mitochondrial

Department of Microbiology, La Trobe University, Melbourne VIC 3086, Australia International Review of Cytology, Volume 263 ISSN 0074-7696, DOI: 10.1016/S0074-7696(07)63005-8

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genome of Dictyostelium is larger than its mammalian counterpart and contains 56 kb (compared to 17 kb in mammals) encoding tRNAs, rRNAs, and 33 polypeptides (compared to 13 in mammals). It produces a single primary transcript that is cotranscriptionally processed into multiple monocistronic, dicistronic, and tricistronic mRNAs, tRNAs, and rRNAs. The mitochondrial fission mechanism employed by Dictyostelium involves both the extramitochondrial dynaminbased system used by plant, animal, and fungal mitochondria and the ancient FtsZ-based intramitochondrial fission process inherited from the bacterial ancestor. The mitochondrial protein-import apparatus is homologous to that of other eukaryote, and mitochondria in Dictyostelium play an important role in the programmed cell death pathways. Mitochondrial disease in Dictyostelium has been created both by targeted gene disruptions and by antisense RNA and RNAi inhibition of expression of essential nucleus-encoded mitochondrial proteins. This has revealed a regular pattern of aberrant mitochondrial disease phenotypes caused not by ATP insufficiency per se, but by chronic activation of the universal eukaryotic energy-sensing protein kinase AMPK. This novel insight into the cytopathological mechanisms of mitochondrial dysfunction suggests new possibilities for therapeutic intervention in mitochondrial and neurodegenerative diseases. Key Words: Dictyostelium, Mitochondrial disease, Mitochondrial biogenesis, AMPK. ß 2007 Elsevier Inc.

1. Introduction The cellular slime mold Dictyostelium discoideum has long been regarded as a valuable and attractive tool for the study of eukaryotic cell biology. The organism combines typical eukaryotic cellular and molecular biology with the experimental tractability of a microorganism in which biochemical, classical, and molecular genetic as well as cell biological approaches are readily adopted. Its developmental life cycle is unique among protists, and at the different stages of development Dictyostelium features both plant- and animal-like characteristics. Completion of the Dictyostelium genome sequencing project has further enhanced the organism’s utility in many aspects (Chisholm et al., 2006; Eichinger et al., 2005; http://www. dictybase.org). Dictyostelium discoideum is one of eight nonmammalian model organisms recognized by the National Institute of Health (NIH) in the United States for their utility in the study of fundamental molecular processes of medical importance (http://www.nih.gov/science/models/). Other developments have also made Dictyostelium an extremely attractive model organism in which to study mitochondrial biogenesis and disease. Not only is

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mitochondrial disease readily created in Dictyostelium, but also its mitochondrial genome has been completely sequenced, and mitochondrial transcription and ribonucleic acid (RNA) processing have been studied in detail. Furthermore, this organism, with its motile unicellular and multicellular stages and multiple cell types, offers a great variety of potentially relevant and readily assayed phenotypes—pinocytosis, phagocytosis, mitosis, extracellular signaling, amoeboid motility, chemotaxis, phototaxis, thermotaxis, differentiation, morphogenetic movement in a multicellular tissue, and so forth. Other mitochondrial disease models are limited either by the range of potential phenotypes that can be analyzed or by the variety and ease of creation of mitochondrial genetic disorders.

2. Mitochondrial Biology 2.1. Mitochondrial genetics The mitochondrial genomes of most organisms exist as circular deoxyribonucleic acid (DNA) molecules, which vary dramatically in size. The largest genomes are found in plants, range from about 200 kb to over 2000 kb and are far more complex than their metazoan and protistan counterparts. The transfer of essential genes from the mitochondrial genome to the eukaryotic nucleus during evolution and numerous rearrangements in the mitochondrial DNA created an extraordinary diversity of the mitochondrial genome among the different species. Despite the variability, a common basic set of genes is present in all mitochondrial genomes sequenced so far. This universal gene set encodes proteins involved in respiration, as well as ribosomal RNAs and transfer RNAs (Schuster and Brennicke, 1994; Wolstenholme, 1992). Mitochondrial genomes from more than 540 eukaryotic species are available in public databases. Most of these are, however, from animals, fungi, and plants. Only a small fraction of the sequence data available (11%) represents those of protists, with the Dictyostelium mitochondrial genome being the second amoebozoan mitochondrial genome to have been sequenced completely. 2.1.1. The Dictyostelium mitochondrial genome: Gene content and organization A high A þ T content and universal codon usage Ogawa et al. presented an overview of the gene content and the organization of the Dictyostelium mitochondrial genome in 2000. The mitochondrial DNA (mtDNA) is a circular molecule and 55,564 base pairs in size. All genes and open reading frames (ORFs) are transcribed in the same orientation (Fig. 5.1). The A þ T content of 72.6% is very high compared to that of the nuclear DNA, which is a characteristic and distinctive feature of mitochondrial DNA in general

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Figure 5.1 Organization, transcription, and transcript processing of the Dictyostelium mitochondrial genome. The individual genes encoded by the Dictyostelium mitochondrial genome as well as intronic and noncoding sequences are indicated. The 55.6 kb genome appears to be transcribed from a single start site (Transcription start) into one large, primary transcript that is rapidly and cotranscriptionally processed into smaller, mature transcripts. Only the secondary transcripts A (3.1 kb), B (4.6 kb), C (5.6 kb), D (9.5), E (6.0 kb), F (6.5 kb), G (3.7 kb), H (8.7 kb), and their smaller derivatives (tertiary transcripts) have been detected in Northern Hybridization studies (Barth et al., 2001).

(Lang et al., 1999). It is comparable to that of mtDNA of the amoeboid protozoon Acanthamoeba castellanii (70.6%; Burger et al., 1995), and is also close to that of fungi (70–82%), Drosophila (79%), and nematodes (76%), but is substantially higher than that of vertebrate mtDNA (54–64%, Wolstenholme, 1992). Due to the high A þ T content, it was initially suspected that some codons of the Dictyostelium mtDNA may be nonuniversal, in line with the fact that deviations from the standard genetic code are evident in the mitochondrial translation system of many eukaryotic groups. Based on the analysis of several protein-encoding genes on a 7.5-kb mtDNA fragment, however, Angata et al. (1995a) concluded that the universal genetic code is used in Dictyostelium for the translation of mitochondrial messenger RNAs. Gene content The Dictyostelium mitochondrial genome codes for 33 proteins and also contains six ORFs, two ribosomal RNA genes, and 18 transfer RNA genes. Most genes are tightly packed and some even overlap

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Table 5.1

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Dictyostelium mitochondrial genes and their function

Respiration NADH dehydrogenase ATP synthase Cytochrome c oxidase Cytochrome bc1-complex Translation Ribosomal RNAs Transfer RNAs Ribosomal proteins

nad1,2,3,4,4L,5,6,7,9,11 atp1,6,8,9 cox1/2,3 cob rns, rnl trnaA,C,E,F,H,I1,I2,I3,K,L1,L2, M,N,P,Q,R,W,Y rps2,4,7,8,11,12,13,14,19, rpl2,5,6,11,14,16

(see following); however, intergenic spacers ranging in size from a few nucleotides to more than 2 kb also exist (see Fig. 5.1). All genes are involved in two major biological processes that take place in mitochondria, namely respiration and translation (Table 5.1). In addition to the standard set of genes, the Dictyostelium mtDNA also contains the genes of some NADH dehydrogenase and ATP synthase subunits (such as nad7, nad9, nad11, and atp1), which are nuclear-encoded in many other organisms and are posttranscriptionally imported into the mitochondria of these organisms (Cole and Williams, 1994; Cole et al., 1995; Iwamoto et al., 1995). The six ORFs of unknown function found in the Dictyostelium mitochondrial genome are not present in other organisms and are presumably not involved in essential, universal functions. However, some of the ORFs in the Dictyostelium mitochondrial genome are found in similar positions in the mitochondrial genome of Polysphondylium pallidum, suggesting that these ORFs represent real genes, although the sequences show only little similarity in pairwise alignments (Gray et al., 2004). Cytochrome oxidase subunit 1 and 2 genes are fused and contain group-I introns Some other notable features of the Dictyostelium mtDNA include the fusion of the adjacent cytochrome oxidase subunit 1 and 2 genes (cox1/2; see Fig. 5.1), which form a single ORF with no apparent stop codon present at the 30 end of cox1 (Ogawa et al., 1997; Pellizzari et al., 1997). The two genes, however, are expressed as two individual proteins that migrate separately on SDS-PAGE (Bisson et al., 1985). In addition to its peculiar gene organization, which is shared by A. castellanii (Lonergan and Gray, 1996), the Dictyostelium cox1/2 gene is interrupted by four introns, that possess the potential to form conserved secondary RNA structures characteristic of group-I introns. For one of the group-I introns, the self-splicing activity was demonstrated in an in vitro self-splicing assay (Ogawa et al.,1997).

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Three out of the four introns also contain freestanding ORFs. Two of the ORFs code for amino acid sequences that show high homology to the aI4 DNA endonuclease (I-SceII) encoded by a mitochondrial group-I intron of Saccharomyces (S.) cerevisiae. When expressed in Escherichia coli, both the Dictyostelium and the S. cerevisiae proteins lead to degradation of the E. coli chromosome and inhibition of growth, indicating that both proteins belong to the same type of homing endonucleases (Delahodde et al., 1989; Ogawa et al., 1997; Sargueil et al., 1990). Prior to the characterization of the cox1/2 gene, another group-I intron has been reported in the rnl gene coding for the large subunit ribosomal RNA (Angata et al., 1995b). Interestingly, this intron is inserted at the same site as the introns found in the mitochondrial rnl gene from the green alga Scenedesmus obliquus (Ku¨ck et al., 1990) and from the colorless alga Prototheca wickerhamii (Wolff et al., 1993). Mitochondrial ribosomes lack a 5S rRNA Dictyostelium mitochondria seem to lack a conventional 5S-type ribosomal RNA that otherwise forms part of all bacterial ribosomes and the cytoplasmic ribosomes of eukaryotes. This is also the case in animal and fungal mitochondria, where the genome does not encode a 5S rRNA, and where it has been demonstrated that a 5S rRNA is absent from ribosomes (Attardi and Ojala, 1971; Gillham, 1994). In contrast, plant mitochondrial genomes have been shown to encode a 5S rRNA, and its presence in mitochondrial ribosomes has been demonstrated (Gray, 1992). A 5S rRNA has also been discovered and characterized in A. castellanii (Bullerwell et al., 2003). For this reason it is noteworthy that Pi et al. (1998) have isolated a novel, small mitochondrial RNA species in Dictyostelium that may be derived from the 5S rRNA in plant mitochondria. Although its primary sequence displays only little homology with plant mitochondrial 5S rRNA, it contains some highly conserved sequences and its proposed secondary structure was demonstrated to be similar to that of conventional 5S rRNAs (Erdmann and Wolters, 1986; Forget and Weissman, 1967). However, the Dictyostelium RNA does not appear to form part of the mitochondrial ribosomes (Pi et al., 1998), nor does it have the characteristic features of a 5S rRNA (Bullerwell et al., 2003). Ribosomal protein genes are arranged in clusters In contrast to animals, in which all mitochondrial ribosomal proteins are encoded by nuclear DNA (Bonen, 1991), a number of the Dictyostelium mitochondrial ribosomal proteins are mitochondrially encoded. Dictyostelium shares this feature with plants and many lower eukaryotes, including protists (Leblanc et al., 1995; Pritchard et al., 1990; Wolff et al., 1993). The number of mitochondrially encoded ribosomal protein genes varies greatly among these species, from only one in S. cerevisiae and other fungi (Burke and Rajbhandary, 1982; Cummings et al., 1989; Zamaroczy and Bernardi, 1986) to 17 in the oomycete Phytophthora infestans (Lang and Forget, 1993).

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Although the single ribosomal protein genes are frequently found within intronic sequences of large ribosomal subunit RNA genes (Burke and Raibhandary, 1982; Cummings et al., 1989), multiple genes are usually arranged in gene clusters. The Dictyostelium mitochondrial DNA contains two such clusters of ribosomal protein genes (see Fig. 5.1). Both clusters are arranged around a set of large ORFs, ORF425 and ORF1740. The first cluster, located upstream of the ORFs, consists of six tightly packed genes, whereas the second cluster lies downstream of the set of ORFs and contains eight ribosomal protein genes. Parts of the ORF sequences show similarities to the S3 ribosomal protein gene sequence of other organisms but are otherwise too big to code for a common ribosomal protein. In addition to the tight gene clustering with intergenic regions ranging in length from 0 to 11 base pairs, the coding regions of 10 pairs of genes even overlap by 1 to 29 base pairs (Iwamoto et al., 1998). The gene arrangement within the clusters is similar to that found in E. coli (Cerretti et al., 1983; Zurawski and Zurawski, 1985) and Marchantia polymorpha (Oda et al., 1992) and is almost identical to that of A. castellanii (Burger et al., 1995). It is noteworthy that the ribosomal protein gene rpl11, which has not been found in the mtDNA of any other organism, is present in the Dictyostelium and A. castellanii mitochondrial genomes (Iwamoto et al., 1995). In addition, the ribosomal proteins rps4 and rps2 are encoded by separate genes that do not form part of the gene clusters in the Dictyostelium mitochondrial DNA (see Fig. 5.1). 2.1.2. Mitochondrial transcription and transcript processing The Dictyostelium mitochondrial genome is transcribed by a bacteriophage-like RNA polymerase Despite the variability in their gene content, arrangement, and patterns of transcription, the mitochondria of different organisms employ a similar strategy for transcribing the genetic information they contain. Surprisingly, in view of the eubacterial origin of mitochondria, the mitochondrial genome in fungi, plants, and animals is transcribed by nuclear-encoded RNA polymerases that share no sequence similarities with the bacterial RNA polymerase (Cermakian et al., 1997). Instead, the mitochondrial RNA polymerases show relatively high sequence similarities to the T3 and T7 bacteriophage RNA polymerases. In contrast to the bacterial multisubunit (a2bb0 ) core RNA polymerase, the bacteriophage-like RNA polymerases are simple, single-subunit proteins, which may have replaced the original, bacterial-like enzyme at an early stage in the evolution of the mitochondrial transcription machinery. The mitochondrial RNA polymerases identified so far are structurally and functionally conserved, suggesting they are the key proteins of mitochondrial transcription. Based on the widespread sequence similarity among mitochondrial RNA polymerases, the nuclear gene for the Dictyostelium mitochondrial RNA polymerase has been identified (Accession No. AAK73754) and cloned (Le and Barth, unpublished). Sequence alignments of the predicted

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protein sequence with sequences of known mitochondrial RNA polymerases from other species revealed extensive homology to mitochondrial RNA polymerases of plants and to some extent to fungal mitochondrial RNA polymerases, with complete conservation of all catalytically essential amino acids. In phylogenetic trees constructed to compare the evolutionary relationships between the different RNA polymerases, the Dictyostelium mitochondrial RNA polymerase formed a subgroup together with plant mitochondrial RNA polymerases. As expected for the enzyme-mediating transcription of the mitochondrial genome, the N-terminus of the Dictyostelium RNA polymerase displays the typical features of mitochondrial targeting sequences (Le and Barth, unpublished). Transcription from a single promoter generates polycistronic RNA molecules Northern hybridization analyses using gene-specific probes directed against all genes present in the Dictyostelium mitochondrial genome revealed the presence of eight large, polycistronic transcripts (see Fig. 5.1). These polycistronic transcripts are further processed to form smaller, monocistronic, dicistronic, or tricistronic mature RNA molecules (Barth et al., 1999). As larger transcripts were not detected in any of the hybridization studies, it was concluded at the time that the Dictyostelium mitochondrial genome is transcribed into eight major transcripts. The 50 ends of these transcripts were mapped by primer extension analysis, and the sequences upstream of the putative transcription initiation sites were aligned in an attempt to identify consensus promoter sequences (Barth et al., 2001). This approach, however, was limited by the fact that some of the 50 ends determined by primer extension analysis may have represented sites of RNA processing rather than transcription initiation. Subsequent investigations carried out to distinguish between genuine transcription initiation sites and 50 ends that have been generated by RNA processing demonstrated that the eight polycistronic transcripts observed previously were derived from processing of a single primary RNA transcript (Le et al., unpublished). These findings locate the start of transcription to a single initiation site in a noncoding region upstream of the rnl gene encoding the large ribosomal subunit RNA (see Fig. 5.1). The processing of the large primary transcript into smaller, mature RNA molecules is of particular interest because it appears to involve mechanisms similar to the ones responsible for RNA transcript processing in human mitochondria where the long, bidirectional transcripts are punctuated by tRNAs (Anderson et al., 1981; Ojala et al., 1981). RNA editing of the small subunit ribosomal RNA Apart from additional processing events such as the removal of group-I intronic sequences from the large ribosomal subunit RNA gene (rnl) and from the cox1 and cox2 genes as mentioned previously, at least one mitochondrial transcript is also subjected to RNA editing. A comparison of the cDNA sequence generated

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from RNA transcribed from the gene coding for the small ribosomal subunit RNA (rns) with the mitochondrial DNA sequence revealed C-toU substitutional editing of the rns transcript at a single site (Barth et al., 1999). The editing of structural RNA molecules is still regarded as uncommon because most of the documented editing events involve mRNAs, in which the editing has created initiation codons or eliminated reading frame shifts. The editing of the Dictyostelium rns transcript was in fact only the third example of editing of a structural RNA reported at the time (Gott and Emeson, 2000). Whether the editing of the rns rRNA is of any importance for RNA function remains unclear. However, because the editing was observed to consistently occur in the whole population of RNA molecules, a certain necessity for the sequence alteration can be assumed. The edited site has been shown to correspond to a nucleotide in the E. coli 16S rRNA located in a highly conserved loop essential for ribosomal tRNA selection and proof reading (Powers and Noller, 1994). Based on this, Barth et al. (1999) postulated a similarly important role for the editing of the mitochondrial rns transcript in Dictyostelium (Fig. 5.2). In addition to the rRNA transcript, the mRNAs of some Dictyostelium mitochondrial genes have also been examined for the occurrence of RNA editing, including the cob gene (Angata et al., 1995) and the genes for subunit I and II of the cytochrome c oxidase (Pellizzari et al., 1997). However, no evidence for editing was found in either of these mRNAs. Apart from this, based on secondary structure modeling, some Dictyostelium mitochondrial encoded tRNAs have been predicted to undergo the same type of 50 editing documented for the mtDNA-encoded tRNAs in A. castellanii (Lonergan and Gray, 1993; Price and Gray, 1999).

2.2. Protein import into mitochondria Because mitochondrial genomes encode only a relatively small number of proteins, most of the proteins present and functioning in the mitochondria are nuclear-encoded and need to be imported after they have been synthesized on cytoplasmic ribosomes. Protein import into mitochondria and subsequent assembly are therefore fundamental mechanisms of mitochondrial biogenesis, and they have been studied for many years in a variety of organisms. Mitochondria consist of four subcompartments: the outer and inner membrane, the intermembrane space, and the matrix (Taylor and Pfanner, 2004; Neupert, 1997). Many components of the machinery for protein import, sorting, and assembly of the imported proteins into these mitochondrial subcompartments have been identified and characterized. The outer membrane harbors the translocase of the outer mitochondrial membrane (TOM complex), which recognizes and translocates preproteins, and the SAM complex, which mediates sorting and protein assembly. In the inner membrane two translocase complexes can be found. The first one,

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459

459

GC C G

GC C G

C

C C G A C

G G U A

C G A C

A

C G U U-A G-C C-G U-G U-A U-G

A A U C 441 GG C-G C-G

C

U

G G U A A

C

C-to-U

G U U-A G-C C-G U-G U-A U-G A A U C 441 GG C-G C-G

Figure 5.2 Editing of the Dictyostelium mitochondrial SSU rRNA. Based on the structure of the 530 loop in the E. coli 16S rRNA in the region of the editing site, presumptive secondary structures are shown for the unedited (left side) and edited (right side) transcripts. Linked shaded boxes show possible higher order base pairing interactions that in the edited molecule allow pseudoknot formation as in the E. coli 16S rRNA. For the unedited molecule, an alternative base pairing arrangement is shown that could compete with and perturb correct pseudoknot formation, thereby interfering with translation (taken from Barth et al.,1999).

TIM23, is predominantly responsible for the import of preproteins into the matrix, whereas the second one, TIM22, is required for the sorting and assembly of those proteins destined for the inner membrane (Gordon et al., 2000). Although most of our knowledge about mitochondrial protein import is based on studies in S. cerevisiae and Neurospora crassa, it is assumed that similar mechanisms function in the mitochondria of other species. Homologs of a number of components of the import machinery are encoded in the genomes of vertebrate and invertebrate animals and in the higher plants Arabidopsis (A.) thaliana and Solanum tuberosum (Hoogenraad et al., 2002; Ja¨nsch et al., 1998; Werhahn et al., 2001). In an attempt to shed light on the evolution of the mitochondrial protein import machinery, Macasev et al. (2004) performed an extensive comparative sequence analysis of eukaryotic genome sequences. Three subunits, Tom40, Tom7, and Tom22, have been identified as common elements of the protein translocase in the mitochondrial outer membrane, which suggests the subunits to be the core of the protein translocase in the earliest mitochondria. During the course of their investigations, the orthologs of these subunits have also been identified in Dictyostelium (Macasev et al., 2004). Tom40 is believed to form a membrane-embedded, b-barrel–shaped translocation pore through

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which all protein import into the mitochondria occurs (Pfanner and Chacinska, 2002). The roles of Tom7 and Tom22 are not as clear; however, their direct and tight association with Tom40 has been demonstrated in yeast (Meisinger et al., 2001). When studying protein import into the mitochondria of Dictyostelium, Ahmed et al. (2006) made the interesting observation that GFP-fusion proteins targeted to mitochondria were present in the cell at much lower levels than nontargeted fusion proteins (Fig. 5.3). These differences were shown to be the result of significantly slower translation rates of the targeted fusion proteins, suggesting import of at least some mitochondrial proteins occurs cotranslationally (see Fig. 5.3). In support of this, the mRNA of the cotranslationally imported proteins has been detected in association with mitochondria isolated from Dictyostelium cells (Ahmed et al., 2006). A cotranslational import of some mitochondrial proteins has been suggested before based on several studies conducted in yeast. In one of these studies, ribosomes synthesizing mitochondrial proteins have been found to be attached to the outer membrane of isolated mitochondria (Kellems et al., 1975). The association of both mRNA and ribosomes with isolated mitochondria clearly supports the view that, apart from posttranslational protein import, a cotranslational protein import machinery must also exist. Whether a mitochondrial protein is cotranslationally or posttranslationally imported seems to depend on whether its mRNA is transported to the mitochondrial surface, and potential cis-acting signals mediating the translocation have been identified on some mRNA molecules ( Jansen, 2001; Sylvestre et al., 2003).

2.3. Mitochondrial morphology and division 2.3.1. Mitochondrial morphology Traditionally, mitochondria are described as spherical organelles floating freely in the cytoplasm. Only in recent years has it become clear that most mitochondria have far more complex morphologies. The organelles undergo continuous cycles of fission and fusion, and these processes control not only the number of the organelles in the cell, but also their size and distribution (Yaffe, 1999). The distribution of mitochondria is also determined by their movement along and attachment to cellular structures such as the actin and microtubule cytoskeletons. Although mitochondria are typically localized to microtubules in mammalian cells and the fission yeast Saccharomyces pombe, mitochondria of the budding yeast S. cerevisiae and of plants rely on the actin cytoskeleton for their movement. Proteins mediating organellar transport and interaction with the cytoskeleton, such as actin- or microtubule-based motor proteins, have been identified and characterized (Heggeness et al., 1978; Sogo and Yaffe, 1994; Svoboda and Slaninova, 1997; Van Gestel et al., 2002).

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Exposure time (s)

15

30

C 45

GFP

Over exposed

Cpn23.GFP

Over exposed

Cpn40.GFP Cpn150.GFP Top82.GFP

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Cpn23GFP

Cpn40GFP

Cpn150GFP

Total GFP Protein

0

1600

1

2

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Time (h)

1400

D

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Cpn23.GFP Cpn40.GFP Cpn150.GFP

1000

Radio of GFP label to total protien label

GFP fluorescence (Rfu per million cells)

B

Radioactivity incorporated ( %)

1

800 600 400 200 0 23 .G FP

AX

2

P

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F 15

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Mitochondrially Cytoplasmic GFP targeted GFP

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Figure 5.3 Translation of mitochondrially targeted GFP is inhibited. (A) Mitochondrially targeted GFP fluorescence is much lower compared to cytoplasmic GFP. Microscopic images of GFP fluorescence were taken at variable exposure times (s), as a measure of GFP activity, from a single Dictyostelium cell representative of the entire population of transformant cells. Dictyostelium transformants expressing either cytoplasmic or mitochondrially targeted GFP were grown to log phase in axenic medium and were fixed on coverslips. Fixed cells were observed under fluorescence microscope and the images of GFP fluorescence were taken at variable exposure times (1, 15, 30, and 45 s). Cells expressing cytoplasmic GFP (GFP or Cpn23.GFP) were overexposed at 45 s (images not shown) due to much stronger GFP fluorescence compared to those expressing mitochondrially targeted GFP (Cpn40.GFP, Cpn150.GFP, and TopA82.GFP). (B) GFP fluorescence on transformant cell populations expressing either nontargeted or targeted GFP fusion proteins. AX2 cells were used as a negative control. Axenically grown Dictyostelium cells were incubated with Lo-Flo HL5 for 2 h, and then GFP fluorescence was measured in a fluorometer. (C) The rate of GFP synthesis is higher for cytosolic than for mitochondrially targeted GFP. Cells were labeled with [35S] methionine for 3 h, and at hourly intervals the incorporated radioactivity was detected in both the whole cell extract and the immunoprecipitated GFP. Radioactive signals were subjected to densitometric analysis using a Storm phosphorimaging system (Amersham Biosciences). In each experiment the intensity of the radioactive GFP signal was normalized as a percentage of the maximum signal obtained (the Cpn23GFP signal at 3 h). The chart includes data from three independent experiments. Error bars represent standard errors of the mean.The inset shows the images of the radioactive signals from both

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In Dictyostelium, mitochondria are commonly found dispersed throughout the cell where they appear as small dark bodies in the cytoplasm when visualized by phase-contrast bright-field microscopy (Gilson et al., 2003). Three classes of shapes can be detected in wild-type cells: spheres, rods, and tubules. Although all three classes can sometimes be found in one and the same cell, the majority of cells contains spherical or rod-shaped mitochondria. It is also noteworthy that, unlike the mitochondria of the budding yeast for example (Nunnari et al., 1997), the Dictyostelium mitochondrial tubules do not seem to be branched (Gilson et al., 2003). The even cellular distribution of mitochondria is disturbed when the gene coding for CluA, a novel protein first identified in Dictyostelium, is disrupted (Zhu et al., 1997). In cluA cells, all of the mitochondria are found in a single cluster near the cell center. The CluA protein does not seem to have motor function itself because it lacks any homology to actin- or microtubule-based motor proteins but may form a link between motor molecules and the mitochondria (Zhu et al., 1997). A functional homolog of the Dictyostelium protein has been characterized in S. cerevisiae (CLU1), where the disruption of CLU1 lead to clustering of the mitochondria similar to the one observed in Dictyostelium cells (Fields et al., 1998), and CluA-like sequences have subsequently been identified in the genomes of many other eukaryotic organisms (Fields et al., 2002; Sawano et al., 2005; Zhu et al., 1997). Apart from being essential for maintaining the normal distribution pattern of mitochondria in the cell, data suggest the Dictyostelium CluA protein to be involved in mitochondrial membrane dynamics. When comparing the ultrastructure of mitochondria in wild-type cells and cluA cells, Fields et al. (2002) found the mutant mitochondria connected to neighboring mitochondria through narrow constrictions. Based on these observations, the authors suggested that in cluA cells mitochondrial division may have been blocked in the final stages (Fields et al., 2002). A phenotype similar to the one previously described has also been observed in Dictyostelium cells lacking dynamin A, a member of the dynamin family, which forms part of the GTP-binding protein superfamily (Wienke et al., 1999). Dynamins have a well-established function in clathrin-mediated endocytosis (Damke et al., 1994; Kosaka and Ikeda, 1983) and play a

the whole cell extract and the immunoprecipitated GFP from one representative experiment. (D) Incorporation of radioactivity into immunoprecipitated GFP relative to the incorporation into total cellular protein. The inset shows the densitometric values for the total protein radioactive signal at hourly intervals. Negative controls using wildtype (AX2) cells (not expressing any GFP isoforms) showed similar levels of incorporation of radioactivity into total protein, but no labeled GFP signal in immunoprecipitates (not shown). (Panels A and B are from Fig. 2 of Ahmed et al. [2006]. Panels C and D are from Fig.4 of Ahmed et al. [2006].)

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potential role in the organization of the cytoskeleton and the regulation and maintenance of cell shape (Urrutia et al., 1997). Dictyostelium cells lacking a functional copy of dymA show, among other phenotypes, alterations in the morphology of the different organelles in the cell, including mitochondria. When visualized by indirect immunofluorescence of fixed dymA cells and by phase contrast microscopy of living dymA cells, the mitochondria appeared in large clusters forming continuous reticular structures (Wienke et al., 1999). Although these alterations may be due to more general defects in membrane transport processes, the formation of the reticular structures also points to a direct role of dynamin A in mitochondrial division, which will be discussed later. 2.3.2. Dictyostelium mitochondria contain orthologs of the prokaryotic cell division protein FtsZ Mitochondria cannot be made de novo and, to maintain their numbers, must divide by fission very much like their bacterial ancestors. It is therefore not surprising that some of the proteins involved in bacterial cytokinesis may also act in the division of mitochondria (and chloroplasts). One of the most widespread and important bacterial division proteins is the cell division protein FtsZ (Addinall and Holland, 2002; Erickson, 2000; Errington et al., 2003; Margolin, 2000). The protein seems to play a key role in the division process: it has the ability to self-assemble into a ring structure on the inner face of the cytoplasmic membrane at the division site, thereby recruiting other proteins of the division machinery (Bramhill, 1997; Ma et al., 1996). FtsZ is a GTPase (Nolgales et al., 1998), and based on its functional and structural homology to the eukaryotic cytoskeletal protein tubulin, it has been proposed to be its prokaryotic ancestor (Erickson, 2000; Margolin et al., 1996). The discovery of the first eukaryotic FtsZ in the chloroplasts of A. thaliana (Osteryoung and Vierling, 1995) led to the presumption that, apart from chloroplasts, mitochondria may also use FtsZ for division. However, with the availability of more completed eukaryotic genome sequences, it became evident that most eukaryotic genomes do not encode a mitochondrial FtsZ. Rather, in yeast (S. cerevisiae), animals (Caenorhabditis [C.] elegans, Drosophila melanogaster, Homo sapiens), and plants (A. thaliana), members of the dynamin family of GTPases have been shown to be responsible for the division of the outer mitochondrial membrane (Arimura and Tsutsumi, 2002; Bleazard et al., 1999; Labrousse et al., 1999; Smirnova et al., 2001). The dynaminrelated proteins (DRPs) are likely to have been recruited early in the evolution of mitochondria, which would explain their widespread phylogenetic distribution (Miyagishima et al., 2003). However, apart from DRPs mediating mitochondrial division in perhaps all eukaryotes, certain groups of protists still contain relics from the mitochondrial ancestor in the form of mitochondrial FtsZs. The first mitochondrial FtsZ was identified in the

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chromophyte alga Mallomonas splendens (Beech et al., 2000), and in 2003, two orthologs of the prokaryotic cell division protein were identified in Dictyostelium (Gilson et al., 2003). The Dictyostelium mitochondrial FtsZ proteins, FszA and FszB, are both nuclear-encoded and are imported into the mitochondrial matrix by means of N-terminal targeting peptides. Disruption of the genes of the two proteins led to tubular elongation of the mitochondria, which are predominantly spherical or rod-shaped in wild-type cells (Gilson et al., 2003). This observation clearly indicated a decreased ability of the mutant cells to divide their organelles, and it provided the first genetic demonstration of the roles of FtsZ homologs in mitochondrial division. However, the exact function of the two proteins in the division process remains to be determined. FszA was localized to an incomplete, equatorial ring consistent with a direct role in mitochondrial division, whereas FszB was found in an electron-dense, submitochondrial body of unknown origin and function (Gilson et al., 2003). With that, the discovery of the two orthologs in Dictyostelium represented the first demonstration of two differentially localized FtsZs within the one organelle. Through the analyses of the fszA /fszB double mutants, it also became clear Dictyostelium must obviously employ other, probably dynamin-like division proteins that retain sufficient fission activity even in the absence of FszA and FszB. Dictyostelium encodes two dynamin-like proteins, DymA and DymB (Gilson et al., 2003; Wienke et al., 1999), with DymA being highly similar to the dynamin-like proteins Dnm1p in S. cerevisiae, DRP1 in C. elegans and mammals, as well as ADL2b in A. thaliana. Dnmp1 and its orthologs have been shown to form rings on the outside of mitochondria at the site of division and are regarded as key proteins in mitochondrial fission (Arimura and Tsutsumi, 2002; Bleazard et al., 1999; Labrousse et al., 1999; Smirnova et al., 2001). In Dictyostelium dymA mutants, mitochondrial division seemed to be more impaired than in the fszA /fszB mutants because the mitochondria were also clustered into interconnected sets of tubules (Wienke et al., 1999). Although many of the proteins involved in mitochondrial division have been discovered and characterized, the actual mechanisms controlling the process of fission and fusion remain vague. However, evidence exists that the same machinery mediating the formation of the mitochondrial network also participates in the process of apoptosis.

2.4. Mitochondria and programmed cell death in Dictyostelium That mitochondria have a direct role in the control of apoptosis has long been known and is well documented (Mohamad et al., 2005; Youle and Karbowski, 2005). Apoptosis is the best-understood form of programmed cell death, a physiological process of cell suicide, which is important for the

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development and homeostasis of multicellular organisms. The process is genetically controlled and is initiated by a wide range of stimuli and signaling pathways (Desagher and Martinou, 2000; Vaux and Korsmeyer, 1999). It is the permeabilization of the outer mitochondrial membrane by the Bcl-2 family members BAX and BAK (Kluck et al., 1999) and the subsequent loss of mitochondrial membrane potential that form a key element in the cascade of events. This leads to the release of cytochrome c from the intermembrane space into the cytosol, where it binds to the apoptotic protease-activating factor 1 (Apaf-1) (Li et al., 1997). Both form a multimeric complex, the apoptosome, which takes part in the activation of caspases, the major effectors of apoptosis (Zou et al., 1999). The caspases rapidly cleave intracellular substrates leading to the typical morphological changes observed during apoptosis, such as cell shrinking, plasma membrane blebbing, chromatin condensation, and the formation of apoptotic bodies (Hengartner, 2000). In addition to the cell death pathway that leads to the activation of caspases, the permeabilization of the outer mitochondrial membrane can also cause the release of caspase-independent factors from the mitochondria such as apoptosis-inducing factor (AIF), which can contribute to an apoptosis-like cell death. Much of our knowledge about mitochondrial participation in apoptosis is based on studies in mammalian systems, whereas mitochondrial activation of the cell death pathways in nonmammalian organisms remains poorly understood. Thus far, these processes have been described in only a few species of unicellular eukaryotes, including Dictyostelium (Cornillon et al., 1994), Trypanosoma cruzi (Ameisen et al., 1995), Tetrahymena thermophilia (Christensen et al., 1995), and others. In these organisms, similar phenotypes to those observed in multicellular organisms have been detected (Ameisen, 1996; Cornillon et al., 1994). However, it was not clear at the time whether these phenotypes were the result of a similar cascade of events. Programmed cell death in Dictyostelium occurs during the normal developmental cycle, leading to the formation of stalk cells, which, together with the spores, form the fruiting bodies. However, this developmentally programmed cell death is of a vacuolar autophagic type, does not require the activation of any caspases (Olie et al., 1998; Roisin-Bouffay et al., 2004), and although progressive cell shrinkage, blebbing of the plasma membrane, and some chromatin condensation can be observed, this form of cell death differs from apoptosis in multicellular organisms because it lacks the typical ladderlike nuclear DNA fragmentation (Cornillon et al., 1994). When multicellular development is inhibited, Dictyostelium cells can undergo another form of caspase-independent programmed cell death, which resembles that of multicellular organisms more closely: apart from the previously mentioned apoptotic phenotypes, this form of cell death also leads to the formation of pseudoapoptotic bodies, which, together with dying cells, are ingested by neighboring Dictyostelium cells (Tatischeff et al., 2001). Both cell

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death pathways therefore allow the recycling of cellular components during starvation (Otto et al., 2003). Both Dictyostelium cell death pathways also involve the disruption of the mitochondrial transmembrane potential, and the cascade of events is preceded by the release of apoptosis-inducing factor (DdAIF) from the mitochondria, indicating a direct involvement of the organelles in this pathway (Arnoult et al., 2001). DdAIF shows high homology to the mammalian AIF (30% identity, 60% similarity) and to AIFs of other organisms, all of which have a mitochondrial localization signal (MLS) and a nuclear localization signal (NLS) in common. At the onset of cell death, the Dictyostelium protein is targeted to the nucleus upon release from the mitochondria into the cytoplasm, similar to its mammalian counterpart. Furthermore, the functional homology between the mammalian and the Dictyostelium protein has been demonstrated in experiments that clearly showed that cytoplasmic extracts from dying Dictyostelium cells triggered the partial degradation of isolated mammalian and Dictyostelium nuclei in a cell-free system (Arnoult et al., 2001). The fact that cell death pathways in unicellular organisms such as Dictyostelium and those reported in multicellular organisms are induced by the release of apoptosis-inducing factors from mitochondria suggests an evolutionarily conserved role of these organelles in the control of cell suicide (Ameisen et al., 1995; Green and Reed, 1998).

3. Mitochondrial Disease 3.1. Mitochondrial disease in humans Human mitochondrial diseases are an eclectic family of genetic disorders with complex, poorly understood pathologies ( James and Murphy, 2002; Lin and Beal, 2006; Maassen et al., 2004; McKenzie et al., 2004; Rossignol et al., 2003). They can arise because of mutations affecting either mitochondrial genes or nuclear genes encoding mitochondrial proteins. Most of the genetically defined mitochondrial disorders involve mutations in the mitochondrial genome. This is because the small size of the mitochondrial genome makes detection of mutations easier and because of the unusual nature of mitochondrial genetics. The mitochondria are maternally inherited, and in general, every cell contains large numbers of mitochondria, each of which carries on average two or three copies of the mitochondrial genome. In addition, mitochondrial fusion and fission events in mammalian cells allow mixing of the genomes of the mitochondria. This makes it possible for a subpopulation of the mitochondrial genomes in a cell to contain what would otherwise be lethal mutations, often without ill effect. This state, in which the cells contain a mixture of wild-type and mutant mitochondrial genomes, is called heteroplasmy, and the phenotypic

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outcome depends upon the mutant load—the proportion of mutant mitochondrial genomes in the cell. During mammalian oogenesis, there is a population bottleneck in which the number of mitochondria in the differentiating oocyte decreases to around one dozen (Cummins, 2001; Poulton and Marchington, 2002). All of the mitochondria in all of the tissues of the offspring are derived ultimately from this dozen, so that for purely stochastic reasons the mutant load in siblings and in different tissues can differ dramatically. In addition, it has been suggested that in proliferating cells and tissues there would be a selective growth advantage for cells with lower mutant loads, so that mutant loads increase over time only in postmitotic nonproliferating tissues (Lin and Beal, 2006). There are also tissue-specific differences in energy demand, in the expression of nuclear-encoded mitochondrial proteins, and in the age-dependent accumulation of mutations in the mitochondrial genome (McKenzie et al., 2004). Together these factors conspire to prevent a consistent clinical outcome of mitochondrial disease in humans. Individuals with the same genetic defect can have very different symptoms, while different genetic defects can produce very similar clinical outcomes. These can include deafness, blindness, epilepsy, strokelike episodes, ataxia, Parkinsonism, muscle weakness, exercise intolerance, diabetes, cardiomyopathy, kidney malfunction, and combinations thereof. Despite the complexity of genotype-phenotype relationships in mitochondrial diseases, the pathological outcomes mostly take the form of degenerative neurological or neuromuscular disorders or heart disease and so affect primarily postmitotic tissues with high energy demands (McKenzie et al., 2004; Rossignol et al., 2003). In fact, it has become clear that mitochondrial dysfunction plays a key role in the pathology of all major neurological disorders, including those caused by mutations affecting normally cytoplasmic proteins (Lin and Beal, 2006). These include Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. In each case, the disease pathology involves mitochondrial damage and dysfunction, while the causative mutant proteins are either normally mitochondrial or have been shown in the mutant form to associate with or be imported into the mitochondria. Depending upon which mitochondrial protein is affected, some but not all mitochondrial mutations lead to impaired electron transport and excess production of reactive oxygen species (ROS) resulting from the transfer of respiratory electrons directly to molecular oxygen from ubiquinone ( James and Murphy, 2002). Because they are so reactive, these partially reduced oxygen species diffuse only short distances, but they are able to damage the mitochondrial membranes and proteins as well as cause further mutations in the mitochondrial genome. ROS damage arising this way is believed to be cumulative and to contribute to the degenerative nature of mitochondrial diseases.

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The bewildering complexity of the pathology of mitochondrial diseases has so far defeated a clear understanding of the disease mechanisms. However some generalizations have emerged (Rossignol et al., 2003). First, all of the disease-causing mutations interfere with the ATP-generating capacity of the mitochondria, and second, disease symptoms indicating cellular dysfunction can occur before the onset of visible cytopathology. In neural tissues the late stages of disease involve programmed cell death, but disease pathology occurs before the cell death pathway is activated (Lin and Beal, 2006). In muscle tissues, runaway mitochondrial proliferation (producing so-called ragged red fibers) and cellular hypertrophy may occur.

3.2. Genetic methods for creating mitochondrial disease in Dictyostelium The study of mitochondrial disease in Dictyostelium required the development of methods for genetically creating sublethal mitochondrial dysfunction. This has been achieved in two ways—disruption of mitochondrial genes in a subset of the mitochondrial genomes (heteroplasmic gene disruption) and antisense or RNAi inhibition of expression of nuclear genes encoding essential mitochondrial proteins. In addition, several genes that appear not to be essential for mitochondrial respiratory function have been knocked out. 3.2.1. Heteroplasmic targeted disruption of mitochondrial genes The first disruption of a Dictyostelium mitochondrial gene was nontargeted (Wilczynska et al., 1997). Cells were transformed with an integrating shuttle vector that cannot replicate extrachromosomally and screened for a phototaxis-deficient phenotype. This was the first time random integration of a plasmid had been used to isolate Dictyostelium mutants, and the original aim of the experiment was to identify new genes essential for photosensory signal transduction. Surprisingly, when the disrupted gene was cloned and sequenced, it was identified as the mitochondrial large ribosomal subunit RNA gene (rnl). Subsequent targeted disruptions of this gene verified that its disruption produced a phototaxis-deficient phenotype. Random integration of plasmids into the Dictyostelium nuclear genome produces phototaxis mutants at a frequency of about 0.15%. These mutants arise by nontargeted insertion into genes essential for normal phototaxis. However, the presence of a portion of the mitochondrial rnl gene in the integrating plasmid was found to target the insertions by homologous recombination into rnl in as many as 40% of the transformants. In subsequent experiments, the frequency of targeted disruption of a variety of other mitochondrial genes has ranged from about 2 to 12% (Francione and Fisher, unpublished). By this means mutants are readily isolated in which any chosen mitochondrial gene has been disrupted. Not unexpectedly, the qualitative strength of signals in

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Southern blots suggested the disruption of these mitochondrial genes had occurred only in a subset of the mitochondrial genomes in the cell. In other words, these mitochondrial gene disruptions are heteroplasmic. Disruption of every mitochondrial genome in the cell is not only unlikely but also would be expected to be lethal. It should be noted that the mitochondrial gene disruptions in these experiments are always accompanied by independent, nontargeted insertions into the nuclear genome (Barth et al., 1998a; Wilczynska et al., 1997). Thus one cannot rely on a single disruptant to determine the phenotypic effects of targeted mitochondrial gene disruption. Instead one or more of the aberrant phenotypes in any given transformant could be caused by the accompanying nontargeted disruption of a chromosomal gene. However, pulsed field gel electrophoresis and Southern blotting showed the additional chromosomal insertions occur in different locations in every transformant. One can therefore be confident that phenotypes consistently observed in targeted mitochondrial disruptants are caused by the mitochondrial event. This technique has provided the means to create in Dictyostelium a model for human heteroplasmic mitochondrial disease in which any chosen mitochondrial gene can be targeted. 3.2.2. Antisense and RNAi inhibition of nuclear genes encoding essential mitochondrial proteins Mitochondrial disorders can result from mutations affecting not only mitochondrial genes, but also nuclear genes that encode mitochondrial proteins. One such gene encodes chaperonin 60 (Cpn60), a universal protein-folding chaperone found in bacteria, mitochondria, and chloroplasts (Lin and Rye, 2006). The mitochondrial chaperonin 60 protein sequence and structure is highly conserved with more than 60% sequence identity (80% similarity) between even distantly related eukaryotes and 55% identity (75% similarity) between the bacterial (E. coli GroEL) and mitochondrial (Dictyostelium) proteins (Kotsifas et al., 2002). Often referred to as Hsp60 (heat shock protein 60) because its expression is induced by heat and other stresses in most organisms (but not in Dictyostelium), Cpn60 is essential for viability because it is required for correct folding of a subset of proteins within the mitochondria. Rare cases have been reported of mitochondrial disease in humans resulting from an undersupply of chaperonin 60 (Agsteribbe et al., 1993; Briones et al., 1997; Huckriede and Agsteribbe, 1994). These patients exhibited severe neurological and developmental disorders, accompanied by multiple respiratory enzyme deficiencies, and died in early infancy. To reproduce this condition in Dictyostelium, researchers took advantage of the ease with which antisense RNA inhibition can be applied in this organism since it was first used in 1985 (Crowley et al., 1985). The 30 half of the Dictyostelium chaperonin 60 gene (hspA) was cloned into an expression vector in the antisense orientation to allow expression in Dictyostelium of a

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corresponding RNA transcript complementary to the normal hspA mRNA (Kotsifas et al., 2002). When Dictyostelium cells are transformed with an integrating expression vector, each independent transformant contains a different, stable number of copies of the integrated plasmid (Barth et al., 1998a). Copy numbers in independent transformants can range from several to several hundred. The use of a construct expressing an antisense RNA directed against a specific target gene therefore produces a large number of transformants, each of which exhibits a characteristic, stable level of antisense inhibition. Inhibition of chaperonin 60 expression in this way progressively reduced the levels of the native mRNA in Northern blots as the copy number for the antisense RNA construct increased (Kotsifas et al., 2002). The advantage of this technique is that sublethal reductions in the expression levels of essential proteins can be achieved in circumstances where gene knockouts would be lethal. Furthermore, there is no need to undertake laborious screening to find the transformants of interest, as is the case when searching for gene knockout strains. The number of copies of the antisense-inhibition construct in each transformant is different, stable, readily determined by several different techniques (Barth et al., 1998b), and correlates with the extent of antisense inhibition of expression of the target gene (Bokko et al., 2007; Kotsifas et al., 2002). The copy number can therefore be used as a ranking index of expression levels so that quantitatively assayed phenotypes can be presented in genetic dose-response curves (Fig. 5.4). Phenotypes that are consistent among the transformants and whose severity varies in a copy number-dependent manner can be confidently ascribed to the effects of the antisense inhibition. Similar considerations apply to RNAi inhibition of expression except that RNAi is considered to be more effective (Kuhlmann et al., 2006; Martens et al., 2002)—sometimes too effective. RNAi constructs contain an inverse duplication of part of the coding sequence, separated by a spacer region so that the expressed RNA will form a stem-loop structure. Transformation appeared to be lethal in the case of an RNAi expression construct designed to knock down expression of sdhA which encodes the catalytic subunit of mitochondrial succinate dehydrogenase (Complex II of the mitochondrial electron transport chain) (Lay and Fisher, unpublished). Transformants could not be isolated when using this construct singly but were obtained when a cotransformation was performed using it in combination with the empty vector. The obtained cotransformants contained eight or fewer copies of the sdhA RNAi inhibition construct, accompanied by large numbers of copies of the empty vector. Previous work had shown that more than eight copies of this vector were required to provide sufficient resistance to G418 for successful selection of transformants (Barth et al., 1998a). As the RNAi construct copy number increased, phototaxis became impaired, and finally, at eight copies, aggregation, slug formation, and migration failed altogether (Lay and Fisher, unpublished).

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Correlation of phenotype with copy number 40

150 Zero copies = normal nondiseased cells Ax2 (Wild type)

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35

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5 0 0 −230 −170 −110 −50 Chaperonin 60 expression index (copy number)

Figure 5.4 Effect of mitochondrial disease (chaperonin 60 antisense inhibition) on Dictyostelium growth and phototaxis. The number of copies of the chaperonin 60 antisense-inhibition construct is used as an index of chaperonin 60 expression levels. Following the convention of Bokko et al. (2007), copy numbers are assigned negative values to reflect that expression of the native chaperonin 60 mRNA is inhibited by the construct. Lowest expression levels are thus represented by the left-most points on the X axis (largest negative numbers). Growth is represented as the generation time (h) in liquid medium (HL-5) measured from the slopes of log-linear regressions of cell numbers versus time during exponential growth.Vertical bars for generation times represent 95% confidence intervals. The accuracy of phototaxis is the maximum likelihood estimate of k, the concentration parameter of the von Mises distribution (a bell-shaped‘‘normal’’distribution for directional data). k ranges from 0 in the case of no orientation (all directions equally probable) to infinity in the case of perfect orientation (all directions identicalçdirectly toward the light).Vertical bars for the accuracy of phototaxis represent 90% confidence intervals.The inset shows digitized trails of the wild-type parental strain AX2, three antisense-inhibited strains, and a control strain expressing the sense strand of the same chaperonin 60 fragment as used in the antisense inhibition construct. The antisense construct expressed an RNA transcript from the 30 half of the chaperonin 60 gene (hspA) (Kotsifas et al., 2002). Phototaxis is impaired only if the fragment is in the antisense orientation and so able to produce an antisense RNA. Data from Kotsifas et al. (2002).

3.2.3. Targeted disruption of nuclear genes encoding nonessential mitochondrial proteins Whereas heteroplasmic disruption of essential mitochondrial genes is possible, it would be lethal to disrupt a nuclear gene encoding an essential mitochondrial protein. This is because Dictyostelium has a haploid genome so that a nuclear gene encoding a mitochondrial protein must supply that polypeptide to every mitochondrion in the cell. However, not all

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mitochondrial proteins are essential for viability. For such proteins knockouts can be isolated by homologous recombination and the resulting phenotypes studied. Examples include the torA (Tortoise), fszA, fszB, and midA genes described later.

3.3. Mitochondrial disease phenotypes and associated signaling pathways One of the advantages Dictyostelium offers for the study of mitochondrial disease is that its life cycle provides a large variety of readily assayed phenotypes. These include amoeboid motility and chemotaxis, mitosis and cytofission, phagocytosis, macropinocytosis, cell and tissue differentiation and pattern formation, phototaxis, thermotaxis, multicellular tissue movement and morphogenesis, and spore germination. The differentiation and morphogenetic movements in the multicellular stages are coordinated by intercellular signals and associated intracellular signaling pathways which involve many elements common to all eukaryotes. This phenotypic richness is accompanied by the advantages of a genetically tractable microorganism whose entire genome sequence has been determined (Chisholm et al., 2006; Eichinger et al., 2005). At the same time, the multicellular stages arise by aggregation of clonally derived, genetically identical cells, thereby avoiding all of the overlaid complexities of mammalian development alluded to earlier, which obscure regularities in the underlying cell biology. Almost every cellular activity requires energy, usually in the form of ATP, sometimes in the form of other energy-rich compounds (e.g., GTP, PEP, NADH) or in the electrochemical gradients across cellular membranes. These different energy sources are interchangeable by various biochemical means so that one might naively have expected all cellular activities to be impaired if mitochondrial ATP-generating capacity were compromised. However, this is not the case. Some cellular functions are affected while others are not. This could reflect the differing energy demands of various cellular functions. However, work in the Dictyostelium model has revealed that mitochondrial disease is a signaling disorder rather than an energy insufficiency per se (Bokko et al., 2007). Some energyrequiring phenotypes are unaffected by mitochondrial dysfunction because they are not subject to regulation by the intracellular signals activated when mitochondrial ATP-generating capacity is compromised. Table 5.2 shows the different phenotypes that have been found to be associated with mitochondrial respiratory dysfunction in Dictyostelium caused either by treatment with pharmacological agents or by genetic manipulation. The first phenotypes found to result from mitochondrial dysfunction in Dictyostelium were phototaxis and thermotaxis (Table 5.2) in the multicellular (‘‘slug’’) stage of the life cycle (see Fig. 5.4) (Kotsifas et al., 2002; Wilczynska et al., 1997). Dictyostelium slugs exhibit extraordinarily sensitive

Table 5.2

Phenotypes associated with mitochondrial dysfunction in Dictyostelium

Method of generating mitochondrial dysfunction

Phenotype Growth on bacteria

Growth in broth

Phagocytosis

Pinocytosis

Phototaxis

Pharmacological, expected to affect respiration Ethidium bromide inhibition of mtDNA replication

Thermotaxis

Pattern formation

Aggregation

Stalky

Nuclear cluA Defective disruption cytokinesis Nuclear torA disruption Nuclear midA disruption Nuclear Dd-TRAP1 RNAi inhibition þ, Wild-type phenotype;

þ

Wilczynska et al., 1997 Inazu et al., 1999; Fisher (unpublished) Kotsifas et al., 2002; Bokko et al., 2007

Stalky

þ

þ

(Defective cytokinesis)

þ

þ

þ

þ

Gilson et al., 2003; Fisher (unpublished) Zhu et al., 1997 van Es et al., 2001

þ

, Aberrant phenotype; þ/ , Mildly aberrant phenotype; Shaded cells, Phenotype not reported.

References Chida et al., 2004

Genetic, expected to affect respiration Heteroplasmic rnl disruption Heteroplasmic rps4 þ/ disruption Chaperonin 60 antisense inhibition Genetic, not known to affect respiration þ (fszA ); Nuclear fszA, fszB þ (fszA ); (fszB ) (fszB ) disruption

Chemotaxis

þ

þ

Torija et al., 2005 Morita et al., 2004

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and accurate phototaxis and thermotaxis responses (Fisher, 1997, 2001). The corresponding signaling pathways converge early so that almost all of the proteins involved are required for both responses. Although not well understood, the photo/thermosensory transduction pathways in Dictyostelium are known to involve a variety of typically eukaryotic signaling molecules, including heterotrimeric and small GTP-binding proteins, certain actinbinding proteins including the scaffolding protein filamin, protein kinases such as Akt/PKB and Erk2, the second messengers cAMP, cGMP, Ca2þ, and inositol triphosphate along with the associated enzymes and target proteins, and cytoskeletal proteins (Bandala-Sanchez et al., 2006; Fisher, 2001). Both phototaxis and thermotaxis are dramatically impaired by heteroplasmic mitochondrial gene disruptions and by antisense inhibition of chaperonin 60 gene expression. Ethidium bromide-mediated depletion of mitochondrial DNA also caused a severe impairment of phototaxis (Chida et al., 2004). The sensitivity of the photosensory and thermosensory signaling pathways to mitochondrial disease led Wilczynska et al. (1997) to suggest mitochondrial dysfunction may impair specific cellular signaling pathways more sensitively than other processes. Subsequent work has verified this and shown that diverse cytopathologies associated with mitochondrial disease in Dictyostelium arise from cellular signaling malfunctions, not from insufficient ATP (Bokko et al., 2007). In fact, the cellular ATP levels remain close to normal even in mitochondrially diseased cells with severe phenotypic abnormalities. This is discussed in more detail in a later section. The second phenotype found to be associated with mitochondrial disease in Dictyostelium is impaired growth (Table 5.2), both on bacterial lawns and in nutrient broth (Fig. 5.5) (Bokko et al., 2007; Kotsifas et al., 2002; Wilczynska et al., 1997). Research has shown that this is not due to defects in phagocytosis and macropinocytosis (Bokko et al., 2007), and it is not associated with any apparent changes in cell size (Bokko et al., unpublished data). This means that the rate of uptake of nutrients from the medium is not limiting the growth of mitochondrially diseased cells and that the slower progression through the cell cycle is accompanied by a correspondingly slower biosynthesis of new cytoplasm. In mammalian cells and in yeast, one of the conserved signaling pathways controlling cell growth and proliferation involves the mTOR protein, which integrates a variety of nutrient- and stress-dependent signals. In metazoa inhibition of growth via this pathway involves, in turn, the activation of TSC2, the stimulation of Rheb GTPase activity (converting Rheb to its inactive GDP-bound form), and the inhibition of the kinase activity of mTOR (Feng et al., 2005; Inoki et al., 2005). This conserved signaling pathway is also encoded in the Dictyostelium genome (Chisholm et al., 2006; Eichinger et al., 2005), but its functional roles in relation to growth control or mitochondrial disease have not yet been defined. Dictyostelium also contains a set of homologs of cell cycle control genes encoding one each of the

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Figure 5.5 Effect of chaperonin 60 and AMPKa expression levels on Dictyostelium growth. Each circle represents a different clonal cell line (strain) carrying the indicated number of copies of either the chaperonin 60 (Cpn60) antisense construct, the AMPKa antisense construct, or the AMPKaToverexpression construct. Lines are fitted only to the data represented by the circles. Each square represents a different strain carrying different numbers of copies of both the chaperonin 60 antisense construct and the AMPKa antisense construct. Data for these strains therefore appears in all panels, in each of which it is plotted according to the copy number relevant to that panel only.Vertical bars are 95% confidence intervals. Negative values indicate the copy numbers of antisense inhibition constructs, whereas positive values indicate copy numbers of the overexpression construct. Copy numbers of zero include both the wild-type strain (AX2) and control strains carrying sense construct controls, but no copies of the relevant antisense or overexpression construct. (A, B) Time taken for a growing Dictyostelium colony (plaque) to expand 5 mm during growth at 21o on an E. coli B2 lawn on SM agar. The growth time was calculated from the slope of the line measured by linear regression analysis of plaque diameter versus time during 5^7 days of growth. (C, D) Generation time for Dictyostelium cells growing in HL-5 liquid medium at 21o, shaken at 150 rpm. Generation times were calculated from growth curve slopes using log-linear regression analysis of cell counts during the exponential phase of growth. (From Fig. 3 of Bokko et al. [2007].)

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cyclins A, B, and D; the cyclin-dependent kinase Cdk1; the retinoblastoma protein Rb1 and several of its targets including the transcription factor E2F, together with its binding partner DP (MacWilliams et al., 2006). Mitochondrial disease could impair cell proliferation through any of these cell cycle control proteins. The third phenotype associated with mitochondrial disease in Dictyostelium is a misdirection of cells into the stalk differentiation pathway and their mislocalization in the migrating slug (Chida et al., 2004; Kotsifas et al., 2002). This occurs both in response to genetic and pharmacological manipulations that would be expected to impair mitochondrial respiration (Table 5.2), and it leads to production of misshapen, thick, short stalks when the fruiting body is formed (Fig. 5.6). Furthermore, Matsuyama and Maeda (1995) reported that virtually all cells enter the stalk pathway in response to inhibitors of mitochondrial cyanide-resistant respiration, a treatment which also impaired mitochondrial membrane potentials. Although the pathways controlling cell type choice and differentiation in Dictyostelium are not fully understood, a number of the signaling molecules and interactions involved have been identified (Coates and Harwood, 2001; Strmecki et al., 2005; Williams, 2006). These molecules, whose levels or activities could be altered in mitochondrial disease, include the following: 1. Extracellular signals—cAMP, DIF-1, and several related polyketides, the signaling peptide SDF-2; 2. Surface receptors—G-protein coupled cAMP receptors CAR3 and CAR4, and the SDF-2 receptor DhkA; 3. Protein kinases—serine-threonine kinases such as PKA, GskA, and Erk2, the tyrosine kinase Zak1, and the histidine kinase DhkA; 4. Transcriptional regulators—three STAT family transcriptional repressors, a basic leucine zipper protein DimA/B, the Myb domain protein MybE, the homeodomain protein Wariai (Han and Firtel, 1998), RblA (MacWilliams et al., 2006), and the b-catenin homolog Aardvark; 5. Adenylyl cyclase ACB and cAMP phosphodiesterase RegA; 6. Cytosolic-free Ca2þ (Schaap et al., 1996). The final phenotype shown to result from mitochondrial respiratory dysfunction in Dictyostelium is the transition from growth to development and subsequent chemotactic aggregation (Table 5.2). Thus Yasuo Maeda and colleagues (2005) showed that aggregation is delayed and the expression of an early developmental gene, carA, is reduced by heteroplasmic disruption or antisense inhibition of the mitochondrial rps4 gene (Hosoya et al., 2003; Inazu et al., 1999). Aggregation is similarly impaired by ethidium bromide-mediated depletion of mitochondrial DNA (Chida et al., 2004), by chaperonin 60 antisense inhibition (Bokko et al., 2007; Kotsifas et al., 2002), and by RNAi inhibition of succinate dehydrogenase expression (Lay and Fisher, unpublished). This impairment of aggregation results in production

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Figure 5.6 Effect of chaperonin 60 and AMPKa expression levels on multicellular morphogenesis in Dictyostelium. Photographs were taken from above (main panels) or from the side (insets) of fruiting bodies formed during growth at 21oC on a bacterial lawn (Klebsiella aerogenes). Apart from (B) the parental wild-type strain (AX2), the strains contained (A) antisense inhibition constructs for both chaperonin 60 (main panelç73 copies; insetç56 copies) and AMPKa (main panelç109 copies; insetç98 copies) or (C) the chaperonin 60 antisense construct only (main panelç48 copies; inset, top rowç67 copies; inset, other rowsç74 copies) or (E) the AMPKa antisense inhibition construct (143 copies) only or (D, F) the AMPKaToverexpression construct only (30 copies and 148 copies, respectively). Mitochondrial disease (chaperonin 60 antisense inhibition) and AMPKaT overexpression both caused the formation of fruiting bodies with thick, short stalks (red arrows in C, D, F). Fruiting body morphology was normal for a mitochondrially diseased strain (chaperonin 60 antisense inhibition) in which AMPKa expression was antisense-inhibited (cyan arrows in A). In otherwise healthy cells, AMPKa antisense inhibition resulted in formation of fewer, smaller fruiting bodies that were morphologically normal (green arrows in E). For comparative purposes, the strains were selected to show the phenotypes at moderate copy numbers of the various constructs. The aberrant phenotypes in panels C, D, E, and F were more severe at higher copy numbers. (From Fig. 5 of Bokko et al. [2007].)

of fewer, smaller aggregates. In the mitochondrial DNA-depleted cells at least, this is manifested both as a delay in the onset of aggregation and the breakup of initially large aggregation streams to produce smaller aggregates (Chida et al., 2004). Aggregation in Dictyostelium is the first visible manifestation of a developmental program initiated in response to the combined effects of two signals—starvation and high cell density. Mitochondrial dysfunction could impair aggregation by acting on one or more of the corresponding signal transduction pathways. Early work suggested the starvation signal is carried primarily by an undersupply of amino acids (Marin, 1976), but the precise molecular nature of the signal is not understood. However, several signals have been identified that inform cells of their density in growing cultures

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and are required to initiate the transition from growth to development (Maeda, 2005). The best understood of these signals is PSF-1 (PreStarvation Factor), a 65 kD glycoprotein that is secreted into the medium and reaches high enough concentrations toward the end of log phase to stimulate development. In the absence of a bacterial food source, PSF-1 stimulates expression of early genes in development in a pathway that involves activation of the protein kinase YakA, inhibition of the RNA-binding translational repressor PufA, and expression of cAMP-dependent protein kinase (PKA). PKA activity indirectly represses growth-specific transcripts and induces aggregation-specific transcripts, including those encoding the major adenylyl cyclase (ACA) and cAMP receptor (CAR1) involved in aggregation. This creates an autocatalytic developmental signal because PKA is activated by cAMP, which is synthesized by ACA in response to CAR1 signaling. The signaling pathway by which extracellular cAMP stimulates ACA-mediated synthesis and secretion of more cAMP is well characterized and involves the G-protein bg heterodimer, PI3 kinases, CRAC (cytosolic regulator of adenylyl cyclase), RasC, RasGEF, Pianissimo, Rip3 (Ras interacting protein), and the MAP kinase ERK2 (Saran et al., 2002). Any of these could be affected pleiotropically by mitochondrial dysfunction.

3.4. Phenotypic thresholds in mitochondrial disease in Dictyostelium Human mitochondrial diseases are characterized by the so-called threshold effect—cellular functions remain unaffected while the proportion of mutant mitochondrial genomes in the cell remain below a certain level (Rossignol et al., 2003). Furthermore, the threshold at which particular cellular functions do become impaired varies from one energy-requiring cellular activity to another and from one cell type to another. It has been suggested that this arises at least in part because of excess ATP-generating capacity in cells combined with tissue-specific differences in energy demand. Rossignol et al. (2003) discussed in-depth the thresholds that exist at each of the stages of expression of mitochondrial genes and their function in respiration and ATP synthesis. Only when all of these layered thresholds are exceeded would ATP generation become compromised. Dictyostelium mitochondrial respiratory disease appears to exhibit similar thresholds in that not all energy-consuming cellular activities are affected equally. In every case that has been examined, manipulations that would impair mitochondrial respiratory dysfunction have resulted in deranged phototaxis and thermotaxis (Bokko et al., 2007; Chida et al., 2004; Kotsifas et al., 2002; Wilczynska et al., 1997; Francione and Fisher, unpublished). However, not every strain has exhibited significant growth defects (Wilczynska et al., 1997). Kotsifas et al. (2002) reported that as the number of copies of the chaperonin 60 antisense RNA construct increased, both

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phototaxis and growth became more severely impaired (see Fig. 5.4). However, the effect on growth was significant only at copy numbers greater than 60. This indicates that photo/thermosensory transduction is more sensitive to mitochondrial dysfunction than growth. At high copy numbers, multicellular morphogenesis also became defective and resulted in the production of fruiting bodies with short, thickened, misshapen stalks (see Fig. 5.6) (Kotsifas et al., 2002). These defects in the multicellular stages of Dictyostelium development are observable only if aggregation can still occur. However, as the copy number increased, fewer, smaller fruiting bodies were also formed, indicating an impairment in aggregation. Yasuo Maeda’s group observed heteroplasmic knockout of the rps4 gene (encoding a mitochondrial small subunit ribosomal protein) produced such a severe aggregation defect that almost no slugs or fruiting bodies formed (Inazu et al., 1999). The few slugs that do form in this mutant exhibit defective phototaxis (Kotsifas et al., 2002). Bokko et al. (2007) found that despite dramatic impairment of growth, both in liquid medium and on a bacterial food source, macropinocytosis and phagocytosis were unaffected by chaperonin 60 antisense inhibition. Together, the data support a regular hierarchy of phenotypes which can be ranked in order of appearance as the underlying mitochondrial disorder becomes more severe. This ranking reflects the sensitivity of the assayed phenotype to the degree of mitochondrial dysfunction and may be written as photo/thermotaxis > growth > multicellular morphogenesis > aggregation >> phagocytosis and phagocytosis.

3.5. Phenotypes associated with mitochondrial defects not known to affect respiration Not every genetic defect that affects mitochondria would necessarily compromise respiratory energy-generating capacity. One example is the absence of the mitochondrial fission proteins FszA and FszB (Gilson et al., 2003). In the metazoan, fungal, and plant lineages, mitochondrial FtsZ appears to have been lost without compromising mitochondrial energy production. Dictyostelium mutants lacking FszA exhibit no discernible aberrant phenotypes apart from elongated mitochondria. In particular, they show none of the deficiencies described in the preceding sections as characterizing mitochondrial disease in Dictyostelium. The mitochondria in the FszA- and FszB-deficient mutants still stain normally with Mitotracker Red, whose ability to stain mitochondria depends upon the mitochondrial membrane potential. Although the mitochondria in these mutants are elongated, the total mitochondrial area per cell is not noticeably altered. It is therefore unlikely that mitochondrial energy production is compromised in these strains. Mutants lacking FszB also fail to show the typical phenotypic patterns just described for Dictyostelium mitochondrial disease. Although FszB mutants do grow more slowly, they are unaffected in aggregation, multicellular morphogenesis, and phototaxis.

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Another nuclear gene that appears to be required for the completion of mitochondrial fission events is cluA (Zhu et al., 1997), homologs of which have since been found in the other major eukaryotic lineages (Logan et al., 2003). As described in preceding sections, CluA-deficient mutants show aberrant mitochondrial morphology and distribution. They grow more slowly because of a defect in cytokinesis that results in enlarged multinucleated cells but apart from this exhibit no overtly abnormal phenotypes and no apparent mitochondrial respiratory dysfunction (Fields et al., 1998). The defect in cytokinesis was suggested to result from the physical occlusion of cytofission by the clumped, incompletely divided mitochondria. The mitochondrial protein TorA (tortoise), on the other hand, is clearly required for normal growth on bacterial lawns and for chemotactic orientation (van Es et al., 2001). The TorA protein is found in the same mitochondrial subpolar body as FszB (Gilson et al., 2003). The slow growth of the TorA-deficient mutant on bacteria was attributed to its defective chemotaxis because phagocytosis rates were normal. However, it could equally be due to direct effects on the pathways controlling cell growth and cell cycle progression. Whether any of the other phenotypic defects typical of mitochondrial respiratory dysfunction are exhibited by TorA mutants has not been reported. Another nuclear-encoded mitochondrial protein to have been studied functionally is midA, which was initially identified as one of 30 genes of unknown function, lacking homologs in Saccharomyces cerevisiae and Schizosaccharomyces pombe, but with high similarity to uncharacterized human genes (Torija et al., 2005). Phagocytosis and macropinocytosis were defective in the midA knockout strain, and this was accompanied by correspondingly smaller cell size and slower growth on bacteria and in liquid medium. In this respect the midA-deficient cells differ from those of chaperonin 60 antisense-inhibited cells which grow more slowly but are unaltered in cell size, phagocytosis, and pinocytosis. Also unlike them, the mutant exhibited no obvious defects in aggregation or fruiting body morphology, but spore viability was compromised. In addition, the midA mutant exhibited a ‘‘slugger phenotype’’—probably because of an accumulation of excess ammonia, it remained in the migratory slug stage and failed to form fruiting bodies on unbuffered, wet filter pads. A similar phenotype is observed in mitochondrial DNA-depleted cells (Chida et al., 2004). The absence of midA caused no changes in mitochondrial membrane potential (Mitotracker Red fluorescence), cellular oxygen consumption (Clark electrode measurements), or mitochondrial ‘‘mass’’ (Mitotracker Green fluorescence). The steady state amount of ATP per cell was reduced, which prompted Torija et al. (2005) to suggest a mitochondrial dysfunction in ATP production. However, once the reduction in cell volume is accounted for, it is clear that the cellular ATP concentrations were not lower than in wild-type cells. There is therefore no reason to believe that mitochondrial

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ATP-generating capacity is compromised in midA-deficient mutant cells. It should be noted that chaperonin 60 antisense inhibition (which would unavoidably compromise mitochondrial ATP-generating capacity) also causes no detectable changes in mitochondrial membrane potential, mitochondrial ‘‘mass,’’ or ATP levels (Bokko et al., 2007, Annesley and Fisher, unpublished). The final mitochondrial protein in Dictyostelium whose function has been studied genetically is DdTRAP-1, a homologue of mammalian TRAP-1 (TNF Receptor Associated Protein 1)—a mitochondrial member of the Hsp90 molecular chaperone family localized in the mitochondria as well as extramitochondrial sites including the nucleus, secretory granules, and cell membranes. Like chaperonin 60 (Kotsifas et al., 2002), the DdTRAP-1 gene is transcribed only in vegetative cells, but the levels of the protein are stable throughout development (Morita et al., 2002). Unusually, DdTRAP-1 is localized primarily to the actin cortex and cell membranes in vegetative Dictyostelium cells, but during the first 6 h of starvation, it is translocated to the mitochondrial matrix by an unknown mechanism that does not require the predicted mitochondrial presequence at its N-terminus. The presequence is not present in the cytoplasmic DdTRAP-1 protein in vegetative cells, so it has been suggested that the nascent polypeptide may be imported transiently into the mitochondria, the presequence removed, and the protein then exported back to the cytosol in vegetative cells (Morita et al., 2002; Yamaguchi et al., 2005). It is also possible that the presequence is removed in the cytosol by proteolytic trimming of the folded protein. This has been observed for GFP fused to incomplete or mutant mitochondrial-targeting presequences (Ahmed et al., 2006; Ni et al., 1999). RNAi inhibition of expression of DdTRAP-1 results in slow vegetative growth and delayed aggregation (Morita et al., 2004). DdTRAP-1 expression is induced by heat stress, and in the RNAi-inhibited cells, morphogenesis and spore differentiation become markedly deranged at high temperatures and the spores that are formed are less heat- and detergent-resistant than wild-type spores (Morita et al., 2005).

3.6. AMPK—the missing link between phenotype and genotype in mitochondrial disease The phenotypes caused by genetic defects affecting the mitochondria suggested that the primary cytopathological effect of mitochondrial disease might be the impairment of specific cellular signal transduction pathways. This was first proposed by Wilczynska et al. (1997), but it was not clear what the molecular link between mitochondrial respiratory dysfunction and cellular signaling might be. This missing link was later identified as AMP-activated protein kinase (AMPK) (Bokko et al., 2007).

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AMPK has received increasing attention as an essential sensor and homeostatic regulator of cellular energy status (Hardie, 2004; Hardie and Hawley, 2001; Hardie and Sakamoto, 2006; Hardie et al., 2003; Kahn et al., 2005). AMPK is a heterotrimeric serine-threonine protein kinase containing a catalytic a subunit and a regulatory g subunit, both of which are bound to the b subunit which serves as a scaffold for assembly of the holoenzyme. Whereas mammalian cells contain three genes encoding isoforms of each of the a and g subunits as well as two b subunit genes, Bokko et al. (2007) found a single gene for each of the three AMPK subunits in a search of the Dictyostelium genome sequence. The a subunit contains an N-terminal catalytic domain which is very highly conserved and a less–well-conserved C-terminal portion responsible for binding to the b subunit and for autoinhibition of the enzyme activity. AMP binding to the two Bateman domains of the mammalian g subunit relieves the autoinhibition and activates the enzyme in three ways—by an allosteric effect, by rendering the a subunit susceptible to activatory phosphorylation by upstream kinases, and by rendering it resistant to inhibitory dephosphorylation by protein phosphatases (Hardie and Sakomoto, 2006). ATP inhibits AMPK by competing with AMP for binding to the g subunit, with the result that AMPK responds primarily to the AMP/ATP ratio. Three upstream AMPK-activating kinases in mammalian cells have been identified on the basis of their similarity with and ability to functionally substitute for the upstream kinases in yeast—LKB1 (Hong et al., 2003; Woods et al., 2003), CAMKKa (Hong et al., 2005; Hurley et al., 2005), and TAK1 (Momcilovic et al., 2006). Of these, it has been reported that LKB1 is the major kinase responsible for the physiological activation of AMPK in mammalian tissues (Woods et al., 2003). The downstream targets of AMPK in mammalian cells are diverse and are collectively responsible for both the acute and chronic physiological roles of the enzyme in activating ATP-yielding pathways and inhibiting energy-requiring pathways. The acute actions of AMPK are mediated by changing the activities (e.g., acetyl CoA carboxylase, Witters and Kemp, 1992) and/or subcellular locations (e.g., translocation of the GLUT4 receptor to the cell surface, Kurth-Kraczek et al., 1999) of the target proteins. The chronic effects of AMPK activity are a result of changes in gene expression (Hardie and Hawley, 2001; Hardie and Sakamoto, 2006; Woods et al., 2000) and include stimulation of mitochondrial biogenesis and proliferation (Bergeron et al., 2001; Zong et al., 2002). The overall result of these AMPK actions in otherwise healthy cells is a return of the cellular ATP/AMP ratios to normal. In its role as central regulator of cellular energy homeostasis in healthy cells, AMPK is activated by a variety of energy-depleting cellular stresses such as ischemia, glucose deprivation, and strenuous exercise. Now it is clear that in mitochondrial disease in Dictyostelium, AMPK is also activated

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chronically and is, in fact, responsible for the diverse phenotypic outcomes described in the preceding sections (Bokko et al., 2007). The evidence for this is as follows: 1. Various phenotypes associated with mitochondrial dysfunction—impaired phototaxis (see Fig. 5.4), growth (see Fig. 5.5), and morphogenesis (see Fig. 5.6)—are mimicked by overexpression of a truncated, constitutively activated form of AMPK containing the entire catalytic domain (AMPKaT). 2. Antisense inhibition of AMPK a subunit expression completely suppresses all of the phenotypic abnormalities of mitochondrially diseased cells (created by antisense inhibition of chaperonin 60 expression) (see Figs. 5.5, 5.6). 3. Phenotypes (phagocytosis and macropinocytosis rates) unaffected by overexpression of AMPKaT or antisense inhibition of expression of the native AMPKa mRNA were also unaffected by mitochondrial disease. 4. The phototaxis deficiency of mitochondrially diseased slugs is mimicked, in an AMPK-dependent manner, by long-term exposure to AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside), a physiological activator of AMPK in mammalian cells. However, it is not known whether the AICAR is actually acting on AMPK itself in Dictyostelium rather than on some other adenosine-binding protein. The various phenotypic abnormalities associated with mitochondrial dysfunction and with AMPKaT overexpression were gene dose-dependent, meaning they became more severe as the copy number of the corresponding expression construct in the genome increased (see Figs. 5.4–5.6). AMPK was also found to stimulate mitochondrial biogenesis and ATP production in otherwise healthy cells—mitochondrial ‘‘mass’’ and ATP levels were higher in AMPKaT overexpressing cells and lower in AMPKa antisense-inhibited cells. However, ATP levels and mitochondrial ‘‘mass’’ were unaffected by mitochondrial disease (chaperonin 60 antisense inhibition). These results led Bokko et al. (2007) to suggest the model in Fig. 5.7 for the cytopathology of mitochondrial respiratory dysfunction. According to the model, antisense-inhibiting chaperonin 60 expression impairs mitochondrial biogenesis and ATP-generating capacity. The resulting chronic AMPK activation reduces cellular ATP consumption by inhibiting some (but not all) energy-consuming cellular activities and feeds back to activate mitochondrial biogenesis and ATP production. This leads to a new steady state at which ATP levels and mitochondrial mass are close to normal, but AMPK is chronically active and continues to impair photosensory signal transduction, multicellular morphogenesis, cell growth, and proliferation. The ATP levels in these mitochondrially diseased cells are clearly sufficient to support normal cellular activities so that reducing the level of AMPK expression by antisense-inhibition is all that is needed to return the aberrant phenotypes of mitochondrially diseased cells to normal.

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Figure 5.7 Model for the role of AMPK signaling in mitochondrial disease. Cpn60 is chaperonin 60, whose undersupply in antisense-inhibited cells causes mitochondrial dysfunction. Arrowheads indicate stimulation, and barred ends indicate inhibition. AMP, ADP, and ATP are interchangeable. Mitochondrial biogenesis and function favor ATP production, whereas the cellular activities in the ellipses in the flame-shaded region consume ATP and favor AMP generation. AMP activates and ATP inhibits AMPK, which in turn stimulates mitochondrial biogenesis and inhibits some energy-consuming cellular activities (growth and cell cycle progression, morphogenesis, photosensory signal transduction) but not others (phagocytosis, macropinocytosis). In mitochondrially diseased cells, AMPK is chronically activated, which homeostatically returns mitochondrial‘‘mass’’and ATP levels to near normal, but also chronically inhibits cell proliferation and impairs multicellular morphogenesis and photosensory behavior (phototaxis). (From Fig. 8 of Bokko et al. [2007].)

Some cellular activities, exemplified by phagocytosis and pinocytosis, are impervious to AMPK signaling and therefore are unaffected by mitochondrial dysfunction even though they are energy-consuming.

3.7. Implications The bewildering complexity of genotype–phenotype relationships in human mitochondrial diseases has limited our understanding of the underlying cytopathological mechanisms which have thus far been assumed to result from ATP insufficiency. In the Dictyostelium model these complexities are avoided, but at the same time the phenotypic richness of the life cycle has provided a range of energy-requiring cellular activities that could potentially have been impaired by shortage of energy. Their study has revealed a regular pattern of mitochondrial disease phenotypes all of which are caused by chronic activation of the cellular energy-sensing alarm protein AMPK.

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There is every indication that this new insight may be true also of human mitochondrial disorders. AMPK is known to stimulate many of the cellular processes (Bergeron et al., 2001; Hardie, 2004; Hardie and Hawley, 2001; Hardie and Sakamoto, 2006; Kahn et al., 2005; Zong et al., 2002) that are characteristic, cytopathological features of mitochondrial disease ( James and Murphy, 2002; Maassen et al., 2004; McKenzie et al., 2004; Rossignol et al., 2003), including mitochondrial proliferation and ragged red muscle fibers, cellular hypotrophy, and induction of apoptotic and autophagic cell death. Cell cycle progression in the developing Drosophila eye is halted specifically by AMPK signaling in response to the ATP-depleting effects of a mitochondrial mutation (Mandal et al., 2005). Furthermore, mitochondrial electron transport uncouplers such as dinitrophenol have been shown, like other cellular stressors, to activate AMPK (Hayashi et al., 2000). Finally, symptoms that are strongly reminiscent of mitochondrial disease appear in a rare human genetic disorder, AICA-ribosiduria, in which an inactive AICAR transformylase causes the AMPK activator ZMP to accumulate in cells (Marie et al., 2004). In addition to dysmorphic features, the affected patient, a female infant, suffered from severe neurological dysfunction, epilepsy, and congenital blindness. There are currently no effective treatments for managing mitochondrial disorders. If the cytopathology in humans is also caused by chronic AMPK activation, treatment efforts aimed at ameliorating a presumed ATP insufficiency are likely to be ineffective (as is the case). In the Dictyostelium model, however, the aberrant phenotypes were completely suppressed by AMPa antisense inhibition. This suggests that pharmacological AMPK inhibition might be a useful therapeutic strategy. Such intervention would not cure the disease and would not be helpful for ameliorating disease symptoms that are genuinely a result of insufficient energy. One such symptom might be exercise intolerance. However, many of the important clinical features of mitochondrial disease may well result from AMPK activation including, for example, apoptotic and autophagic neuronal cell death. In fact, mitochondrial dysfunction is believed to play an important pathogenic role in all major neurodegenerative disorders, not just those that are overtly mitochondrial (Lin and Beal, 2006). The complete suppression of mitochondrial disease phenotypes by AMPKa antisense inhibition in Dictyostelium offers hope that AMPK inhibitors might be found to ameliorate the devastating consequences of mitochondrial and other major neurodegenerative disorders.

4. Concluding Remarks Dictyostelium has proven to be a powerful model for studying mitochondrial biology and disease. The organization of its mitochondrial gene expression system seems to be closer to that of metazoa than the yeast

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counterparts, and the organism has already yielded valuable insights into aspects of mitochondrial evolution and biogenesis. The phenotypic studies of mitochondrially diseased cells have revealed that the normal functioning of the mitochondria is of greater importance for some cellular functions than it is for others. Growth and cell cycle progression, the growth to development transition, cell type choice in the multicellular stages, slug photosensory and thermosensory responses are all impaired if mitochondrial ATP-generating capacity is compromised, some more sensitively than others. Phagocytosis and pinocytosis, on the other hand, are unaffected. These phenotypic patterns reflect the extent to which particular intracellular signal transduction pathways are susceptible to chronic AMPK signaling, the cause of the diverse cytopathologies arising from mitochondrial dysfunction. Still unclear are the points at which AMPK signaling pathways intersect those that control the affected cellular activities. In each case, there are many possible direct or indirect targets for AMPK, including the elements of the YakA, PKA, DdTOR, filamin, and RasD pathways previously described. These should all be rich and productive areas for future investigation.

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Oxytocin and the Human Prostate in Health and Disease Helen D. Nicholson* and Kate Whittington†

Contents 1. Introduction 2. The Human Prostate 2.1. Structure 2.2. Functions 3. Oxytocin and Oxytocin Receptor 3.1. Regulation and structure 3.2. Signaling pathways used by the oxytocin receptor 4. Oxytocin and the Prostate 4.1. Overview of oxytocin and neurophysin in the male reproductive tract 4.2. Local production of oxytocin and neurophysin in the prostate 4.3. Functions of oxytocin in the prostate 4.4. Oxytocin and prostate disease 5. Possible Roles of Oxytocin in the Pathophysiology of Prostate Disease 5.1. Benign prostatic hyperplasia 5.2. Carcinoma of the prostate 5.3. Problems with animal models of human prostate disease 5.4. Possible therapeutic roles for oxytocin 6. Concluding Remarks References

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Abstract Oxytocin is a peptide hormone produced by the neurohypophysis. The discovery that the peptide is produced locally within the male and female reproductive tracts has raised the possibility that oxytocin may have paracrine and autocrine actions outside of the nervous system. Oxytocin and its receptor have been identified in the human prostate. The prostate is an androgen-dependent organ

* {

Department of Anatomy and Structural Biology, University of Otago, New Zealand Clinical Science South Bristol, University of Bristol, Bristol BS8 1TH, United Kingdom

International Review of Cytology, Volume 263 ISSN 0074-7696, DOI: 10.1016/S0074-7696(07)63006-X

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2007 Elsevier Inc. All rights reserved.

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whose function is to secrete components of the seminal fluid. Oxytocin has been shown to modulate contractility of prostate tissue and also to regulate local concentrations of the biologically active androgens. Oxytocin has also been shown to regulate cell growth. Prostate disease is common and results from abnormal growth of the gland. Oxytocin concentrations are altered in both benign and malignant prostate diseases and in vitro studies suggest that the peptide may be involved in the pathophysiology of these diseases. Key Words: Oxytocin, Oxytocin receptor, Prostate, Prostate cancer, Benign prostatic hyperplasia. ß 2007 Elsevier Inc.

1. Introduction The prostate is an enigmatic gland whose function is poorly understood. Interest in the gland has increased because of the increased incidence of benign and malignant diseases of the prostate in the population. Oxytocin (OT) is a peptide hormone produced by the neurohypophysis. It has classically been considered a hormone involved with pregnancy and lactation. The discovery that the peptide is produced locally within the male and female reproductive tracts has raised the possibility that OT may have paracrine and autocrine roles outside of the nervous system. Here we consider the evidence that OT may play a role in the pathophysiology of the human prostate gland.

2. The Human Prostate Before discussing the role of OT, it is necessary to consider the structure and function of the prostate.

2.1. Structure 2.1.1. Gross structure The human prostate is one of the male accessory sex glands. It lies at the base of the bladder and completely encircles the urethra. It is shaped like an inverted cone and in the young male is about the size of a walnut and weighs between 15–20 g. The ejaculatory ducts, which carry secretions from the seminal vesicles together with fluid and spermatozoa from the ductus deferens, run through the cranial part of the gland and open into the urethra at the veru montanum (Fig. 6.1). The prostate consists of glandular tissue responsible for secreting fluid, stromal tissue, and a fibromuscular capsule.

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Traditionally, the prostate has been described as consisting of five lobes: an anterior, middle or median, posterior, and two lateral lobes. This division of the gland was based on the presence in the embryo of five sets of ducts draining five groups of acini that enter the embryonic urethra (Lowsley, 1912). However, in the absence of disease, the outer surface of the prostate does not have any obvious lobes. Studies by McNeal (1968) revealed that histologically the gland can, however, be divided into four zones: peripheral, central, transition, and periurethral (see Fig. 6.1). The peripheral zone comprises around 70% of the total glandular tissue of the normal prostate and surrounds the distal portion of the urethra. The central zone occupies the area around the ejaculatory ducts and consists of 25% of the prostatic glandular tissue (Coakley and Hricak, 2000). Lying around the proximal urethral segment are the transition and periurethral zones. The transition zone is the larger of these two areas, comprising 5% of the glandular tissue in young men. This division of the gland into zones is important clinically because the peripheral zone is the main site of carcinoma of the prostate and prostatitis (McNeal, 1969). Benign hyperplasia, however, occurs predominantly in the transition and periurethral zones of the prostate (McNeal, 1978). Surrounding most of the external surface of the prostate is a fibromuscular capsule. Muscular sphincters are also present within the prostate. The preprostatic sphincter runs down from the bladder neck and surrounds the proximal urethra and periurethral zone. Its contraction at ejaculation is thought to prevent retrograde flow of seminal fluid into the bladder (Blacklock, 1947). A second sphincter which is continuous with the external urethral sphincter is found around the distal urethra (McNeal, 1998). 2.1.2. Microscopic anatomy The function of the prostate is to produce components of the seminal fluid and then expel these secretions into the urethra at the time of ejaculation. Its structure in the form of multiple distensible acini, or glands, connected to the urethra by ducts and surrounded by fibromuscular stromal tissue reflects its function (Fig. 6.2). Five types of cell have been described within the epithelial layer of the acini (Peehl, 2005). These include basal, neuroendocrine, secretory, stem, and transit amplifying epithelial cells. The secretory epithelial cells are the most abundant cells found in the acini. They are columnar in shape and contain many secretory granules. They express the androgen receptor and are responsible for the production and secretion of a variety of proteins including prostate-specific antigen (PSA) and prostatic acid phosphatase (Luke and Coffey, 1994). These secretory cells are dependent on androgen for the maintenance of their structure and for secretory function. The epithelial cells are also dependent on the neighboring stromal cells, and it

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A Bladder Ejaculatory zone Transition zone Periurethral zone

Central zone

Peripheral zone

Verumontanum

B

Prostatic urethra

Bladder

Detrusor muscle

Periurethral glands Central zone Transition zone

Urethra Verumontanum

Peripheral zone

Figure 6.1 Diagrammatic view of the human prostate showing the peripheral, central, transition, and periurethral zones. (A) Sagittal view showing the passage of the ejaculatory duct. (B) Coronal view showing drainage of the glands into the urethra.

appears that interaction between these cell types is necessary for expression of both the androgen receptor and PSA by the epithelial cells. Neuroendocrine cells are scattered throughout the epithelium. These cells contain a variety of neuropeptides including serotonin and somatostatin. Their biological role is unclear, but it is possible they modulate growth and

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Figure 6.2 Photomicrograph of the human prostate stained with mallory trichrome showing a single acinus surrounded by stromal tissue. Epith, epithelial tissue; Str, stromal tissue. Bar represents 50 mm.

differentiation of the surrounding cells (Abrahamsson and Lilja, 1989; di Sant’Agnese, 1986). Basal cells are cuboidal in shape and in man form a single layer that lies on the basement membrane and separates the stromal cells from the epithelium. The basal layer also contains stemlike cells (Schalken and van Leenders, 2003). It has been suggested that these cells comprise 1% of the basal layer and have the capacity to divide and differentiate into other epithelial cell types. Transit amplifying cells have also been identified. These cells are proposed to be stemlike cells in the process of differentiating. The stroma of the prostate consists of smooth muscle, connective tissues, and neurovascular tissues.

2.2. Functions 2.2.1. Normal prostate The prostate contributes approximately 12% of the seminal fluid volume. Fluid accumulates within the acini and is expelled into the urethra at the time of ejaculation. The prostate secretes a variety of factors, some of which have defined roles in the coagulation and liquefaction of seminal fluid and others whose function is less clear. Among the secretory products are citric acid, zinc, and polyamines and a variety of proteins including PSA and prostatic acid phosphatase (Luke and Coffey, 1994). PSA is a glycoprotein that is only found in the epithelium of the prostate. It is a member of the kallikrein family and is thought to be important in the liquefaction of semen (Bilhartz et al., 1991) within the female reproductive tract. It is, perhaps, better known as a factor used in screening for prostate cancer. In the healthy prostate when the basal cell layer and basement membrane is intact, most

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PSA is secreted into the seminal plasma. However, with the development of prostate cancer, the basement membrane may be disrupted, and PSA is then able to pass into the circulation resulting in increased serum concentrations. Prior to the identification of PSA, prostatic acid phosphatase was used to screen for prostate disease. It is an enzyme that may be involved in the cleavage phosphorylcholine present in the seminal fluid (Bilhartz et al., 1991). 2.2.2. Regulation of prostatic growth The human prostate undergoes a characteristic pattern of growth that can be separated into four stages (Xia et al., 2002). From birth to 9 years of age, the prostate grows slowly. There is then a period of rapid growth (0.84 g/year) which lasts until around 30 years of age. Between 30 and 50 years of age, a slow rate of growth is again seen. In some men, growth increases after the age of 50, leading to the development of benign prostatic hyperplasia (see following). As with any organ, the size of the prostate is dependent upon the balance between cell proliferation and cell death. The regulation of this process is complex and is not completely understood; however, some factors are known to be involved. Of these, probably the best known are the androgen family. Studies by Huggins and Hodges (1941) demonstrated that atrophy of the prostate occurred after castration and that this could be prevented by the administration of testosterone. It was subsequently revealed that testosterone is not the biologically active androgen involved in this process, but that conversion of testosterone by the enzyme 5 a-reductase into dihydrotestosterone (DHT) is required (Bruchovsky and Wilson, 1968). In the prostate, most of this conversion occurs in the stromal tissue (Russell and Wilson, 1994). The action of DHT is mediated by the androgen receptor. DHT binds to the intracellular receptor which is then translocated to the cell nucleus where it binds to DNA and stimulates the synthesis of new proteins. Several other hormones have been implicated in the regulation of prostate growth including prolactin and growth hormone, but perhaps the most important of these is estrogen. Estrogen is produced within the prostate from testosterone by the enzyme aromatase. Aromatase is present mainly in the stromal cells (Matzkin and Soloway, 1992), and activity of the enzyme increases with age (Hemsell et al., 1974). Both forms of the estrogen receptor have been identified in the human prostate (Lau et al., 2000). Estrogens have a direct effect to increase stromal growth. They also act indirectly by increasing expression of the androgen receptor (Moore et al., 1979) and stimulating 5 a-reductase activity (Isaacs et al., 1983). Although androgens are considered a key driver of prostate growth, it is clear that other factors are integral to this regulation and may themselves be controlled by androgens (Davies and Eaton, 1991). Considerable evidence suggests factors produced within the prostate itself are involved in this ‘‘fine-tuning’’ of androgen-driven growth. Studies in the rat suggest androgens may act on

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androgen receptors in the stroma to produce factors which then modulate growth of the epithelial cells. A variety of growth factors have been identified in the prostate and many of these, such as EGF, FGF, IGF, and TGFa, act as mitogens on epithelial and stromal cells (Steiner, 1993). Only TGFb has thus far been demonstrated to inhibit growth (Moses et al., 1990). Further understanding of the role of such paracrine factors is important because it is likely to help unravel the complex molecular changes that result in prostate disease. As such, the paracrine regulation of prostate growth is a rapidly expanding area of prostate research, and this includes factors such as oxytocin.

3. Oxytocin and Oxytocin Receptor OT is part of a large family of neurohypophysial peptides that share a high degree of homology. It is a nonapeptide, and the presence of a disulfide bridge between cysteine residues 1 and 6 results in a peptide with a six amino-acid cyclic ring and a three residue tail as the COOH terminal (du Vigneaud et al., 1953) (Fig. 6.3). Differences in the amino-acid residue at position 8 determine whether the resulting peptide belongs to the OT or vasopressin (Vp) side of this family, with basic amino acids (Lys, Arg) denoting the Vp-related peptides and neutral amino acids denoting OT-related peptides (Gimpl and Fahrenholz, 2001). It is likely that this single amino acid difference contributes to determining the affinity of each peptide for its respective receptor (Barberis et al., 1998). The genes for OT and Vp are located on the same chromosomal locus, 20p13 (Rao et al., 1992), and are similar in their intron-exon structure, but are transcribed in opposite directions (Gimpl and Fahrenholz, 2001) (see Fig. 6.3). Both OT and Vp are synthesized as part of larger prohormone molecules that contains an OT-neurophysin I (OT-NpI) or Vp-neurophysin II (Vp-NpII), respectively (Robinson, 1987) (see Fig. 6.3). Cleavage of OT from its neurophysin involves an endopeptidase which acts at a specific cleavage site between OT and neurophysin I (Clamagirand et al., 1986; 1987). The OT prohormone and its resultant cleavage products have been studied most widely in axons of the magnocellular neurones arising from the supraoptic and paraventricular nuclei of the hypothalamus. This work has suggested the function of neurophysin relates to the packaging and storage of OT within secretory granules—a necessity for OT’s transport from its hypothalamic synthesis site to its storage site in the posterior pituitary (Gimpl and Fahrenholz, 2001). In terms of the regulation of OT synthesis, the 50 flanking region of the OT gene contains regulatory elements which are targets for transcription factors of the nuclear hormone receptor family (Burbach et al., 1995). Within this region is a classic palindromic estrogen response element (ERE) (Richard and Zingg, 1990). Studies in the hypothalamus (Nomura et al., 2002), uterus (Larcher

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Vp Gene

OT Gene Ex 1

–19 1 9 1 Signal OT peptide

Ex Ex 2 3

10

Ex 3

76

Ex 2

Ex 1

95

Neurophysin I

Cleavage site Tyr

IIe Gln

Cys Cys

Asp

Pro Leu

Gly

Figure 6.3 Diagrammatic illustration of the gene, located on chromosome 20p13, encoding for oxytocin (OT) and vasopressin (Vp) showing the arrangement of exons (Ex) and the direction of transcription.The contribution of each exon sequence to the prohormone is shown together with the cleavage site that results in the release of free OT, whose amino acid sequence and structure is shown (modified and used with permission from Gimpl and Fahrenholz, 2001).

et al., 1995), and chorio-decidua (Chibbar et al., 1995) suggest a role for this steroid in regulating OT synthesis, although this appears to be a speciesspecific effect. The thyroid hormone receptor is known to be able to bind to an ERE (Scott et al., 1997), and in vivo experiments in rats have shown thyroid hormone can also increase hypothalamic OT mRNA levels (Adan et al., 1992). Functional retinoic acid (RA) response elements have been identified in this 50 region, and in the rat uterus at least, in vivo administration of RA results in up regulation of OT gene expression (Larcher et al., 1995). Nuclear orphan receptors, such as COUP-TFI (Gimpl and Fahrenholz, 2001), COUP-TFII (Chu et al., 1998), and Ear-2 (Chu et al., 1998), have also been shown to bind to 50 regulatory elements and affect OT gene expression.

3.1. Regulation and structure The cDNA sequence encoding the human oxytocin receptor (OTR) was first isolated and identified in myometrial tissue by Kimura et al. in 1992. The encoded receptor is a 388 amino acid polypeptide with seven

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transmembrane domains common to many G-protein coupled receptors (GPCR). A single copy of the OTR gene is present in the human genome and has been mapped to the gene locus 3p25–3p26.2 (Inoue et al., 1994; Michelini et al., 1995). In total, the gene is 17 kb and is composed of 4 exons interspaced by three introns. Transcription is initiated 618 and 621 bp upstream of the initiation codon, and in humans a TATA-like motif and potential SP-1 binding site are in close proximity (Gimpl and Fahrenholz, 2001 and references contained within). A number of binding sites, namely c-Myb, AP-2, and AP-1, are contained within the 50 flanking region. Gonadal steroids appear to play a key role in regulating the transcription of the OTR gene, although a high degree of tissue- and species-specific effects are observed. Estrogens have been shown to stimulate OTR expression in the uterus (Engstrom et al., 1999; Larcher et al., 1995; Soloff et al., 1979), pituitary (Breton et al., 1995), kidney (Breton et al., 1996; Ostrowski and Lolait, 1995), and the hypothalamus (ventromedial nucleus) (Bale et al., 1995; Breton and Zingg, 1997; Young et al., 1997). However, no complete ERE has been identified in the OTR gene, although three half-palindromic motifs have been mapped (Inoue et al., 1994). It has been suggested that estrogens may act to promote OTR transcription via a novel, nonclassical pathway involving a high-affinity binding site for nuclear orphan receptors (Koohi et al., 2005). Progesterone has also been shown to regulate OTR expression but, unlike estrogen, it induces down regulation of the OTR in the uterus (Larcher et al., 1995). However, to date, no progesterone response elements have been identified within the OTR promoter region (Ivell and Walther, 1999). There is a paucity of published information describing mechanisms by which transcription of OTR is regulated within the prostate. Our own data (C. Johnson and H.D. Nicholson, personal communication) on the ventral lobe of the rat prostate show an increase in the intensity of the OTR signal by Western blotting after treatment with ethan1,2-dimethanesulphonate (EDS). This treatment reduces testosterone production via a loss of Leydig cells, and although it indicates the involvement of gonadal steroids in prostatic OTR regulation, it does not differentiate between an androgenic or estrogenic effect. Given the dependency of the prostate on androgens, however, a role for this gonadal steroid in the regulation of the OTR may seem likely. However, a previous study by Filippi and associates suggested that even in androgen-dependent organs such as the rabbit epididymis, estrogens rather than androgens regulate OTR expression (Filippi et al., 2002). As mentioned previously, structurally the OTR is a typical GPCR and forms part of a larger family of heptahelical membrane receptors. Varying degrees of homology exist between the OTR and receptors for other peptides from the isotocin-mesotocin-oxytocin family (Gimpl and Fahrenholz, 2001). This results in homology with the vasopressin V1 (50%) and V2 (40%) receptors and indicates the possibility of a level of

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cross-affinity between OT and Vp (Gimpl and Fahrenholz, 2001). In terms of OT binding, the cyclic portion of OT interacts with OTR’s transmembrane domains 3, 4, and 6, with the linear portion of OT interacting with transmembrane domains 2 and 3 together with the first extracellular loop (Zingg and Laporte, 2003). In addition to the seven transmembrane domains, the OTR contains a short N-terminal extracellular domain and a relatively long C-terminal intracellular domain. The receptor contains several sites for potential posttranslational modification, including N-glycosylation sites prior to the first transmembrane domain and several possible phosphorylation sites in the third cytoplasmic loop and C-terminal region (Kimura et al., 1992). In humans, three N-glycosylation sites have been identified but in vitro manipulation suggests full glycosylation is not essential for full OTR activity (Kimura et al., 1997). Indeed the level of glycosylation observed is suggested to be both species- and tissue-specific (Ivell et al., 2001) and is likely to explain the slight variations reported in OTR’s molecular weight (Table 6.1). Like most GPCR, the OTR undergoes rapid desensitization following stimulation with an agonist (Evans et al., 1997). This internalization process is clathrin dependent (Smith et al., 2006), and the receptor does not appear to be recycled back to the cell surface (Gimpl and Fahrenholz, 2001).

3.2. Signaling pathways used by the oxytocin receptor The wide range of roles ascribed to the peptide OT (see following) are intriguing when it is remembered that only one isoform of the OTR has been identified. It is likely, therefore, that although each function requires the initial binding of the OT ligand to a single OTR isoform, the subsequent signaling pathways activated are diverse and will determine the final outcome of OTR activation (Bussolati and Cassoni, 2001). Currently only some of these signaling pathways and the intermediates involved in their activation have been determined, as illustrated in Fig. 6.4, although the majority of this work has been performed in cell systems other than those derived from the prostate. The involvement of the OTR in contraction has been most widely studied in the myometrium where sustained and coordinated contractions are essential for successful parturition. In this case the OTR is coupled, via proteins of the Gaq/11 family, to phospholipase C-b (PLC-b) which catalyses the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate and diacylglycerol (DAG). Inositol 1,4,5-triphosphate will stimulate the release of intracellular calcium stores from the sarcoplasmic reticulum, leading to contraction (Lopez Bernal, 2003). The DAG pathway can activate protein kinase C which has multiple effects including phosphorylation of the mitogen-activated protein kinase (MAPK) pathway (Hoare et al., 1999), which is known to be a regulator of cell proliferation. The OTR has also been shown to interact with Gai in a number of cellular systems (Strakova and Soloff, 1997; Strakova et al., 1998),

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INSIDE CAVEOLAE1

OUTSIDE CAVEOLAE1

MOVEMENT OF OTR

Coupling to

Coupling to VpV1/2 or OTR?6

1, 2

Gα q11

Transactivation3, 4

EGF Receptor

Transactivation3, 4

PLC βγ 3

PLC β 3, 7

IP3

PIP2

Cai

10 PKC

6

G?

cAMP 8, 9

PKA 8, 9

DAG ERK PHOSPHORYLATION Transient

CONTRACTION 7

2, 3, 5

Gα1

2, 3

PROLIFERATIVE 2, 3

Sustained

2, 3

ANTIPROLIFERATIVE 2, 3, 9

Figure 6.4 Schematic illustration proposing likely routes for OTR signaling upon ligand binding. 1Guzzi et al., 2002; 2Strakova et al., 1998; 3Rimoldi et al., 2003; 4Zhong et al., 2003; 5Strakova and Soloff,1997; 6Reversi et al., 2005; 7Lopez Bernal, 2003; 8Cassoni et al.,1997; 9Cassoni et al., 2004; 10Hoare et al.,1999.)

resulting in the phosphorylation of ERK (Reversi et al., 2005). In studies using transfected cells, activation of the OTR has been shown to produce opposing effects on cell proliferation depending upon the system studied, and this is likely to be due to variations in the profile of ERK phosphorylation with a more sustained phosphorylation pattern being linked to an antiproliferative effect (Guzzi et al., 2002; Rimoldi et al., 2003), whereas more transient ERK phosphorylation is thought to stimulate proliferation (Guzzi et al., 2002; Rimoldi et al., 2003). Additionally, in some cellular systems coupling of the OTR to Gai leads to an antiproliferative outcome via the action of protein kinase A (PKA) which is activated via an increase in cAMP (Cassoni et al., 1997, 2004). Whether this occurs in cells which endogenously express the OTR is not yet clear. The mechanism that determines which G-protein the OTR couples to is unclear but may be decided in terms of geography, via compartmentalization. The plasma membrane contains regions, called caveolae, that are rich in caveolin proteins, glycosphingolipids, and cholesterol (Williams and Lisanti, 2004). Initially thought to be important in cell transport processes, more recent evidence suggests caveolae are also involved in signal transduction due to the concentration of many receptors and signaling molecules within them (Liu et al., 2002; Smart et al., 1999). Evidence suggests that movement of the OTR into and out of caveolae can determine the profile of ERK phosphorylation upon OTR activation and hence the ultimate

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effect of OT on cell proliferation. The localization of the OTR within caveolae appears to result in transient (i.e., less than 30 min) ERK phosphorylation (Rimoldi et al., 2003), which stimulates proliferation (Guzzi et al., 2002). In contrast, localization of the OTR outside caveolae results in more sustained (greater than 3 h) ERK phosphorylation (Rimoldi et al., 2003), which was antiproliferative (Guzzi et al., 2002). To date, little work has been done investigating the OTR signaling pathways in the human prostate. This is surprising because OTR activation affects proliferation, and the human prostate has a high prevalence of growth-related pathologies, suggesting that identification of such signaling pathways has potential clinical relevance.

4. Oxytocin and the Prostate 4.1. Overview of oxytocin and neurophysin in the male reproductive tract It has been known for more than 40 years that OT affects the male reproductive tract. Milovanov et al. (1962) showed that intravenous administration of large doses of OT to bulls increased both the volume of the seminal fluid and the number of sperm ejaculated (Milovanov et al., 1962), and studies by Kihlstrom and Melin (1963) and Knight (1974) showed that OT had similar effects in the rabbit and ram, respectively. Circulating OT concentrations rise around the time of ejaculation (Carmichael et al., 1987; Ogawa et al., 1980), and it is postulated that this rise in peripheral OT acts to stimulate the contractility of the seminiferous tubules (Niemi and Kormano, 1965), epididymis, and ductus deferens (Knight, 1974) to increase sperm number in the ejaculate. It was not until the 1980s that the possibility OT might be acting in a paracrine, as well as an endocrine, manner was considered. This became a possibility with the demonstration of OT and its associated neurophysin in human and rat testes in 1984 (Nicholson et al., 1984). Since then OT has been identified in the testes of a variety of species including bull (Ungefroren et al., 1994), marmoset (Einspanier and Ivell, 1997), bandicoot (Gemmell and Sernia, 1989), and quail (Douthwaite et al., 1989). The demonstration of OT mRNA (Foo et al., 1991; Ivell et al., 1997) and secretion of the peptide in vitro confirmed that indeed OT was produced locally within the testis. Oxytocin has also been demonstrated in the epididymis (Harris et al., 1996; Knickerbocker et al., 1988) and prostate (see Section 5). 4.1.1. Oxytocin and contractility The identification of OT within the testis reawakened interest into possible roles of the peptide within the male reproductive tract. The contractile effects of OT in the female reproductive tract are well established, and OT

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has been shown to stimulate contraction of both the myometrium and the myoepithelial cells in the breast. Spermatozoa produced by the testis are not initially motile and need to be actively transported into the epididymis where sperm maturation occurs. This transport is facilitated by the flow of fluid within the seminiferous tubules and contractile activity of these tubules (Hargrove et al., 1977). Roosen-Rungen (1951) was the first to demonstrate that seminiferous tubules exhibited spontaneous contractility, and Niemi and Kormano (1965) showed that this contractile activity could be increased by OT, in vitro. The advent of time-lapse video micrography allowed the identification of two types of tubular contractility: type A and B (Worley et al., 1988). Type A contractility consists of high-frequency movements resembling ripples which affect sections of the tubule. Type B contractility consists of bigger movements which affect larger areas of the tubules and are associated with movement of the tubular contents. It is thought that these type B movements are important in transporting spermatozoa from the testis to the epididymis (Worley et al., 1985). The type A movements, however, have been implicated in spermiation, the process by which spermatozoa are shed from the seminiferous epithelium into the lumen. OT may also play a role here. Seminiferous tubules are quiescent at birth, and activity begins around day 8 postpartum in the rat, soon after the onset of spermatogenesis, and increases until puberty (Worley et al., 1985). Testicular OT concentrations mirror the increase in contractility ( Worley et al., 1985). Furthermore, depletion of testicular OT concentrations using the drug ethane dimethane disulfonate results in decreased tubular movements (Nicholson et al., 1987), but contractility can be restored by the administration of exogenous OT. In vitro, OT affects contractility at specific stages of the spermatogenic cycle and has its largest effect at stages VII–VIII in the rat, the period when spermatozoa are shed. There is also evidence from in vivo studies to support such a role for OT. In the peripubertal rat, treatment with OT brings about the onset of spermiation, whereas administration of a specific OT antagonist delays spermiation (Frayne et al., 1996). In the ram, injection of OT into the testicular artery produces an acute increase in the number of spermatozoa shed into the rete testis tubular fluid (Whittington et al., 2001). Similarly, in transgenic mice that overexpress OT, spermiation is advanced whereas in OT knockout mice the process is delayed (Assinder et al., 2002). These mice do, however, undergo spermiation, suggesting that OT is not the only factor involved in this process. Evidence also shows OT can modulate contractility in the epididymis. The epididymis is the site of sperm maturation, and the transit time is regulated to facilitate this process. Thus, factors that modulate contractility can potentially affect the maturation of spermatozoa. In vitro, OT increases both the frequency and amplitude of contractions of the epididymal wall (Hib, 1974; Jaakkola and Talo, 1981; Knight, 1974). In vivo, OT increases

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epididymal contractions (Knight, 1974) in the ram. This effect is specific, being inhibited by an OT antagonist, and is accompanied by an increase in both fluid output and spermatozoa (Nicholson et al., 1999). The effects of OT augment those seen with adrenaline (Knight et al., 1974), and thus the rise in circulating OT during sexual arousal may act synergistically with the activation of the sympathetic nervous system that occurs at ejaculation to promote sperm transport out of the epididymis. Furthermore, OT also promotes the release of another potent stimulator of epididymal contractility, endothelin-1 (Filippi et al., 2002). Interestingly, it appears that some of the locally produced OT from the testis, epididymis, and/or prostate may be secreted as OT is present in the tubular fluid of the testis (Nicholson et al., 1994), epididymis (Knickerbocker et al., 1988), and also in seminal plasma (Goverde et al., 1998). The precise role of such secreted OT is unclear. It has been shown that paracrine OT from the female reproductive tract has a role in stimulating contractions of the fallopian tube, thereby aiding gamete movement (Kunz et al., 2007; Wildt et al., 1998). It is possible that OT secreted from the male tract may contribute to such a role. A small number of studies have provided conflicting data on the effects of OT, or its antagonists, on sperm motility (Fuchs et al., 1989; Pierzynski et al., 2007; Sliwa, 1994). However, as yet, no receptors for OT have been identified on spermatozoa from any species. 4.1.2. Oxytocin and steroidogenesis As well as affecting contractility in the male reproductive tract, OT also plays a role in regulating steroidogenesis. Adashi and Hsueh (1981) were the first to show OT and other neurohypophysial peptides could inhibit Leydig cell testosterone production in vitro. This was followed by a time of confusion when various groups reported conflicting data on the effects of OT on testicular steroidogenesis (Sharpe and Cooper, 1987; Tahri-Joutei and Pointis, 1988). The reason for some of the conflicting data became apparent in 1991 (Nicholson et al., 1991) when it was shown that although OT might affect testosterone synthesis it also increased its subsequent conversion to the more biologically active androgen, dihydrotestosterone (DHT). Testosterone is converted to DHT by the enzyme 5 a-reductase, and this reaction is important because in all regions of the male reproductive tract, apart from the testis, DHT is the biologically active androgen. Two isoforms of the 5 a-reductase exist, type I and type II (Normington and Russell, 1992). Although both forms are present in the male reproductive tract, type II is the more abundant. Oxytocin increases the activity of both isoforms of the enzyme but appears to do so by different mechanisms. In the case of type I, OT acts by increasing the activity of existing enzyme whereas OT increases synthesis of the type II isoform (Assinder et al., 2004).

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4.2. Local production of oxytocin and neurophysin in the prostate The first report of localization of OT in the prostate was in 1985 when Nicholson et al. used radioimmunoassay and HPLC to identify the peptide in the human prostate. Since then OT has been identified in the prostate of various mammals including the rat (Nicholson, 1996), dog (Nicholson and Jenkin, 1995), and brush-tailed possum (Fink et al., 2005). Immunohistochemistry has localized OT to both the epithelial and stromal cells of the prostate. However, although most of the secretory epithelial cells express OT, localization of the peptide in stromal cells is more sporadic. Neurophysin has also been identified in the prostate and appears to have a similar cellular localization to that of OT. Expression of mRNA for OT has been demonstrated in the possum (Fink et al., 2005). The presence of mRNA and the demonstration of the colocalization of OT and its associated neurophysin support local synthesis of the peptide in the prostate (Fink et al., 2005). In man, OT (Farina-Lipari et al., 2003) and neurophysin have been identified in the prostate ( Whittington et al., 2004). Although immunoreactive OT is more consistently found in epithelial cells, some staining is detected in stromal cells in sections of human prostatic tissues. OT and neurophysin are present in isolates of both epithelial and stromal cells (Whittington et al., 2004). Furthermore, OT is secreted by human prostate tissue explants when they are cultured in vitro (Assinder and Nicholson, 2004). Prostatic OT secretion/production is regulated by gonadal steroids. In the rat, treatment with either testosterone or DHT reduces OT concentrations within the ventral prostate, and this effect is inhibited by administration of an antiandrogen ( Jenkin and Nicholson, 1999). Similarly, castration or treatment with an antiandrogen alone results in increased concentrations of the peptide ( Jenkin and Nicholson, 1999). Estrogen has been shown to affect OT production in the brain (Bale et al., 1995) and appears to have similar effects in the prostate, where it increases local OT concentrations (Jenkin and Nicholson, 1999) (Fig. 6.5). Regulation of OT secretion in the human prostate does not appear to be the same as in the rat. Experiments investigating the effects of gonadal steroids on explants of prostatic tissue taken from men undergoing transurethral resection of the prostate for benign prostatic hyperplasia (BPH) show that, as in the rat, estrogen stimulates OT secretion (Assinder and Nicholson, 2004). However, both testosterone and DHT also increase OT secretion, an effect that is blocked by concomitant treatment with an antiandrogen (Assinder and Nicholson, 2004). Thus, in man, both androgen and estrogen increase OT secretion with no obvious feedback inhibition present (Fig. 6.6). As yet, it is unclear whether this situation also occurs in prostatic tissue from young healthy men or whether it is a feature of BPH.

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Rat

Estrogen +ve

−ve Oxytocinase

Oxytocin

+ve

+ve

−ve

−ve 5 α-reductase +ve DHT

Testosterone

Growth

Figure 6.5 The effects of OTon the regulation of androgens in the rat prostate. DHT, dihydrotestosterone; þve, positive feedback effect; ve, negative effect. Human prostate with BPH Estrogen +ve Oxytocin

+ve +ve

+ve 5 α-reductase

+ve Testosterone

DHT Growth

Figure 6.6 The role of OT in the regulation of androgens in human prostatic tissue. Unlike the rat, in the presence of BPH androgens stimulate rather than inhibit growth. DHT, dihydrotestosterone; þve, positive feedback effect; ve, negative effect.

There is very little information concerning other factors which may regulate OT production in the prostate. Evidence raises the possibility that the availability of OT in the tissue may be modulated by enzymes that break down the peptide. An insulin-regulated aminopeptidase, which is structurally similar to the oxytocinase found in the plasma, has been identified in the ventral prostate of the rat (Arenas and Perez-Marquez, 2002). This putative oxytocinase is up regulated by androgens and down regulated following castration and may provide another mechanism to modulate local tissue concentrations of OT.

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Table 6.1 Summary of the published evidence regarding the localization and molecular weight of OTR in the prostate for different species Evidence for the localization of OTR Species

Epithelial

Stromal

Molecular weight

Rat Human Marmoset Macaque Wallaby Possum

þ5 þ 1,2 (þ) 4

þ5 þ 1–3 þ4 þ3

55 kDa6–60 kDa5 55 kDa3–66 kDa1 Not determined 55kDa3 Not determined 60kDa8

3

þ7 þ8

7

occasional (þ) 8

þ detected, (þ) weak detection, ( ) not detected. 1Whittington et al., 2004; 2Cassoni et al., 2004; 3 Frayne and Nicholson, 1998; 4Einspanier and Ivell, 1997; 5Assinder et al., 2004; 6Zhang et al., 2005; 7 Parry and Bathgate, 1998; 8Fink et al., 2005.

Although the OTR has been identified in the prostate of a range of species, there appears to be a degree of variation with regard to its cellular localization (Table 6.1). In the human prostate, a number of studies have suggested OTR is localized to both epithelial and stromal cells, with weaker expression in the stroma (Cassoni et al., 2004; Whittington et al., 2004). An earlier study, by Frayne and Nicholson (1998), suggested OTR expression was limited to stromal cells only in this species. The reason for this discrepancy likely resides in the use of various antibodies raised against the OTR and the utilization of different techniques for immunovisualization and fixation. The ventral lobe of the rat prostate shows an OTR localization pattern similar to that observed in humans with strong immunoreactivity in the epithelial layer and weaker expression in the stromal cells (Assinder et al., 2004). Interestingly, more intense staining was apparent on the luminal edge of the epithelial cells suggesting activation of these receptors by OT present in the prostatic secretions. In nonhuman primates, a different OTR distribution pattern has been described with immunoreactivity mainly present in the stromal cells, especially those surrounding the ducts (marmoset—Einspanier and Ivell, 1997; macaque—Frayne and Nicholson, 1998). In contrast, the marsupial prostate appears to mainly express its OTR equivalent, the mesotocin receptor, in the epithelial layers with minimal staining in the stroma (Fink et al., 2005; Parry and Bathgate, 1998). It is likely that the differential localization of OTRs in the prostate represents variations in OT’s function in these species. As discussed later, OT is known to stimulate contractions of the prostate and maintain its resting tone (Thackare et al., 2006). Localization of OTRs to the stromal compartment of the prostate supports such a function because a proportion of this tissue will contain smooth muscle cells, especially in the area immediately surrounding the glandular acini (Humphrey, 2003). The other functions ascribed to OT in the prostate are the modulation of the activity

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and expression of 5a-reductase (Nicholson and Jenkin, 1994, 1995) and the regulation of cell proliferation (Plecas et al., 1992). Both functions are likely to be important in stromal and epithelial cells and are supported by the localization of OTRs to these cell types. The proposed role of OT in regulating proliferation and modulating androgen activity leads to questions concerning possible up regulation or down regulation of OTR with the development of prostate pathology. A study by Cassoni et al. (2004) suggests an increase in OTR expression occurs in neoplastic epithelial cells when compared with benign hyperplastic cells. However, our own study (Whittington et al., 2004) does not support such a change in receptor expression and instead suggests OT levels decrease with the development of malignancy (see Section 5.4.2 for further discussion).

4.3. Functions of oxytocin in the prostate 4.3.1. Modulation of contractility Several functions have been postulated for OT in the prostate. As in the female and other areas of the male reproductive tract, OT acts on smooth muscle within the prostate. Studies by Bodanszky and colleagues (1992) have demonstrated that OT can increase the resting tone of the gland as well as stimulating contractile activity in vitro in a variety of species, including man. It is likely that this contractile response is mediated by OTRs being coupled, via proteins of the Gaq/11 family, to phospholipase C-b (PLC-b) (see Section 4.2). Furthermore, OT is more effective than noradrenaline and methacholine (a cholinergic agonist), factors traditionally thought to be involved in stimulating prostatic contractility (Caine et al., 1975). Circulating concentrations of OT rise at the time of sexual arousal and ejaculation (Murphy et al., 1987) and thus OT may play a physiological role by stimulating contraction of the smooth muscle cells in the capsule and surrounding the prostatic acini facilitating expulsion of prostatic secretions. 4.3.2. Regulation of steroidogenesis As mentioned earlier, OT regulates the conversion of testosterone to DHT by increasing activity of the enzyme 5 a-reductase in the testis (Nicholson and Jenkin, 1994). Oxytocin increases 5 a-reductase activity and, as a consequence, DHT concentrations in the prostate (Nicholson and Jenkin, 1995). There are two isoforms of 5 a-reductase present within the human prostate. Type II is the more abundant (Thigpen et al., 1993) and is localized predominantly to the stroma (Silver et al., 1994). Oxytocin increases the activity of both type I and type II isoforms of 5 a-reductase; however, the peptide’s mechanism of action differs for each isoform (Assinder et al., 2004). Treatment of rats with OT for 3 d stimulates transcription of the type II isoform, resulting in the production of more enzyme. The effect of OT on the type I isoform appears to be related to activation of existing

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enzyme because mRNA for the type I isoform is down regulated following OT administration. Although OT stimulates 5 a-reductase activity in both the testis and the prostate of the rat, there are some differences in the response to the peptide in the two organs (Nicholson and Jenkin, 1995). In the testis, long-term administration of OT, in vivo, is accompanied by a sustained increase in 5 a-reductase activity. In the prostate, however, although OT treatment initially produces an increase in enzyme activity, after 7 d, 5 a-reductase activity and prostatic DHT concentrations return to control values (Nicholson and Jenkin, 1995). In the rat, prostate size is maintained relatively constant, and thus a feedback mechanism may exist to ensure longterm changes in DHT concentrations and excess growth do not occur. The regulation of OT production by androgens may facilitate this, with increasing concentrations of the biologically active androgen DHT down regulating local OT production and possibly also increasing concentrations of oxytocinase to further reduce local concentrations of the peptide ( Jenkin and Nicholson, 1999) (see Fig. 6.5). Increased concentrations of OT and/or androgen may also down regulate OTR expression. 4.3.3. Oxytocin and growth In the rat, OT treatment results in increased growth of the prostate (Hristic et al., 1985; Popovic et al., 1982). When OT is administered at the time of castration, animals treated with OT retain larger prostatic acini and epithelial volumes than untreated, castrate animals (Popovic et al., 1990). These changes have been shown to be due to an increase in mitotic activity, as well as a decrease in cell death (Plecas et al., 1992). It has been suggested that these changes reflect a direct action of OT because they occur in the absence of changes in circulating gonadotrophin levels (Hristic et al., 1985). We have shown that treatment with OT also results in an increase in the height of the epithelial cells and that this effect is inhibited by treatment with the OT antagonist desGly-NH2d(CH2)5[D-Tyr2,Thr4]OVT (Manning et al., 1995) (Fig. 6.7), again suggesting this is a specific OT effect. It is unclear in the rat whether the effects of OT are mediated by DHT or whether the peptide directly stimulates cell proliferation, but in the human, it appears that OT may have a direct effect on proliferation. Although OT promotes growth in the rat, it inhibits the proliferation of normal human prostate cells in vitro. When human stromal cells are grown either alone or cocultured with epithelial cells in the presence of DHT and estradiol, physiological concentrations of OT result in a decrease in cell proliferation (King et al., submitted for publication). No effect of OT is seen when prostate epithelial cells are grown alone, but when they are incubated in the presence of stromal cells, OT again inhibits proliferation (Whittington et al., 2007). The fact that both cell types are necessary for OT’s effect on epithelial cells suggests that paracrine factors secreted by the stromal cells

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The effect of oxytocin on epithelial height in the prostate of the rat 16 ***

Epithelial height µm

14

***

12 10 8 ***

***

6 4 2 0

Control

OT 3 days

OTA 3 days

OT 7 days

OTA 7 days

Figure 6.7 The effects of OT on epithelial height in the prostate of the rat. Groups of rats (n ¼ 6) were treated with OTor a specific antagonist for 3 or 7 d. OT, oxytocin treatment; OTA, treatment with an oxytocin antagonist. *** P < 0.001when compared with control animals.

may be involved in this process. The finding that the effect can be reproduced by culturing epithelial cells in media from stromal cells grown in the presence of OT would support this hypothesis. In summary, both OT and its receptor are present within the human prostate, and the localization of the receptors support its actions in promoting contractility and modulating cell proliferation within the normal prostate.

4.4. Oxytocin and prostate disease Diseases of the human prostate are common and include benign prostatic hyperplasia (BPH), carcinoma of the prostate, and prostatitis. Benign prostatic hyperplasia affects more than 50% of men over the age of 60 (Berry et al., 1984), and prostate cancer is the most commonly diagnosed malignancy and the second most common cause of cancer-related deaths in American men (Greenlee et al., 2001). Both of these diseases result from abnormal growth of the prostate gland. In view of OT’s effects on growth, it has been hypothesized that the peptide may play a role in the pathophysiology of prostate disease. Before discussing the evidence which has led to this hypothesis, a brief overview of BPH and prostate cancer is included. 4.4.1. Benign prostatic hyperplasia Benign prostatic hyperplasia is a disease related to aging. It most commonly affects the transition and periurethral zones of the prostate and results in obstruction of the bladder neck and urethra, causing difficulty in passing

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urine. Enlargement of the gland is initially due to overgrowth of the stromal tissue, although with time hyperplasia of the epithelial cells also occurs (Foster, 2000). Early hyperplastic changes may begin around 30 years of age, it affects 50% of men aged 51–60, and by the age of 80 more than 80% of men will be afflicted (Berry et al., 1984). Clinically, the obstructive symptoms are related to the increase in volume of the stromal tissue (Shapiro et al., 1992). The symptoms of BPH are not solely related to the increase in size of the gland as the increase in stromal smooth muscle tone adds a dynamic component to the disease (Caine et al., 1975). One of the major factors regulating prostatic tone is the a-adrenergic system, and treatment with an a-adrenergic blocker often provides some symptomatic relief (Roehrborn et al., 1996). The etiology of BPH is unclear and several theories have been proposed (Isaacs and Coffey, 1989). It is clear that there is a significant relationship between stromal and epithelial cells (Cunha et al., 1987). This relationship is important in the development of the gland, and it has been proposed that with age changes in this relationship reawaken the ability of the stromal cells to grow or to produce growth factors that promote both stromal and epithelial growth (Isaacs and Coffey, 1989). An alternative, or more likely complementary, theory proposes that changes in the ratio of androgens and estrogens may be important in the development of the disease (Isaacs and Coffey, 1989). Androgens are essential for the development of BPH because the disease does not occur in men castrated prior to puberty. However, circulating and prostatic concentrations of the biologically active androgen DHT decrease with age (Pirke and Doerr, 1975; Shibata et al., 2000), suggesting that the relationship may not be straightforward. Furthermore, experiments in the dog have shown that treatment with DHT alone does not induce BPH and the addition of estrogen to DHT is necessary (Habenicht et al., 1986), suggesting that a change in the ratio of DHT: estrogen is important. In men, aromatase activity (Hemsell et al., 1974) and circulating estrogen concentrations increase with age (Shibata et al., 2000). In vitro studies support the hypothesis that altered ratios of DHT:estrogen may be involved in BPH. These studies demonstrate that in the presence of DHT, increasing concentrations of estrogen stimulate stromal cell growth (King et al., 2006). As mentioned earlier, the effects of androgens and possibly estrogens are mediated by growth factors, and there is growing evidence that production of growth factors and their receptors are altered in BPH (Steiner, 1993). 4.4.2. Carcinoma of the prostate Prostate cancer is also a disease of the aging male, with 96% of all cases occurring in men over the age of 60 (Majeed et al., 2000). More than 95% of cancers affecting the prostate are adenocarcinomas, that is, epithelial cell derived, and most of these (70%) occur in the peripheral zone of the gland.

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There are several stages in the development of prostate cancer. Initially, focal lesions of high-grade prostatic intraepithelial neoplasia (PIN) develop. These are characterized by proliferation of secretory epithelial cells within existing acini and fragmentation of the basal epithelial cell layer. The proliferating epithelial cells may also exhibit enlarged nuclei and abnormal differentiation (Humphrey, 2003). Focal lesions are common and increase with age but in many men do not proceed any further. However, in some men these lesions develop into localized cancer and then may progress further to metastatic and then to hormone refractory disease. The reason why progression of focal lesions occurs is not fully understood, although it appears racial and environmental factors are involved (Platz et al., 2000). As with BPH the etiology of prostate cancer has not been fully elucidated. Androgens are necessary for the maintenance and growth of prostate cancer, at least in its early stages, and androgen withdrawal is one of the major treatments of the disease. It is unclear, however, whether androgens play a role in the initiation of the disease or the progression of PIN to localized cancer. Prostate cancer rarely occurs in men who have been castrated, but there is conflicting evidence as to whether circulating or prostatic concentrations of androgen are altered in men who develop prostate cancer. It may be that androgens need to be present but play a permissive role in this process. Changes in expression of growth factors and their receptors and cell adhesion proteins within the prostate are also seen with the development of cancer. Expression of growth factors, such as TGFa, that promote growth increase (Leav et al., 1998), while concentrations of TGFb, which in the normal prostate inhibits growth, may be diminished in malignant tissue (Moses et al., 1990). Alterations in hormones such as estrogen and prolactin and their receptors also occur. In the case of estrogen, for example, only ERa is expressed in normal epithelial cells, but in carcinoma of the prostate, the malignant cells express both ERa and ERb (Lau et al., 2000). As with androgens, it is unclear whether changes in expression of these various factors are a consequence of the malignant transformation of the cells or whether they initiate the disease.

5. Possible Roles of Oxytocin in the Pathophysiology of Prostate Disease As mentioned in Section 4.3.3, OT may play a physiological role in regulating normal growth of the prostate of the rat and man. There is also growing evidence that OT may be involved in the pathophysiology of the gland. Prostatic concentrations of OT are higher in dogs with BPH compared with animals with normal prostates (Nicholson and Jenkin, 1995), and in man, higher levels of OT are found in tissue from men with

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BPH compared with those with prostate cancer (Farina-Lipari et al., 2003; Nicholson, 1996). Furthermore, OT expression appears to be lost with the progression of prostate cancer (Whittington et al., 2004). Whittington and colleagues (2004) did not detect any change in expression of the OT receptor; however, Cassoni et al. (2004) suggest there may be increased expression of the receptor in malignant tissue.

5.1. Benign prostatic hyperplasia The etiology of BPH is not completely understood. One theory suggests that the increasing ratio of estrogen to androgen promotes/facilitates the increase in stromal growth (Isaacs, 1984; King et al., 2006). Data suggest that OT may be involved in this process. In patients with BPH, increased prostatic concentrations of estrogen and OT are observed (Nicholson, 1996; Shibata et al., 2000). When normal stromal cells are cocultured with epithelial cells in the presence of increased concentrations of OT plus estrogen, the normal inhibitory effect of OT on cell proliferation is completely lost (Whittington et al., 2007). Furthermore, in these conditions the inhibitory effect of OT on epithelial cell proliferation is also decreased. The increased concentrations of estrogen seen in BPH alter the inhibitory response of OT, thereby resulting in growth of both stromal and epithelial components of the prostate. The symptoms related with BPH are not only due to the physical enlargement of the prostate but also reflect a dynamic change due to an increase in prostatic tone. Oxytocin is a potent stimulator of prostatic tone and smooth muscle contraction. It is therefore possible that increased local concentrations of OT may contribute to the increased prostatic tone observed in BPH.

5.2. Carcinoma of the prostate The involvement of OT in prostate cancer is less clear. Expression of OT has been shown to decrease with the progression of carcinoma of the prostate (Farina-Lipari et al., 2003, Whittington et al., 2004), while conflicting data on OTR expression have been reported (Cassoni et al., 2004; Whittington et al., 2004). When normal human epithelial cells are cultured with low concentrations of OT (