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Role of FKBP51 and FKBP52 in Glucocorticoid Receptor Regulated Metabolism

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Role of FKBP51 and FKBP52 in Glucocorticoid Receptor Regulated Metabolism Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Sciences Role of FKBP51 and FKBP52 in Glucocorticoid Receptor Regulated Metabolism Submitted by: Manya Warrier In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences Examination Committee Major Advisor: Edwin Sanchez, Ph.D. Academic Sonia Najjar, Ph.D. Advisory Committee: Linda Dokas, Ph.D. Zi-Jian Xie, Ph.D. Cynthia Smas, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: July 23, 2008 Role of FKBP51 and FKBP52 in Glucocorticoid Receptor Regulated Metabolism Manya Warrier The University of Toledo 2008 DEDICATION I dedicate this work to my husband who has given me the courage, support and freedom to reach my dreams, my parents and grandparents who have always given their unconditional love and to Dr. Sonia M. Najjar who helped me immensely during the course of this work. ii ACKNOWLEDGEMENTS I wish to express my deep sense of gratitude to Dr. Edwin R. Sanchez, for giving me an opportunity to do this project, his timely advice and support. Our discussions and his constructive criticisms have greatly helped to improve my scientific thinking. I am grateful to Dr. Weinian Shou and members of his laboratory for providing us with the animals to initiate this project. I am greatly obliged to Dr. Sonia M. Najjar, for her keen interest and valuable guidance throughout this work. I would also like to thank other members of my committee, Drs. Linda A. Dokas, Cynthia M. Smas and Zijian Xie for their insightful suggestions. Special thanks to Dr. Sumudra Periyasamy, Dr. Dapei Li and other members of my lab for their help throughout my doctoral program. I am also deeply indebted to the members of Dr. Najjar’s lab, especially Ms. Payal Patel. It would have been extremely difficult to complete this study without their help. I truly enjoyed working with them and am always grateful for their co-operation. Last but not least, my sincere gratitude to all members of the Pharmacology Department who have made this arduous journey truly enjoyable. iii TABLE OF CONTENTS Dedication …………………………………………...................... ii Acknowledgements …………………………………………….. iii Table of Contents ……………………………………………….. iv Introduction ………………………………………….....................1 Literature ………………………………………………………….7 Materials and Methods …………………………………………..44 Results……………………………………………………………49 Discussion………………………………………………………..69 Conclusions………………………………………………………85 Summary…………………………………………………………87 Bibliography……………………………………………………..91 Abstract…………………………………………………………124 iv INTRODUCTION Steroid receptors (SR) are hormone-activated transcription factors that belong to the family of nuclear receptors (NR) (Hollenberg et al., 1985). Hormone-free receptors reside either in the cytoplasm or nucleus in association with several proteins, each of which assists the receptor to optimize its functions. In its steady state, the receptor complex contains one molecule of receptor, heat shock protein 90 (HSP90) dimer, p23 and a tetratricopeptide repeat (TPR) protein. Several TPR proteins may exist, but to date only four such proteins have been shown to be incorporated into the steroid receptor complexes. They are FK506-binding proteins (FKBP51 and FKBP52), cyclophilin-40 (Cyp40) and protein phosphatase 5 (PP5). The first three are also recognized as larger members of the immunophilin family because of their ability to bind immunosuppressive drugs, such as FK506 and cyclosporine A (CsA). Nevertheless, all four proteins possess a common TPR domain, through which they bind to the single acceptor site (TPR binding site) created by the HSP90 dimer. Besides the TPR site, immunophilins harbor a PPIase domain that has intrinsic peptidyl-prolyl isomerase (PPIase) enzymatic activity (Galat, 1993). Immunosuppressive drugs may bind to this region and inhibit its activity (Schreiber and Crabtree, 1992). Protein phosphatase 5 lacks PPIase activity but is a serine/threonine phosphatase. Three of these proteins, except Cyp40, also possess a PPIase-like region of unknown function. During protein synthesis, peptide bonds on the amino side of the proline residues are formed in cis-conformation. PPIase enzymes catalyze the conversion of cis-bonds to their energy favorable trans-form, thereby altering protein structure (Galat, 1993). Due to this special feature, proteins with PPIase activity, including immunophilins, are known to 1 regulate transcriptional activities by changing protein-protein interactions (Wu et al., 2000). However, such reports for TPR-containing immunophilins are rather limited (Mamane et al., 2000). The major role identified so far for these proteins is in mediating hormone responsiveness and localization of steroid receptors. Evidence to support the latter is plentiful with many reports showing an ability of FKBP52, Cyp40 and PP5 to interact with the motor protein, dynein, to facilitate transport of receptor along cytoskeletal tracts (Galigniana et al., 2002; Galigniana et al., 2004b; Galigniana et al., 2001) In regulating receptor hormone-binding affinity and, in turn, transcriptional activity, FKBP51 and FKBP52 are perhaps better researched over other TPR proteins. The first such evidence came from studies that identified FKBP51 as the cause of glucocorticoid (GC) resistance in squirrel monkeys. Squirrel monkey lymphocytes (SML) express elevated amount of FKBP51 that is incorporated into glucocorticoid receptor (GR) complexes and lowers its hormone-binding affinity (Reynolds et al., 1999). Further, using African green monkey COS-7 cells, Denny et al (2005) demonstrated a critical requirement of FKBP51 interaction with HSP90 but not PPIase activity for this inhibition. The fact that FKBP51 gene expression itself is induced by glucocorticoids provides a potential but excellent feedback system to attenuate and tightly control GR functions. The inhibitory effect of FKBP51 is also established for progesterone receptor (PR) (Hubler et al., 2003). In contrast, elevated levels of this protein have been shown to increase androgen receptor (AR) transcriptional activity (Febbo et al., 2005). Over-expression of human FKBP52 in yeast enhanced hormone-mediated GR signaling by altering hormone potency. Similar to FKBP51, HSP90-binding ability of 2 FKBP52 is important for potentiation (Riggs et al., 2003). Mutating several residues vital for PPIase activity, Riggs et al (2007) discovered that enzymatic activity itself is dispensable, but the domain does play a crucial role in potentiating receptor activity. This mutational analysis further revealed an important role for the amino acid proline in position 119 (P119). FKBP51 has a leucine in this position and mutating it to proline confers a potentiation effect in both yeast and murine cells. Regions outside this residue may also contribute towards the potentiation effect. Enhancement of transcriptional activity by FKBP52 however, is not specific for GR, but also includes AR and PR. Estrogen (ER) and mineralocorticoid (MR) receptors, however, are not affected by FKBP52 in this way (Gallo et al., 2007; Riggs et al., 2007). The mechanisms by which FKBP51 inhibits and FKBP52 stimulates steroid receptor activity are still far from clear. But it was speculated that the notch formed by P119 makes a specific contact with the ligand-binding domain (LBD) of the receptor- HSP90 complex and stabilizes a conformational state that is otherwise transient for hormone-binding. FKBP51 with leucine at residue 119 is not able to do so (Riggs et al., 2007). Even though the exact molecular mechanisms are not yet understood, it is now evident that distinct interactions of two different TPR proteins to steroid receptors via HSP90 give diversity to receptor functions. In order to translate these cell-based findings to physiology, mice lacking FKBP51 and FKBP52 have been created (Tranguch et al., 2005; Yang et al., 2006; Yong et al., 2007b). While FKBP51 deficiency initially showed no apparent phenotypes (Yong et al., 2007b), FKBP52 mutants were defective in select reproductive organs. Male mutant mice were infertile due to penile hypospadias and prostate dysgenesis. Further 3 analysis of affected organs and mouse embryonic fibroblasts (MEFs) derived from knockout mice revealed compromised AR transcriptional activity as the cause for the above mentioned developmental defects. Even though FKBP52 is present in other reproductive organs, interestingly, loss of this protein only seems to affect certain organs (Yong et al., 2007b). FKBP52 -/- females were sterile due to selective reduction in PR transcriptional activity especially in the uterus. Uteri of FKBP52 mutant females were unable to support implantation. Molecular analyses of uterine lysates and MEFs from knockout animals were able to show that FKBP52 specifically regulates PR in this organ. Estrogen responses in the uterus were not down-regulated and were normal or nearly normal (Tranguch et al., 2005; Yang et al., 2006). Thus, these data provide evidence for the selective targeting of steroid receptor signaling through TPR proteins. It is noteworthy that in contrast to previous in vitro findings, ablation of FKBP52 did not change hormone-binding affinity or translocation of steroid receptors (AR or PR), revealing a novel mechanism by which this protein controls SR functions. Taken together, these studies suggest that the TPR proteins are integral part of steroid receptor complexes and their fundamental role is to provide receptors with specificity
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