The transcriptional coactivator PGC-1α as a modulator of ERRα and GR signaling: function in mitochondrial biogenesis Inauguraldissertation Zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Sylvia Nicole Schreiber aus Remseck, Deutschland Basel, 2004 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. M.N. Hall (Fakultätsverantwortlicher) Dr. N.Kralli (Supervisor) Prof. Dr. U.A.Meyer (Koreferent) Basel, den 06.04.2004 Dekan Prof. Dr. Marcel Tanner Table of contents Table of contents Page Table of contents i Abbreviations v Abstract vi Chapter I: Introduction Overview of transcriptional regulation by nuclear receptors 1 Nuclear receptors 4 Nuclear receptor structure 4 Classification of the nuclear receptor family 5 The glucocorticoid receptor 8 The ERR family 11 Coregulators of transcription 15 Types of coactivators 17 The PGC-1 family: inducible, tissue-specific coactivators 22 Aim of my thesis 30 References 31 i Table of contents Chapter II: The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor ERRα Summary 52 Introduction 52 Experimental procedures 55 Results 58 Discussion 68 Chapter II: supplementary data Results and Discussion Mechanism of ERRα induction 71 References 75 Chapter III: The estrogen-related receptor alpha (ERRα) functions in PGC-1α - induced mitochondrial biogenesis Summary 80 Introduction 80 Experimental procedures 82 Results 85 Discussion 96 Supplementary tables 99 ii Table of contents Chapter III: supplementary data Results and Discussion Regulation of mitochondrial biogenesis by PGC-1β 105 References 108 Chapter IV: Analysis of PGC-1α and GR expression profiles Results and discussion 112 Part 1: Introduction 113 Part 2: Genes that are regulated by PGC-1α in the absence of glucocorticoids 119 Part 3: Genes regulated by PGC-1α and glucocorticoids 128 A) Genes that were induced by PGC-1α but repressed by GR 128 B) Genes that were induced by PGC-1α dependent on GR 132 Part 4: Genes that were regulated by GR independent of PGC-1α 139 References 144 Chapter V: Discussion Part 1: The function of ERRα in PGC-1α signaling 152 Part 2: Analysis of the PGC-1α and GR expression profiles 157 Summary and conclusions 161 References 163 iii Table of contents Appendix I: Table 1: Genes induced by PGC-1α in the absence of GR 168 Table 2: Genes induced by PGC-1α but repressed by GR 176 Table 3: Genes induced by both PGC-1α and GR 177 Table 4: Genes induced by GR in the absence and presence of PGC-1α 178 Appendix II: The PGC-1–related protein PERC is a selective coactivator of estrogen receptor α Summary 185 Introduction 185 Experimental procedures 186 Results 187 Discussion 189 References 192 Appendix III: Material and Methods 193 Appendix IV: Curriculum vitae 201 Appendix V Acknowledgements 203 iv Abbreviations Abbreviations Aa amino acids AF activation function DBD DNA binding domain ER estrogen receptor ERE estrogen response element ERR estrogen related receptor ERRE estrogen related receptor response element GFP green fluorescent protein GR glucocorticoid receptor GRE glucocorticoid response element HNF hepatocyte factor LBD ligand binding domain LRH liver receptor homologue LXR liver x receptor NLS nuclear localization signal MAPK mitogen activated kinase MCAD medium-chain acyl-coenzyme A dehydrogenase MEF myocyte enhancing factor MR mineralocorticoid receptor NID nuclear receptor interacting domain NRF nuclear respiratory factor OXPHOS oxidative phosphorylation PPAR peroxisome proliferator-activated receptor PERC PGC-1 related estrogen receptor coactivator PGC peroxisome proliverator-activated receptor coactivator PR progesterone receptor PRC PGC-1 related coactivator PEPCK phosphoenol pyruvate carboxykinase RAR retinoic acid receptor RS arginine serine rich domain RRM RNA recognition motif RXR retinoid x receptor TR thyroid receptor siRNA small interfering RNA SHP small heterodimer partner VDR vitamin D receptor v Abstract Abstract The nuclear receptor family represents a large class of transcription factors that regulate metabolism, differentiation and development. Most of the nuclear receptor family members are orphan receptors, so called because no ligands were known when they were identified. Although for some receptors ligands have been identified by now, many receptors, such as the estrogen related receptor α (ERRα), still remain orphan. The activity of all nuclear receptors requires the recruitment of coregulators, which are able to enhance or repress their activity. Our work has focused on PGC-1α, which responds to physiological signals, such as cold, fasting and exercise, and was characterized as an important factor in the regulation of energy homeostasis and metabolic pathways. In my thesis work, we demonstrate that PGC-1α regulates the expression and activity of the orphan nuclear receptor ERRα. Our findings suggest that PGC-1α may act as a protein ligand, substituting for the lack of small lipohilic ligands for this receptor. The expression of PGC-1α and ERRα is parallel in tissues with high energy demand, and induced in vivo when animals are exposed to cold. Furthermore, our studies demonstrate that ERRα is important for PGC-1α signaling, since diminished ERRα levels significantly reduce the induction of mitochondrial biogenesis by PGC-1α. Binding sites for ERRα are observed in many genes encoding for mitochondrial proteins, and in vitro studies suggest that ERRα activates the transcription of at least a subset of the genes by binding to their promoters. Furthermore, ERRα fused to the potent VP16 activation domain is sufficient to induce mitochondrial biogenesis. We suggest that PGC-1α and ERRα regulate the transcription of genes encoding mitochondrial proteins in response to metabolic requirements. Previous studies from our lab identified PGC-1α as a potent regulator of glucocorticoid receptor (GR) function in vitro. In support of our studies, other groups have shown that PGC-1α coactivates GR on the PEPCK promoter, the key enzyme of gluconeogenesis. Further data has shown that glucocorticoids and glucagon regulate the expression of PGC-1α. This led us to investigate the role of PGC-1α in GR signalling in SAOS2 cells. Our preliminary data suggest that glucocorticoids strongly vi Abstract influence PGC-1α signaling, enhancing some PGC-1α pathways and suppressing others. Finally, our data provide support to the hypothesis that PGC-1α is not a general enhancer of glucocorticoid responses, but rather provides specificity to GR signalling. PGC-1α expression leads to the activation of a distinct set of genes by GR. Future studies should provide more insight into this relationship. vii Chapter I: Introduction Chapter I: Introduction Overview of transcriptional regulation by nuclear receptors All cellular processes involved in development, differentiation, cell growth and metabolism are constantly regulated by the transcriptional activation or repression of many different genes. The misregulation of even a single component often leads to disease, such as obesity, diabetes or cancer (Rosmond, 2002; Smith and Kantoff, 2002; Spiegelman and Flier, 2001; Tenbaum and Baniahmad, 1997). Therefore, the control of gene expression and the mechanisms of achieving specificity in transcriptional pathways have attracted increasing amount of attention in the last two decades (reviewed in Orphanides and Reinberg, 2002). The transcription of genes is regulated in a highly organized fashion to ensure protein expression in a spatially and temporally defined manner. Mechanisms must exist that allow cells to integrate intracellular and extracellular signals to their differentiation state, cell cycle stage or metabolic state, and ensure appropriate transcriptional responses. One class of extracellular signals are steroid hormones. These are small lipophilic molecules that are produced by endocrine glands and transported through the blood, and that can diffuse through the plasma membrane to the cell interior. They exert their transcriptional effects by binding and activating nuclear receptors, which are among the most intensively studied and probably best-understood transcription factors to date (reviewed in Aranda and Pascual, 2001; Mangelsdorf et al., 1995). Several facts have established nuclear receptors as valuable tools for studying the mechanisms that provide specificity in transcriptional regulation. First, nuclear receptors are important modulators of all aspects of physiology. Second, the expression of many nuclear receptors, for example the glucocorticoid receptor (GR), is ubiquitous (Jenkins et al., 2001), yet the responses they elicit are cell type- or physiological state-dependent. Third, nuclear receptors are regulated through small lipophilic ligands, which are good experimental tools for turning on and of the activity of the receptors, as well as have therapeutic applications. 1 Chapter I: Introduction For several years, the regulation of transcription by nuclear receptors was imagined in a simple way. Hormones ‘slip’ into the cells and ‘waken’ up the inactive receptor, which then binds to DNA and activates transcription. However, things are not as simple as they seem. First, nuclear receptors activate or repress transcription mostly, but not always, in a ligand-dependent manner. The identification of the first steroid- related receptors, the estrogen related receptors (ERRs), for which no ligand was known, founded the subgroup of