Ligand-independent activation of steroid hormone receptors by gonadotropin-releasing hormone by Junling Chen Bachelor of Medicine, Xi’an Medical University, 1993 Master of Science, University of Edinburgh, 2000 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Reproductive and Developmental Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November, 2010 © Junling Chen, 2010 Abstract Nuclear receptors including estrogen receptors (ERs) and progesterone receptors (PRs) are activated by their ligands as well as by signaling pathways in response to peptide hormones and growth factors. In gonadotrophs, gonadotropin releasing hormones (GnRHs) act via the GnRH receptor (GnRHR). Both GnRH-I and GnRH-II activate an estrogen response element (ERE)-driven luciferase reporter gene in Lf3T2 mouse pituitary cells, and GnRH-I is more potent in this regard. The ERc is phosphorylated at Ser’18 in the nucleus and at Ser167 in both nucleus and cytoplasm after GnRI-I treatments, and this coincides with increased ERct binding to its co-activator, the P300/CBP-associated factor (PCAF). Most importantly, both GnRH subtypes robustly up-regulate expression of the immediate early response gene, Fosb, while co-treatment with ERa siRNA or PCAF siRNA attenuates this effect. This appears to occur at the transcriptional level because co-recruitment of ERa and PCAF to an ERE within the endogenous Fosb promoter is increased by GnRH treatments, as shown by chromatin immunoprecipitation assays. Furthermore, cross-talk between GnRH-I and PR accentuates gonadotropin production. GnRH-I activates a progesterone response element (PRE)-driven luciferase reporter gene and gonadotropin a subunit (Gsua) gene expression in two mouse gonadotroph cell lines, aT3-1 and L13T2. Up-regulation of the PRE-luciferase reporter gene by GnRH-I is attenuated by pre-treatment with protein kinase A (H89) and protein kinase C (GF109203X) inhibitors, while only GF109203X inhibits GnRH-1-induced Gsua mRNA levels. In both cell lines within the same time-frame, knockdown of PR levels by siRNA reduces GnRH-I activation of Gsua mRNA levels by approximately 40%. Both GnRH-I and GnRH-II also increase mouse Gnrhr-luciferase promoter activity and this is significantly reduced by knockdown of PR in L3T2 cells. We conclude that the effects of GnRH-I on Fosb 11 and Gsua expression, as well as mouse Gnrhr promoter activity in mouse gonadotrophs are mediated by ligand-independent activation of ERc and PR. These ligand-independent effects of GnRHs on steroid hormone receptor function may influence the magnitude of changes in the expression of specific genes in the pituitary during the mouse estrous cycle, which in this context may serve as a model in the human menstrual cycle. 111 Preface 1. A version of chapter 2 has been published. Chen J, An BS, Cheng L, Hammond GL, Leung PC 2009 Gonadotropin-releasing hormone-mediated phosphorylation of estrogen receptor-alpha contributes to Fosb expression in mouse gonadotrophs. Endocrinology 150:4583-4593. Contributions An BS and I designed and performed the experiments. I drafted the manuscript. Leung PC, Hammond GL and Cheng L were responsible for supervision of this work. Hammond GL and Leung PC critically revised the manuscript. All authors read and approved the final manuscript. Presentations Third Annual Scientific Meeting of Chinese Society of Reproductive Medicine, Chinese Medical Association. Guangzhou, China. February 2009. Oral presentation. Gonadotropin releasing hormone-mediated phosphorylation of the estrogen receptor alpha induces expression of the early response gene Fosb in mouse pituitary cells. Chen J, An BS, Cheng L, Hammond GL, Leung PC. CIHR Research Poster Competition at the Canadian Student Health Research Forum. Winnipeg. June 2009. Poster presentation. Gonadotropin-releasing hormone-mediated phosphorylation of estrogen receptor-alpha contributes to Fosb expression in mouse gonadotrophs. Chen J, An BS, Cheng L, Hammond GL, Leung PC. CIHR-IHDCYH Scientific Forum and Poster Session. UBC. May 2010. Poster presentation. Gonadotropin-releasing hormone-mediated phosphorylation of estrogen receptor-alpha contributes to Fosb expression in mouse gonadotrophs. Chen J, An BS, Cheng L, Hammond GL, Leung PC. 2. A version of chapter 3 has been published. Chen J, An BS, So WK, Cheng L, Hammond GL, Leung PC 2010 Gonadotropin-releasing hormone-I-mediated activation of progesterone iv receptor contributes to gonadotropin alpha-subunit expression in mouse gonadotrophs. Endocrinology 151:1204-1211. Contributions An BS, So WK and I participated in the design and performance of the study, as well as the discussion of the results. I drafted the manuscript. Leung PC, Hammond GL and Cheng L were responsible for supervision of this work. Hammond GL and Leung PC critically revised the manuscript. All authors read and approved the final manuscript. An BS and I contributed equally. Presentation The 4th Annual Conference of the Chinese Society of Reproductive Medicine and the 1St China-Association of Southeast Asian Nations Forum in Reproductive Medicine. Nanning, China. February 2010. Oral presentation. Lignd-independent activation of progesterone receptor contributes to the self-priming effect of gonadotropin releasing hormone-I in the pituitary. Chen J, An BS, So WK, Cheng L, Hammond GL, Leung PC. V Table of Contents Abstract. ii Preface iv Table of Contents vi List of Figures ix List ofAbbreviations xi Acknowledgements xiii Chapter 1 Introduction 1 1.1 Overview 1 1.2 GnRH and the GnRH receptor 3 1.2.1 GnRH-I and GnRH-II 3 1.2.2 GnRI-1 receptor 8 1.2.3 Signal transduction mechanism of type I GnRHR 10 1.2.4 Regulation of type I GnRHR 12 1.3 Estrogen receptor a 13 1.3.1 Estrogen receptor 13 1.3.2 ERE-dependent genomic actions 16 1.3.3 ERE-independent genomic actions 17 1.3.4 ERa. phosphorylation 18 1.3.5 Nuclear coactivators 19 1.3.6 Ligand-independent activation of ER 21 1.4 Progesterone receptor 23 1.4.1 Progesterone receptor 23 1.4.2 Ligand-dependent activation of PR 25 1.4.3 PR phosphorylation 26 1.4.4 Ligand-independent activation of PR 26 1.5AP-1 30 1.6 Hypothesis and objectives 34 Chapter 2 GnRH-mediated phosphorylation of estrogen receptor a contributes to Fosb expression in mouse gonadotrophs 43 vi 2.1 Introduction .43 2.2 Materials and methods 44 2.2.1 Cells and cell culture 44 2.2.2 Plasmid and ERE-luciferase reporter gene assays 45 2.2.3 Nuclear extraction, immunoblotting and immunoprecipitation 46 2.2.4 Real-time RT-PCR 47 2.2.5 Chromatin immunoprecipitation (ChIP) 48 2.2.6 Data analysis 49 2.3 Results 49 2.3.1 GnRH-I and GnRH-II rapidly and transiently activate ERa in L13T2 cells 49 2.3.2 GnRHR is required for GnRH-mediated ERE-luciferase activation 50 2.3.3 GnRH treatments affect ERa phosphorylation and promote ERa interactions with PCAF 51 2.3.4 GnRH treatments promote the co-recruitment of ERa and PCAF to the Fosb promoter ERE 52 2.3.5 GnRH treatments increase Fshb expression 54 2.4 Discussion 54 Chapter 3 GnRJ-I-I-mediated activation of progesterone receptor contributes to Gsua expression in mouse gonadotrophs 74 3.1 Introduction 74 3.2 Materials and methods 76 3.2.1 Cells and cell culture 76 3.2.2 Plasmids, siRNA and transient transfection assay 76 3.2.3 Immunoblotting 77 3.2.4 Real-time RT-PCR 77 3.2.5 Data analysis 78 3.3 Results 78 3.3.1 GnRH-I rapidly and transiently activates PR in aT3-l and L13T2 cells .... 78 3.3.2 GnRI-I-I enhances Gsua gene expression in aT3-1 and L3T2 cells 79 3.3.3 Activation of PR and its effects on Gsua expression involves PKC 80 VII 3.3.4 GnRH-I-induced Gsua gene expression requires PR 80 3.4 Discussion 81 Chapter 4 Cross-talk between GnRH and PR in the induction of Gnrhr in mouse gonadotrophs 94 4.1 Introduction 94 4.2 Materials and methods 96 4.2.1 Cells and cell culture 96 4.2.2 Plasmids, siRNA and transient transfection assay 97 4.2.3 Immunoblotting 98 4.2.4 Real-time RT-PCR 98 4.2.5 Data analysis 98 4.3 Results 99 4.3.1 GnRI-I-I and GnRH-II transiently activate mouse Gnrhr in L13T2 cells....99 4.3.2 GnR}I-I rapidly and transiently activates the AP-1 luciferase gene in L13T2 and aT3-1 cells 99 4.3.3 GnRH-I-induced mouse Gnrhr promoter activity requires PR 100 4.4 Discussion 100 Chapter 5 Conclusion and future work 110 5.1 Physiological relevance of the experimental conditions design 111 5.2 GnRH activates specific signaling pathways in the transactivation of ERcL and PR 112 5.3 GnRH-I has more robust effects than GnRH-II in mouse gonadotrophs 113 5.4 Single mutations (S118A or S167A) of ERa phosphorylation sites are not sufficient to affect Fosb protein Levels 114 5.5 PCAF regulates gene expression in the pituitary after interactions with ERa.. 116 5.6 GnRH-I activates PR in a ligand-independent manner to induce Gsua expression 117 5.7 The physiological importance of transactivation of ERa and PR by GnRI-1-I.. 119 References 127 viii List of Figures Figure 1. 1 Hypothalamic-pituitary-gonadal axis 36 Figure 1. 2 Genomic structures of human GnRH-I and GnRH-II genes 37 Figure 1. 3 Genomic structures of human and mouse GnRI-IR type I genes 38 Figure 1. 4 Structural organization of human ERcL and ER 39 Figure 1. 5 Schematic representation of ER phosphorylation sites 40 Figure 1.