Regulation of FOXO transcription factors by gonadotropin-releasing hormone
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G protein-coupled receptors (GPCRs) are a large family of trans-membrane receptors that transmit signals from extracellular stimuli to target intracellular signal transduction pathways. The gonadotropin-releasing hormone receptor (GnRH-R) is a GPCR which binds the decapeptide GnRH. In the pituitary gonadotrope, GnRH stimulates gonadotropin (LH and FSH) biosynthesis and secretion to regulate reproduction. GnRH and the GnRH-Rs are also present in many extra-pituitary tissues, although their role at these sites remains largely undetermined. GnRH-Rs are known to recruit a diverse array of signalling pathway mediators in different cell-types. These include; Gq/11-PLCβ-IP3/DAG-Ca2+/PKC signalling, monomeric G-proteins and integrins to mediate cell adhesion and migration, the activation of the major members of the mitogen-activated protein kinase (MAPK) super-family (extracellular signal-regulated kinase (ERK), c-Jun N-terminal Kinase (JNK) and p38MAPK), and β-catenin and other mediators of the canonical Wnt signalling pathway. This thesis describes the regulation of Forkhead Box O (FOXO) transcription factors by GnRH. The mammalian FOXO transcription factors, FOXO1, FOXO3a and FOXO4, are emerging as an important family of proteins that modulate the expression of genes involved in cell-cycle regulation, induction of apoptosis, DNA damage repair and response to oxidative stress. In this thesis, emphasis is placed on delineating the novel role of FOXO transcription factors in mediating two important and widely-researched areas of GnRH biology. Firstly, the role of FOXO transcription factors in mediating cell-growth inhibition in response to GnRH treatment is assessed in a heterologous HEK293/GnRH-R expressing cell line. Secondly, the role of transcription factors in regulating luteinising hormone-β (LHβ)-subunit expression is investigated in the LβT2 gonadotrope cell line. Activation of the GnRH-R can inhibit cell proliferation and induce apoptosis in certain tumour-derived cell lines. Several studies have reported that these events can occur as a result of changes in the expression profiles of specific cell-cycle regulatory and apoptotic genes, many of which are FOXO-target genes, including GADD45, FasL, p21Cip1 and p27Kip1. In this thesis, a role for FOXOs in targeting the expression of several of these genes in response to GnRH is assessed, highlighting a specific role for FOXO3a in mediating GADD45 and FasL expression. The signalling mechanisms through which FOXO3a regulates GADD45 expression in response to GnRH is also described. Finally, a stable FOXO3a-knock-down cell line was generated in order to further examine FOXO3a involvement in GnRH-induced cell-growth inhibition. GnRH is an essential regulator of the reproductive process by stimulating the synthesis of LH and FSH in pituitary gonadotropes, thereby regulating gametogenesis and steroidogenesis. Diverse signalling pathways have been reported to regulate LHβ-subunit expression in response to GnRH, including the ERK/JNK/p38MAPK cascades and factors such as Egr1, SF1 and β-catenin. In the second part of this thesis, the role of FOXOs in regulating LHβ-subunit expression in response to GnRH is described. The data presented suggests that GnRH can regulate LHβ-subunit expression through both indirect and direct FOXO3a-mediated mechanisms. Firstly, FOXO3a was found to regulate Egr1 expression to indirectly target LHβ-promoter activity. Secondly, a role for β-catenin as a FOXO3a co-factor to directly regulate LHβ-subunit expression, together with Egr1 and SF1, is also proposed. FOXO3a expression and sub-cellular localisation was assessed and demonstrated in LβT2 cells and in adult human male pituitary sections. The research presented in this thesis adds to the diversity of signalling pathways and mediators that GnRH can target in different cellular backgrounds in order to mediate a variety of cellular processes. The antiproliferative and apoptotic effects of GnRH on tumour-derived cell lines are well-documented, and this research highlights a novel role for FOXO3a in mediating these events. The regulation of gonadotropin synthesis remains an important topic of research, and the novel implication of FOXO3a in mediating LHβ-subunit expression adds further complexity to gonadotrope physiology.