Hormone Receptors and Enzymes Regulating Gonadal and mammary function

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The LH Receptor (LHR). The transcription of the LHR gene is subject to repression/ derepression through a complex modulation at both genetic and epigenetic levels. Several modes of regulation centered on the Sp1/Sp3 binding domains of the LHR gene promoter have been identified in cancer cells that express the LHR. Furthermore, our studies have determined signalling pathways involved in the regulation of this gene. LHR transcription is subject to an epigenetic regulatory mode whereby the proximal Sp1 site acts as an anchor to recruit HDAC 1/2 and the mSin3A corepressor complex, resulting in promoter-localized histone hypoacetylation and limited Pol II recruitment which partly contributes to gene silencing. Site-specific methylations of H3/H4 act in concert with changes in histone acetylation levels to regulate expression of this gene. The methylation status of the LHR gene promoter provides another layer of cell specific modulation. We found that PI3K/PKC-zeta signaling pathways and Ser/Thr phophatases PP1 and PP2A, are key participants in TSA-induced derepression of the LHR. Phosphorylation of Sp1 at Ser 641 by PI3K/PKC-zeta causes dissociation of repressor protein p107 from the LHR gene promoter, inducing gene activation. On the other hand, TSA-mediated chromatin changes release PP1 or PP2A from the promoter, serving as an 'on' mechanism for Sp1 phosphorylation by PI3K/PKC-zeta, leading to p107 de-recruitment and LHR activation. The coordinated balanced between PI3K-zeta and phosphatase(s) is critical for up- or down- regulation of LHR gene expression by its effect on Sp1 phosphorylation. We also showed participation of the PKC-alpha/Erk pathway, where its endogenous activation increases LHR gene expression through induction of Sp1 phosphorylation by PKC-alpha/Erk at Ser residue(s). This causes dissociation of the HDAC1/mSin3A complex from the promoter, and transcriptional activation. This effect was independent of the promoter-associated chromatin changes. These findings indicate that LHR gene expression at the transcriptional level is regulated by complex and diverse networks, in which coordination and interaction among these diverse regulatory effectors are crucial for silencing/activation of LHR expression. Gonadotropin-regulated testicular genes: We previously identified Gonadotropin-Regulated Testicular Helicase (GRTH/Ddx25), which is present in the nucleus and cytoplasm of pachytene spermatocytes and round spermatids. GRTH is a component of mRNPs which transport target mRNAs for storage in chromatoid bodies of spermatids, to be released for translation during spermatogenesis. GRTH is also found in polyribosomes, where it selectively regulates the translation of mRNAs encoding spermatogenic factors. GRTH-null male (KO) mice are sterile due to spermatid arrest and failure to elongate. Transcription of messages in spermatids was not altered, but their translation was selectively abrogated. Two GRTH species were identified: the 56 kDa nuclear species interacts with CRM1 and participates in mRNA transport; the phosphorylated cytoplasmic 61 kDa species, associates with polyribosomes. We demonstrated that GRTH/Ddx25 is a multifunctional RNA helicase that is an essential regulator of sperm maturation. In KO mice the striking apoptosis in spermatocytes entering the metaphase of meiosis indicated its important role in the survival and apoptotic fate of adult germ cells. Pro- and anti-apoptotic factors were found to be under GRTH regulation. KO mice have decreased Bcl-2 and Bcl-xL (anti-apoptotic), increased Bid, Bak, and Bad (pro-apoptotic), reduced phospho-Bad, release of cytochrome c an increase of Smac. These changes caused increase cleavage of caspases 9 and 3 and PARP and activation of caspase 3. GRTH had a negative role on caspase 3 mRNA stability, selectively associated with certain pro- and anti-apoptotic factor mRNAs, and with those of caspase 9, 3 and PARP, IB, p300 and HDAC1. I B was elevated and its phospho-form was reduced in KO mice. The increase of HDAC1 and abolition of p300 expression in KO mice indicated its nuclear action on NF- B-mediated transcription of anti-apoptotic genes. GRTH also regulates the death pathway and caspase 8-mediated events. These studies demonstrated that GRTH is a negative regulator of tumor necrosis factor receptor 1 and caspase pathways, and promotes NF- B function to control apoptosis in spermatocytes of adult mice. Our investigation of potential GRTH-mediated actions in male infertility identified SNPs - a missense mutation at exon 8 and a silent mutation at exon 11 that might be associated with non-obstructive azoospermia in a Japanese population. The unique heterozygous missense mutation Arg242His in exon 8 could impair the post-translational modification of the expressed protein. The mutant protein does not undergo phosphorylation and consequently could affect translational regulation of essential genes during germ cells development. Infertility is associated with a silent mutation (exon 10) of the GRTH gene in a population from West China, and might alter RNA splicing and increase the risk of impaired spermatogenesis. In contrast, we found no statistical significance of this allele change in Japanese men with non-obstructive azoospermia. Interestingly, it was also noted that the SNPs of exons 8 and 11 found in Japanese patients were not apparent in Chinese patients. Thus, SPNs of the GRTH gene might be associated with an ethnic background of male infertility among Asian men. Prolactin receptor (PRLR) : Short forms (SF) of the PRLR can silence PRL-induced activation of gene transcription by the long form (LF). This results from LF/SF heterodimerization and the absence of cytoplasmic (C) sequences in the SF partner, which are essential for STAT activation. Compared to the LF, the SF, S1b does not contain the conserved cytoplasmic motif structure beyond the Box 1 JAK2 site. We also found that hetero- and homo-dimerization of hPRLR can occur independent of ligand, and that PRL is a conformational modifier inducing JAK/STAT signaling. In recent studies both functional analysis and computer simulations revealed that the four conserved Cys residues in the D1 subdomain of the extracellular domain (EC) are essential to maintain the correct conformation of dimerized PRLR and the dominant negative action of the SF S1b on ligand-induced LF-mediated STAT5-dependent transcriptional activation. The inhibitory action of S1b on LF function was abolished by disruption of intra-molecular disulfide bridges (S1bx). This results from the ability of S1bx to form higher-order kinetic homodimer associations than S1b, and their reduced affinity to form heterodimers with the LF, contrasted with S1b propensity to form heterodimers with LF. This in turn facilitates the formation of LF homodimers competent to mediate PRL-induced signaling. Other studies examined the impact of the Cys-mutation on JAK2 phosphorylation. In the absence of hPRL, a basal level of JAK2 phosphorylation was observed in cells transiently transfected with S1b but not with the Cys-mutant (S1b4x) or cells stably expressing LF. Co-IP studies showed loss of JAK2 association with S1b4x compared to S1b. This revealed a functional link between the EC domain conformation and JAK2 association with Box-1 that compromised basal phosphorylation. Overall, our studies demonstrated the relevance of the intramolecular disulfide bridges of the PRLR for S1b inhibitory action on PRL-induced LF-mediated STAT5-dependent action, and for cytoplasmic events related to JAK2 association/activity.