Hormone Receptor/Enzyme Control--Gonad /Mammary Function

Child Health And Human Development

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Abstract

The luteinizing hormone receptor (LHR): We have previously demonstrated that transcription of the LHR gene is subject to repression by histone deacetylation at its promoter region, where a histone deacetylase (HDAC )/mSin3A complex is anchored at a proximal Sp1 site. Our more recent studies have shown that epigenetic silencing and activation of the LHR is achieved through coordinated regulation at both histone and DNA levels. The studies characterized the LHR gene status in JAR and MCF-7 cells where basally the transcription of this gene is markedly silenced, and in normal placenta PLC cells, where LHR expression is in active state. Singly HDAC, inhibitor trichostatin A (TSA), evoked lower activation of the LHR gene in JAR than in MCF7 cells (40 fold vs 150 fold). This effect was localized to the 176bp promoter region, which is highly methylated in JAR and lightly methylated in MCF7 cells, and DNA demethylating reagent 5-Aza C caused marked synergistic activation of the LHR gene in JAR cells but not in MCF7 cells. The activated state of the of the LHR gene induced by TSA and 5-AzaC in JAR and MCF7-cells was observed basally in LHR-expressing PLC cells in which the promoter is unmethylated and associated with hyperacetylated histones are unresponsive to drug treatment. Histone modifications have a dominant role in the control of silencing or activation of LHR gene expression, and DNA methylation are operative under the architecture set by histone modifications. Alteration of the chromatin structure within the promoter ( methylation or acetylation of H3 at K9 present at the silenced and derepressed LHR promoter, respectively) is concurrently necessary for DNA demethylating process and for subsequent dissociation of inhibitory complex (HDAC/ Sin3A and HDAC associated protein,MDB2), association of activators and basal transcriptional components (TFIIB, Pol II). DNA methylation did not affect the histone code of the LHR promoter, while demethylation of the LHR CpG sites was necessary for maximal stimulation of this gene. The studies have revealed the existence of a novel silencing/derepression mechanism which pertains to a different class than that derived from epigenetic studies on tumor suppressor genes. Gonadotropin-regulated testicular genes: Gonadotropin-Regulated Acyl CoA synthetase (GR-LACS) is a 79 kD protein cloned in our laboratory from a rat cDNA library. This protein which is transcriptionally down-regulated by gonadotropin and is capable of activating long-chain fatty acids (LACS), is a new member of the fatty acyl-CoA synthetase family. GR-LACS aside from two conserved regions shares low amino acid sequence similarity with other family members. GR-LACS which is abundantly expressed in Leydig cells and scarcely in germinal cells of the adult testis may contribute to the provision of energy requirements and biosynthesis of steroid precursors and participate in the regulation of the male gonad. Comparative analysis of GR-LACS expression in rat and mice, have determined its cell-, tissue-, and species-specific expression. GR-LACS protein is expressed in the rodent brain and gonads, and only in the mouse in the adrenal cortex. It is found in most regions of the brain and is highly expressed in the hippocampal region. In the ovary it is associated with follicles undergoing atresia (i.e. those in transition between preantral to antral stages, and subordinate antral follicles not selected for ovulation). Thus, GR-LACS could serve as a marker for atresia to further explore mechanisms associated with follicle development. GR-LACS is present in the newborn and immature testis tubules and after puberty only in the Leydig cells. Its presence at premeiotic stages, at a time of intense proliferation of Sertoli cells and spermatogonia, could contribute to the development of normal adult spermatogenesis. A distinct protein species of 64 kDa that was more abundant that the 79 kDa form, and also a minor form of 73 kDa species, were observed in the rat brain and mouse ovary. In other studies, to gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determine the structural requirements of the mouse gene. The minimal promoter domain of this TATAless gene resides within -254/-217 bp 5' of the ATG codon with four transcriptional start sites within this region. Transcription of GR-LACs gene requires an Sp1/Sp3 binding element located downstream of the TSS within the exon 1, which is essential for basal promoter activity. We have previously identified a novel Gonadotropin-Regulated Testicular Helicase Helicase (GRTH/Ddx25). This enzyme is present in the nucleus and cytoplasm of pachytene spermatocytes and round spermatids, binds mRNA species as an integral component of messenger RNP particles, with storage in chromatoid bodies located in the cytoplasm of spermatids. GRTH-targeted null male mice are sterile due to spermatid arrest (step 8), with failure to elongate. The transcription of messages in spermatids of these mice was not altered, but their translation was abrogated in a selective manner. We are now defining the function of this helicase as an RNA binding protein and its storage and translational function during sperm progression. A cytoplasmic species of 61 kDa and a nuclear species of 56 kDa were recognize by N- and C- terminal antibodies, and the 61 kDa species is phosphorylated. In addition to its storage function, the cytoplasmic species of GRTH associates polyribosomes to regulate the translational activity of specific subsets of expressed genes. Prolactin receptor: Prolactin acts through the long form of the receptor (LF) to cause differentiation of mammary epithelium and to initiate and maintain lactation through activation of the Jak2/Stat5 pathway and subsequent transcriptional events. Prolactin is a potent mitogen in human normal and cancerous breast tissues/cells and the evidence suggests that prolactin has a role in the development of human breast tumors. Our laboratory identified two novel short forms (SF) with abbreviated cytoplasmic domain (S1a, and S1b) that are products of alternative splicing and inhibit the activation induced by PRL through the LF. A significant decrease in the ratio of SF is observed in the tumor tissue and breast cancer cell lines compared to normal breast and control mammary cells. The relatively reduced expression of short forms in cancer could cause gradations of unopposed prolactin-mediated long form stimulatory function, and contribute to breast tumor development/progression. In the light of the dimerization requirement for initiation of signal transduction by prolactin through the long form, and since the inhibitory action of the SF (S1a and S1b) could result from the heterodimerization, we have studied the association between and among LF and short forms S1a and S1b of hPRLR from different prospectives using 3' tagged constructs which exhibited activities comparable to the wild type, to investigate homodimer and heterodimer formation. The formation of homodimers of a fraction of the total receptors was revealed in Western blots only under reducing conditions. Initial co-immunoprecipitation studies using combinations of specific anti-tag antibodies demonstrated the presence of heterodimerized complexes between LF and the individual SFs. Neither homodimer nor heterodimer formation required the presence of hormone. BRET analysis further demonstrated the physical association of hPRLR variants in HEK293 cells and its occurrence in vivo,. Surface biotinylation demonstrated that formation of dimers occurs at the cell membrane and further supported the existence of constituitive homodimers and heterodimers and explained the nature of the inhibition by the short of the long form activating function by the hormone.