The Luteinizing Hormone receptor (LHR). 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. This Sp1 site was shown to be an essential mediator of trichostatin A (TSA)-induced LHR gene activation. We have recently shown that epigenetic silencing and activation of the LHR is achieved through coordinated regulation at both histone and DNA levels in cancer cells. The LHR gene promoter is markedly silenced in JAR and MCF-7 cells, while is in an active state in transformed normal placenta PLC cells. The HDAC inhibitor (TSA) evoked robust but significantly 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. Consequently, TSA and the DNA demethylating reagent 5-Aza C caused marked synergistic activation of the LHR gene in JAR cells (increased from 40- to 150-fold) but not in MCF7 cells. Multiple site-specific lysine acetylation of H3/H4 is associated with such activation. The maximal activated state of the LHR was observed basally in LHR-expressing PLC cells in which the promoter is unmethylated, associated with hyperacetylated histones, and unresponsive to drug treatment. While DNA methylation did not affect the histone code of the LHR promoter, demethylation of the LHR CpG sites was necessary for maximal stimulation of this gene. Release of the repressive HDAC/mSin3A complex required both histone modifications and, concurrently, a demethylated promoter. The fact that TSA-induced histone acetylation and gene activation in JAR cells prevailed in the absence of changes in Sp1/Sp3 expression, their binding activity, disassociation of the HDAC/mSin3A complex from the Sp1 site or demethylation of the promoter indicated a different mechanism involved in TSA-induced derepression. Our most recent studies have revealed that phosphatidylinositol 3-kinase/protein kinase C zeta (PI3K/PKCzeta)-mediated Sp1 phosphorylation accounts for Sp1site-dependent LHR activation. TSA caused marked phosphorylation of Sp1 at serine 641 in JAR and MCF7 cells. Blockade of PI3K or PKCzeta activity abolished the effect of TSA on Sp1 phosphorylation. PKCzeta was shown to associate with Sp1 and this association was enhanced by TSA. Sp1 phosphorylation was required for the release of the pRB homologue p107 from the LHR promoter, while it acted as a repressor of the LHR gene. Inhibition of PKCzeta activity blocked the dissociation of p107 from the LHR gene promoter and markedly reduced Sp1 phosphorylation and transcription. These findings demonstrated that phosphorylation of Sp1 by PI3K/PKCzeta is critical for TSA-activated LHR gene expression, and revealed a novel mechanism of TSA action through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKCzeta-induced Sp1 phosphorylation-dependent manner. ? ? Gonadotropin-Regulated Genes: Gonadotropin Regulated-Acyl CoA synthetase (GR-LACS) is a 79 kD protein which is transcriptionally down-regulated by gonadotropin and is capable of activating long-chain fatty acids. GR-LACS has sequence identity with two conserved regions of the LACS family, but shares low overall sequence similarity (23-28%). It is abundantly expressed in Leydig cells and minimally in germinal cells of the adult testis. In the ovary it is associated with follicles undergoing atresia. GR-LACS protein is expressed in most regions of the rodent brain and is highly expressed in the hippocampus. A GR-LACS protein species of 64 kDa that was more abundant that the 79 kDa long form species, and also a minor form of 73 kDa species, were observed in the rat brain and mouse ovary. Two species resulting from alternative splicing of the GR-LACS gene were identified in a rat brain cDNA library: a short form 1 (S1) lacking exon 8 and short form 2 (S2) lacking exons 6-8. Expression studies revealed that the sizes of the S1/S2 proteins are comparable to those of the endogenous variant species. Neither S form contains FACs activity, suggesting that exon 8 is essential for the enzymatic function. In addition,the S1 exhibited a dominant negative effect on the FACS activity of the long-form (LF) GR-LACS, while S2 displayed a lesser but significant inhibitory effect. However, because of its endogenous abundance in the brain, this variant form could be a more effective regulator of LF activity than S1. GR-LACS variants may regulate the LF?s activity in the brain. Gonadotropin Regulated Testicular Helicase (GRTH/Ddx25), a member of the DEAD-box protein family, is essential for completion of spermatogenesis. Our current studies are defining the function of this helicase as an RNA binding protein and its storage and translational function during sperm progression. We have recently determined its subcellular distribution of the GRTH in germ cells. A cytoplasmic and a nuclear species of 61 and 56 kDa, respectively, are both recognized by N- and C-terminal antibodies. The observed differences in molecular weight species are due to Thr phosphorylation of the 61 kDa cytoplasmic form. Other studies have indicated that cAMP- PKA participates in the post-translational modification. The association of these protein species with specific cellular compartments could be of importance for GRTH-dependent regulatory functions. ? ? Prolactin Receptors Prolactin: (PRL) 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. PRL is a tumor promoter in rodents and has been implicated in the development of breast cancer. We have identified two short forms (SF) of the human prolactin receptor (hPRL) with abbreviated cytoplasmic domain (S1a, and S1b) that inhibit the activation induced by PRL through the LF. The relatively reduced expression of SF in breast cancer could cause unopposed PRL-mediated LF stimulatory function, and contribute to breast tumor development/progression. Inhibition by SFs may result from hormone independent heterodimer formation. Although SF heterodimers with LF bind hormone and mediate JAK2 activation, the SF partner lacks cytoplasmic sequences essential for activation of the JAK2/STAT5 pathway and prevents the LF from mediating the activation of PRL-induced genes. In breast cancer cells, E2 increases PRLR hE13 transcripts directed by hPIII which lack an ERE. This promoter contains functional Sp1 and C/EBP sites that bind Sp1/Sp3 and CEBPbeta, respectively. Abolition of the E2 effect by mutation of either element indicated the cooperation of these transfactors in E2-induced transcription of the hPRLR. E2 activated-estrogen receptor alpha(ERalpha) through interaction with Sp1/Sp3 and C/EBPbeta bound to DNA caused transcriptional activation of the promoter and of hPRLR expression in cancer cells. The ligand binding domain of ERalpha was essential for its physical interaction with C/EBPbeta and E2 promoted this association, and its DNA binding domain was required for transactivation of PIII. Other studies revealed tethering of C/EBPbeta to Sp1 by the E2-activated ERalpha, favouring interaction with its cognate element, and recruitment of coactivators to the complex, with consequent region-specific changes in histone acetylation. These hormone/receptor-induced associations and chromatin changes favored TFIIB and RNA Pol II recruitment and the activation of PIII directed hPRLR transcription. Stromal and adipose tissue, which are major sources of estrogen in post-menopausal women, could exert paracrine control of PRL and PRLR expression in adjacent mammary epithelial cells and stimulate breast tumor growth.

Project Start
Project End
Budget Start
Budget End
Support Year
31
Fiscal Year
2006
Total Cost
Indirect Cost
Name
U.S. National Inst/Child Hlth/Human Dev
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Sheng, Yi; Tsai-Morris, Chon-Hwa; Li, Jie et al. (2009) Lessons from the gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) null mouse model: a role in steroidogenesis, but not result in X-ALD phenotype. J Steroid Biochem Mol Biol 114:44-56
Xie, Y-L; Hassan, S A; Qazi, A M et al. (2009) Intramolecular disulfide bonds of the prolactin receptor short form are required for its inhibitory action on the function of the long form of the receptor. Mol Cell Biol 29:2546-55
Dufau, Maria L; Tsai-Morris, Chon-Hwa (2007) Gonadotropin-regulated testicular helicase (GRTH/DDX25): an essential regulator of spermatogenesis. Trends Endocrinol Metab 18:314-20
Tsai-Morris, Chon-Hwa; Koh, Eitetsu; Sheng, Yi et al. (2007) Polymorphism of the GRTH/DDX25 gene in normal and infertile Japanese men: a missense mutation associated with loss of GRTH phosphorylation. Mol Hum Reprod 13:887-92
Dong, Juying; Tsai-Morris, Chon-Hwa; Dufau, Maria L (2006) A novel estradiol/estrogen receptor alpha-dependent transcriptional mechanism controls expression of the human prolactin receptor. J Biol Chem 281:18825-36
Qazi, Aamer M; Tsai-Morris, Chon-Hwa; Dufau, Maria L (2006) Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization. Mol Endocrinol 20:1912-23
Li, Jie; Sheng, Yi; Tang, Pei Zong et al. (2006) Tissue-cell- and species-specific expression of gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) in gonads, adrenal and brain. Identification of novel forms in the brain. J Steroid Biochem Mol Biol 98:207-17
Sheng, Yi; Tsai-Morris, Chon-Hwa; Gutti, Ravi et al. (2006) Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is a transport protein involved in gene-specific mRNA export and protein translation during spermatogenesis. J Biol Chem 281:35048-56
Sheng, Yi; Li, Jie; Dufau, Maria L et al. (2005) The gonadotropin-regulated long-chain acyl CoA synthetase gene: a novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity. Gene 360:20-6
Zhang, Ying; Fatima, Naheed; Dufau, Maria L (2005) Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription. Mol Cell Biol 25:7929-39

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