Hormone Receptors and Enzymes Regulating Gonadal and Mam

促黄体激素受体（LHR）。LHR基因在启动子区域受到组蛋白去乙酰化的抑制，其中组蛋白去乙酰化酶(HDAC)/mSin3A复合物锚定在Sp1位点的近端。这个Sp1位点被证明是曲古抑素A （TSA）诱导的LHR基因激活的重要介质。我们最近的研究表明，LHR的表观遗传沉默和激活是通过癌细胞组蛋白和DNA水平的协调调节实现的。LHR基因启动子在JAR和MCF-7细胞中明显沉默，而在转化的正常胎盘PLC细胞中则处于激活状态。与MCF7细胞相比，HDAC抑制剂（TSA）在JAR细胞中激发了较强的LHR基因激活（40倍比150倍）。这种效应定位于176bp启动子区域，该区域在JAR中高度甲基化，在MCF7细胞中轻度甲基化。因此，TSA和DNA去甲基化试剂5-Aza C在JAR细胞中引起了LHR基因的显著协同激活（从40倍增加到150倍），但在MCF7细胞中没有。H3/H4的多位点特异性赖氨酸乙酰化与这种激活有关。LHR的最大激活状态基本上是在LHR表达的PLC细胞中观察到的，其中启动子未甲基化，与高乙酰化组蛋白相关，并且对药物治疗无反应。虽然DNA甲基化不影响LHR启动子的组蛋白编码，但LHR CpG位点的去甲基化对于最大程度地刺激该基因是必要的。抑制性HDAC/mSin3A复合体的释放既需要组蛋白修饰，同时也需要去甲基化启动子。事实上，在Sp1/Sp3表达没有变化的情况下，tsa诱导的组蛋白乙酰化和基因激活在JAR细胞中普遍存在，它们的结合活性、HDAC/mSin3A复合物与Sp1位点的解离或启动子的去甲基化表明，tsa诱导的抑郁有不同的机制。我们最近的研究表明，磷脂酰肌醇3-激酶/蛋白激酶Czeta （PI3K/PKCzeta）介导的Sp1磷酸化可解释Sp1位点依赖性LHR激活。TSA在JAR和MCF7细胞中引起Sp1丝氨酸641位点的明显磷酸化。阻断PI3K或PKCzeta活性可消除TSA对Sp1磷酸化的影响。PKCzeta被证明与Sp1相关联，这种关联被TSA增强。Sp1磷酸化是pRB同源物p107从LHR启动子释放所必需的，而它作为LHR基因的抑制因子。抑制PKCzeta活性阻断了p107与LHR基因启动子的分离，显著降低Sp1磷酸化和转录。这些发现表明，PI3K/PKCzeta磷酸化Sp1对TSA激活的LHR基因表达至关重要，并揭示了一种新的TSA作用机制，即通过PI3K/PKCzeta诱导Sp1磷酸化依赖的方式从LHR基因启动子中募集抑制因子。

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.