Hormone Receptors and Enzymes Regulating Gonadal and mammary function

黄体生成素 (LH) 受体 (LHR)。 LHR 基因的转录通过遗传和表观遗传水平上的复杂调节受到抑制/去抑制。 在表达 LHR 的癌细胞中已经鉴定出几种以 LHR 基因启动子 Sp1/Sp3 结合域为中心的调节模式。此外，我们的研究已经确定了参与该基因调节的信号通路。 LHR 转录受到表观遗传调控模式的影响，其中近端 Sp1 位点充当锚定点来募集 HDAC 1/2 和 mSin3A 辅阻遏物复合物，导致启动子局部组蛋白低乙酰化和有限的 Pol II 募集，这在一定程度上导致了基因沉默。 H3/H4 的位点特异性甲基化与组蛋白乙酰化水平的变化协同作用，调节该基因的表达。 LHR 基因启动子的甲基化状态提供了另一层细胞特异性调节。我们发现 PI3K/PKC-zeta 信号通路以及 Ser/Thr 磷酸酶 PP1 和 PP2A 是 TSA 诱导的 LHR 去抑制的关键参与者。 PI3K/PKC-zeta 对 Sp1 Ser 641 的磷酸化导致阻遏蛋白 p107 从 LHR 基因启动子上解离，从而诱导基因激活。另一方面，TSA 介导的染色质变化从启动子中释放 PP1 或 PP2A，作为 PI3K/PKC-zeta 磷酸化 Sp1 的“开启”机制，导致 p107 去招募和 LHR 激活。 PI3K-zeta 和磷酸酶之间的协调平衡对于通过影响 Sp1 磷酸化来上调或下调 LHR 基因表达至关重要。我们还显示了 PKC-α/Erk 通路的参与，其中其内源激活通过 PKC-α/Erk 在 Ser 残基处诱导 Sp1 磷酸化来增加 LHR 基因表达。这会导致 HDAC1/mSin3A 复合物从启动子上解离，并导致转录激活。这种效应与启动子相关的染色质变化无关。这些发现表明转录水平的LHR基因表达受到复杂多样的网络的调节，其中这些不同的调节效应器之间的协调和相互作用对于LHR表达的沉默/激活至关重要。 促性腺激素调节的睾丸基因：我们之前鉴定了促性腺激素调节的睾丸解旋酶（GRTH/Ddx25），它存在于粗线期精母细胞和圆形精子细胞的细胞核和细胞质中。 GRTH 是 mRNP 的一个组成部分，mRNP 运输靶 mRNA 并储存在精子细胞的类染色体中，并在精子发生过程中释放进行翻译。 GRTH 也存在于多核糖体中，它选择性地调节编码生精因子的 mRNA 的翻译。 GRTH 缺失的雄性 (KO) 小鼠由于精子细胞停滞和无法伸长而无法生育。 精子细胞中的信息转录没有改变，但它们的翻译被选择性地废除。鉴定出两种 GRTH 物种：56 kDa 核物种与 CRM1 相互作用并参与 mRNA 转运；磷酸化的细胞质 61 kDa 种类，与多核糖体相关。我们证明 GRTH/Ddx25 是一种多功能 RNA 解旋酶，是精子成熟的重要调节因子。 在 KO 小鼠中，进入减数分裂中期的精母细胞发生显着的凋亡，表明其在成体生殖细胞的存活和凋亡命运中发挥着重要作用。 发现促凋亡因子和抗凋亡因子受到 GRTH 的调节。 KO小鼠的Bcl-2和Bcl-xL（抗凋亡）减少，Bid、Bak和Bad（促凋亡）增加，磷酸Bad减少，细胞色素c的释放和Smac增加。这些变化导致 caspase 9 和 3 以及 PARP 的裂解增加以及 caspase 3 的激活。GRTH 对 caspase 3 mRNA 稳定性具有负面作用，选择性地与某些促凋亡因子和抗凋亡因子 mRNA 以及 caspase 9、3 和 PARP、IB、p300 和 HDAC1 相关。在 KO 小鼠中，IB 升高，其磷酸形式减少。 KO 小鼠中 HDAC1 的增加和 p300 表达的消除表明其核对 NF-B 介导的抗凋亡基因转录有作用。 GRTH 还调节死亡途径和 caspase 8 介导的事件。这些研究表明，GRTH 是肿瘤坏死因子受体 1 和 caspase 通路的负调节因子，并促进 NF-B 功能来控制成年小鼠精母细胞的凋亡。 我们对 GRTH 介导的男性不育症潜在作用的研究发现了 SNP——外显子 8 的错义突变和外显子 11 的沉默突变，可能与日本人群的非梗阻性无精子症有关。外显子 8 中独特的杂合错义突变 Arg242His 可能会损害表达蛋白的翻译后修饰。该突变蛋白不发生磷酸化，因此可能影响生殖细胞发育过程中必需基因的翻译调节。不孕症与中国西部人群中 GRTH 基因的沉默突变（外显子 10）有关，可能会改变 RNA 剪接并增加精子发生受损的风险。相比之下，我们发现日本非梗阻性无精症男性的这种等位基因变化没有统计学意义。有趣的是，人们还注意到，在日本患者中发现的外显子 8 和 11 的 SNP 在中国患者中并不明显。 因此，GRTH 基因的 SPN 可能与亚洲男性不育的种族背景有关。 催乳素受体（PRLR）：PRLR 的短形式（SF）可以抑制长形式（LF）PRL 诱导的基因转录激活。 这是由于 LF/SF 异二聚化和 SF 伴侣中缺乏细胞质 (C) 序列造成的，而这对于 STAT 激活至关重要。与 LF 相比，SF，S1b 不包含 Box 1 JAK2 位点之外的保守胞质基序结构。 我们还发现 hPRLR 的异二聚和同二聚化可以独立于配体发生，并且 PRL 是诱导 JAK/STAT 信号传导的构象修饰剂。在最近的研究中，功能分析和计算机模拟表明，细胞外结构域（EC）的 D1 子结构域中的四个保守的 Cys 残基对于维持二聚化 PRLR 的正确构象以及 SF S1b 对配体诱导的 LF 介导的 STAT5 依赖性转录激活的显性负作用至关重要。 S1b 对 LF 功能的抑制作用通过分子内二硫键 (S1bx) 的破坏而被消除。这是由于 S1bx 能够形成比 S1b 更高阶的动力学同二聚体关联，并且它们与 LF 形成异二聚体的亲和力降低，这与 S1b 与 LF 形成异二聚体的倾向形成鲜明对比。 这反过来又促进了能够介导 PRL 诱导信号传导的 LF 同二聚体的形成。其他研究检查了 Cys 突变对 JAK2 磷酸化的影响。在不存在 hPRL 的情况下，在瞬时转染 S1b 但未转染 Cys 突变体 (S1b4x) 的细胞或稳定表达 LF 的细胞中观察到 JAK2 磷酸化的基础水平。 Co-IP 研究显示，与 S1b 相比，JAK2 与 S1b4x 的关联性丧失。这揭示了 EC 结构域构象和 JAK2 与 Box-1 关联之间的功能联系，从而损害了基础磷酸化。总体而言，我们的研究证明了 PRLR 的分子内二硫桥与 PRL 诱导的 LF 介导的 STAT5 依赖性作用的 S1b 抑制作用以及与 JAK2 关联/活性相关的细胞质事件的相关性。

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.