Hormone Receptors and Genes Regulating Gonadal and mammary function

调节性腺和乳腺功能的激素受体和基因

• 批准号: 8351088
• 负责人: MARIA DUFAU
• 金额: $ 157.13万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8351088
• 关键词: Acetylation; Androgens; Beryllium; Boxing; Breast; Breast Cancer Cell; Cell Nucleus; Cell physiology; Cells; Cholesterol; Chromatin; Complex; Coupled; Cytokine Receptors; DNA; DNA Binding Domain; DNA Methylation; DNA Polymerase II; Development; Distal; Elements; Endocrine; Enzymes; Epigenetic Process; Epithelium; Estrogens; Event; Exclusion; Failure; Family; Gametogenesis; Gene Expression; Generic Drugs; Genes; Genetic Transcription; Germ Cells; Gonadal structure; Gonadotropins; Half-Life; Histone Acetylation; Histone Deacetylase; Histones; Hormone Receptor; Human; Human Chorionic Gonadotropin; Immunoprecipitation; In Vitro; Inner mitochondrial membrane; Knockout Mice; LH Receptors; Laboratories; Lactation; Link; Lipids; Luteinizing Hormone; Maintenance; Mammary gland; Mediating; Mediator of activation protein; Messenger RNA; Methylation; Mitochondria; Molecular; Mus; Nuclear; Nuclear Export; Nuclear Orphan Receptor; Ovarian; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Polyribosomes; Pregnenolone; Process; Production; Prolactin; Prolactin Receptor; Proteins; RNA Helicase; Rattus; Receptor Gene; Recruitment Activity; Regulation; Reporting; Role; SP1 gene; Second Messenger Systems; Serum; Signal Transduction; Site; Small Interfering RNA; Spermatids; Spermatogenesis; Spermiogenesis; Sterility; Steroid biosynthesis; Steroids; Structure; Swelling; Testis; Testosterone; Tissues; Transcription Factor TFIIB; Transcriptional Activation; Translations; Wild Type Mouse; autocrine; base; cholesterol biosynthesis; chromatin immunoprecipitation; cofactor; demethylation; derepression; dimer; gonad function; helicase; in vivo; inhibitor/antagonist; insight; knock-down; leydig interstitial cell; male; malignant breast neoplasm; member; novel; peptide hormone; prevent; promoter; protein transport; receptor; receptor expression; second messenger; sperm cell

The Luteinizing Hormone Receptor (LHR): The LHR is a G-coupled seven-transmembrane receptor which is expressed primarily in the gonads where it mediates luteinizing hormone signals that regulate cyclic ovarian changes and testicular function. Our findings demonstrated that LHR gene expression at the transcriptional level is regulated by complex and diverse networks, in which coordination and interactions between these regulatory effectors are essential for silencing/activation of LHR expression. The proximal Sp1 site of the LHR promoter recruits histone deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcriptional expression. Site specific acetylation/methylation induce cell-specific phosphatase release. This serves as an on mechanism for Sp1 phosphorylation by PI3K/PKCzeta at Ser641 causing p107 repressor decruitment from Sp1 and LHR transcriptional activation. Maximal derepression of the LHR gene is dependent on complete DNA demethylation of the promoter, histone hyperacetylation and release of repressors (p107 and HDAC/Sin3A). This causes recruitment of TFIIB and Pol II and transcriptional activation. Our more recent studies demonstrated that Positive Cofactor 4 (PC4) has an important role in the formation/assembly of general transcriptional machinery in TSA-mediated LHR transcription. It is recruited by Sp1 following TSA treatment and acts as a coactivator of Sp1. The coactivator domain of PC4 and DNA binding domain of Sp1 are essential for their interaction. However, PC4 does not participate in TSA induced chromatin structural alteration since silencing of PC4 does not prevent release of HDAC/Sin3A and phosphatase from Sp1. These two events were demonstrated to be dependent on DNA methylation and chromatin decompactation resulting from histone acetylation at the promoter. Although TFIIB recruitment is largely dependent on PC4 we have ruled out TFIIB as target to link Sp1 to the transcriptional machinery. Since interaction with other members of this initiation and or/mediator complex is anticipated we are currently investigating PC4s linking function using various strategies. We have excluded acetylation of PC4 either basally or TSA- induced in the transcriptional activation process. However, immunoprecipitation studies demonstrated that TSA specifically induced acetylation of two PC4 interacting proteins (Mr 10 and 15 kDa). We are proceeding with their identification to determine their role in LHR gene transcription. Gonadotropin regulated genes: Gonadotropin Regulated Testicular RNA helicase (GRTH/DDX25) GRTH /DDX25 discovered in our laboratory is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and germ cells. It is a multi-functional protein that is essential for the completion of spermatogenesis. Males lacking GRTH are sterile due to the absence of sperm resulting from failure of round spermatids to elongate. Normal basal levels of testosterone in serum and LCs excluded abnormal steroidogenesis as responsible for the arrest of spermiogenesis. However, testosterone production was highly magnified in LCs of GRTH null mice (KO) compared to WT upon stimulation by gonadotropin both in vivo and in vitro. Our recent studies have revealed that GRTH has an important regulatory role in gonadotropin-induced androgen production by LCs. These cells in KO mice had reduced lipid droplets and swollen mitochondria (site of conversion of cholesterol,supplied by lipid droplets, to pregnenolone) with increased cholesterol content at the inner mitochondrial membrane. This resulted from the increase in StAR, a protein that transports cholesterol to the inner mitochondrial membrane and HMGCR a key enzyme in cholesterol biosynthesis. The half-life of StAR mRNA was significantly increased in the KO mice vs Wild type (WT) and association of StAR mRNA with GRTH protein was observed in WT mice. Major testosterone increases in KO over WT was observed upon hCG stimulation which resulted from the availability in KO of basal accumulated cholesterol as substrate of P450scc for pregnenolone production and other distal enzymes of the androgen pathway. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis provides insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male. GRTH participates in the export of specific mRNAs relevant to spermatogenesis from nuclear to cytoplasmic sites of germ cells including the Chromatoid body of spermatids (storage/processing site) and polyribosomes where it participates in translation of relevant messages. In recent studies we demonstrated the involvement of GRTH in the export of its own message from the nucleus to cytoplasmic sites in addition to previously reported mRNAs of spermatogenic genes. Association of GRTH mRNA with GRTH protein was observed in testis extracts of WT mice. Blockade of nuclear export with inhibitor leptomicin b caused significant nuclear accumulation of GRTH mRNA, with mayor decrease at the cytoplasmic level and its exclusion from the CB. This study has demonstrated the essential participation of the GRTH export/transport function to the CB and other cytoplasmic sites. Prolactin receptor (PRLR): The prolactin receptor is a member of the lactogen/cytokine receptor family which mediates the diverse cellular actions of prolactin (PRL) in several target tissues. PRL is a major factor in the proliferation and differentiation of breast epithelium and is essential in the stimulation and maintenance of lactation. It has been also implicated in the development of breast cancer. In humans hPRLR expression hPRLR is controlled at the transcriptional level by multiple promoters (one generic, also present in rat and mouse PIII, and five human specific hE1N1-hE1N5) that were defined and characterized in our laboratory. The transcription of PRLR in breast cancer cells by the preferentially utilized promoter PIII which lacks an estrogen responsive element is directed by E2/ERa through complex formation with SP1 and C/EBPb that associate with cognate elements and TFIIB and Pol II recruitment. BRET revealed ERa constitutive homodimers. E2 enhances through stabilization of the dimer, E2/ERa-dimer interaction with SP1 and C/EBPb. Chromatin immunoprecipitation and small interfering RNA knockdown of members of the complex in breast cancer cells (ERa+) demonstrated the endogenous recruitment of components of the complex onto the PIII promoter of the hPRLR gene. SP1 is the preferred transfactor for the recruitment of ERa to the complex that facilitates the C/EBPb association. The E2/ERa-induced hPRLR transcription was demonstrated in ERa-negative cells. Recruitment of ERa or C/EBPb but not Sp1 was E2 inducible. Cells with depletion of C/EBPb showed the recruitment of Sp1 and ERa. Only SP1 was recruited to PIII when ERa was knocked down. The ERa-SP1-C/EBPb complex was formed by the association of the DNA binding domain of ERa with ZF motifs of SP1 followed by recruitment of C/EBPb through its LeuZipper (LZ). Interactions between ZF of SP1 and LZ of C/EBPb stabilizes the ERa-SP1-C/EBPb complex. Our studies have demonstrated that the enhanced complex formation of ERa dimer with SP1 and C/EBPb dimers at the PIII promoter by E2 has an essential role in the transcriptional activation of the hPRLR gene in breast cancer cells

黄体生成素受体 (LHR)：LHR 是一种 G 偶联七次跨膜受体，主要在性腺中表达，介导黄体生成素信号，调节卵巢周期性变化和睾丸功能。我们的研究结果表明，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。 LHR 启动子的近端 Sp1 位点募集组蛋白脱乙酰酶和 Sin3A 辅阻遏物复合物，有助于 LHR 转录表达的沉默。位点特异性乙酰化/甲基化诱导细胞特异性磷酸酶释放。这是 PI3K/PKCzeta 在 Ser641 位点磷酸化 Sp1 的开启机制，导致 Sp1 和 LHR 转录激活中的 p107 阻遏蛋白缺失。 LHR 基因的最大去抑制取决于启动子的 DNA 完全去甲基化、组蛋白高度乙酰化和阻遏物（p107 和 HDAC/Sin3A）的释放。这会导致 TFIIB 和 Pol II 的募集以及转录激活。我们最近的研究表明，阳性辅因子 4 (PC4) 在 TSA 介导的 LHR 转录中的一般转录机制的形成/组装中具有重要作用。 它在 TSA 处理后被 Sp1 招募，并充当 Sp1 的共激活剂。 PC4 的共激活子结构域和 Sp1 的 DNA 结合结构域对于它们的相互作用至关重要。 然而，PC4 不参与 TSA 诱导的染色质结构改变，因为 PC4 的沉默不会阻止 Sp1 释放 HDAC/Sin3A 和磷酸酶。这两个事件被证明依赖于启动子处组蛋白乙酰化导致的 DNA 甲基化和染色质解致密化。尽管 TFIIB 募集很大程度上依赖于 PC4，但我们排除了将 TFIIB 作为将 Sp1 连接到转录机制的目标。由于预期与该起始和/或介体复合物的其他成员相互作用，我们目前正在使用各种策略研究 PC4 的连接功能。我们排除了转录激活过程中基础性或 TSA 诱导的 PC4 乙酰化。然而，免疫沉淀研究表明，TSA 特异性诱导两种 PC4 相互作用蛋白（Mr 10 和 15 kDa）的乙酰化。我们正在对其进行鉴定，以确定它们在 LHR 基因转录中的作用。 促性腺激素调节基因：促性腺激素调节睾丸 RNA 解旋酶 (GRTH/DDX25) 我们实验室发现的 GRTH /DDX25 是 Leydig 细胞 (LC) 和生殖细胞中存在的 RNA 解旋酶 DEAD-box 家族的睾丸特异性成员。它是一种多功能蛋白质，对于完成精子发生至关重要。缺乏 GRTH 的雄性是不育的，因为圆形精子细胞无法伸长，导致缺乏精子。血清和 LC 中睾酮基础水平正常排除了导致精子发生停滞的异常类固醇生成。然而，与 WT 相比，GRTH 缺失小鼠 (KO) 的 LC 在体内和体外受到促性腺激素刺激后，睾酮产生高度放大。我们最近的研究表明，GRTH 在 LC 促性腺激素诱导的雄激素产生中具有重要的调节作用。 KO 小鼠中的这些细胞的脂滴减少，线粒体（由脂滴提供的胆固醇转化为孕烯醇酮的位点）肿胀，线粒体内膜的胆固醇含量增加。 这是由于 StAR（一种将胆固醇转运至线粒体内膜的蛋白质）和 HMGCR（胆固醇生物合成中的关键酶）的增加所致。与野生型 (WT) 小鼠相比，KO 小鼠中 StAR mRNA 的半衰期显着延长，并且在 WT 小鼠中观察到 StAR mRNA 与 GRTH 蛋白的关联。在 hCG 刺激后观察到 KO 中的睾酮较 WT 显着增加，这是由于 KO 中可利用基础积累的胆固醇作为底物 P450scc 用于孕烯醇酮生产和雄激素途径的其他远端酶。 GRTH 与促性腺激素介导的类固醇生成相关的抑制作用的发现，为该解旋酶在调节男性类固醇生成中的新型负自分泌分子控制机制提供了见解。 GRTH 参与与精子发生相关的特定 mRNA 从生殖细胞的核到细胞质位点的输出，包括精细胞的染色质体（存储/加工位点）和多核糖体，其中它参与相关信息的翻译。在最近的研究中，我们证明除了先前报道的生精基因的 mRNA 之外，GRTH 还参与将其自身的信息从细胞核输出到细胞质位点。 在 WT 小鼠睾丸提取物中观察到 GRTH mRNA 与 GRTH 蛋白的关联。用抑制剂leptomomicin b 阻断核输出导致GRTH mRNA 显着在核内积聚，细胞质水平明显减少，并且被排除在CB 之外。 这项研究证明了 GRTH 输出/运输功能对 CB 和其他细胞质位点的重要参与。 催乳素受体 (PRLR)：催乳素受体是催乳素/细胞因子受体家族的成员，可介导催乳素 (PRL) 在多个靶组织中的多种细胞作用。 PRL 是乳腺上皮增殖和分化的主要因素，对于刺激和维持泌乳至关重要。它也与乳腺癌的发展有关。在人类中，hPRLR 的表达 hPRLR 在转录水平上由我们实验室定义和表征的多个启动子（一种通用启动子，也存在于大鼠和小鼠 PIII 中，以及五个人类特异性 hE1N1-hE1N5）控制。 PRLR 在乳腺癌细胞中的转录由优先利用的缺乏雌激素反应元件的启动子 PIII 进行，由 E2/ERa 通过与 SP1 和 C/EBPb 形成复合物来指导，SP1 和 C/EBPb 与同源元件以及 TFIIB 和 Pol II 招募相关。 BRET揭示了ERa组成型同二聚体。 E2 通过稳定二聚体、E2/ERa-二聚体与 SP1 和 C/EBPb 的相互作用来增强。乳腺癌细胞 (ERa+) 中复合物成员的染色质免疫沉淀和小干扰 RNA 敲低证明复合物成分内源性募集到 hPRLR 基因的 PIII 启动子上。 SP1 是将 ERa 招募到促进 C/EBPb 结合的复合物的首选转因子。 ERa 阴性细胞中证实了 E2/ERa 诱导的 hPRLR 转录。 ERa 或 C/EBPb 而非 Sp1 的募集是 E2 可诱导的。 C/EBPb 耗尽的细胞显示出 Sp1 和 ERa 的募集。当 ERa 被击倒时，只有 SP1 被招募到 PIII。 ERa-SP1-C/EBPb 复合物是通过 ERa 的 DNA 结合域与 SP1 的 ZF 基序结合形成的，然后通过其 LeuZipper (LZ) 招募 C/EBPb。 SP1 的 ZF 和 C/EBPb 的 LZ 之间的相互作用稳定了 ERa-SP1-C/EBPb 复合物。 我们的研究表明，E2 在 PIII 启动子处增强 ERa 二聚体与 SP1 和 C/EBPb 二聚体的复合物形成，对于乳腺癌细胞中 hPRLR 基因的转录激活具有重要作用