Hormone Receptors and Enzymes Regulating Gonadal and Mammary Function

调节性腺和乳腺功能的激素受体和酶

• 批准号: 7594114
• 负责人: MARIA DUFAU
• 金额: $ 106.58万
• 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/7594114
• 关键词: 1-Phosphatidylinositol 3-Kinase; Accounting; Affinity; Amino Acids; Binding; Bioluminescence; Boxing; COS-1 Cells; Caseins; Cell Nucleus; Cell physiology; Cells; Chromatin Structure; Complex; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; DNA Methylation; Development; Dominant-Negative Mutation; Dose; Down-Regulation; Endocrine; Energy Transfer; Enzymes; Epigenetic Process; Equilibrium; Event; Exons; Failure; Gametogenesis; Gene Activation; Gene Expression; Gene Targeting; Generations; Genes; Genetic Screening; Genetic Transcription; Germ Cells; Gonadotropins; HMGB2 Protein; Heterodimerization; High Mobility Group Proteins; Histone Acetylation; Histone Deacetylase; Histone Deacetylation; Histones; Hormone Receptor; Human; Infertility; JAK2 gene; Japanese Population; Knockout Mice; LH Receptors; Left; Leucine; Ligands; Localized; Luteinizing Hormone; MCF7 cell; Mammary gland; Mediating; Mediator of activation protein; Messenger RNA; Missense Mutation; Modification; Molecular; Molecular Conformation; Mus; Mutation; N-terminal; Nuclear; Nuclear Export; Nuclear Localization Signal; Nuclear Orphan Receptor; Pathway interactions; Patients; Peptidyl-Dipeptidase A; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Polyribosomes; Process; Prolactin; Prolactin Receptor; Promoter Regions; Protein Overexpression; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; RNA; RNA Helicase; RNA-Binding Proteins; Receptor Activation; Receptor Gene; Recruitment Activity; Regulation; Repression; Role; SP1 gene; Second Messenger Systems; Signal Transduction; Site; Sperm Maturation; Spermatids; Spermatocytes; Spermatogenesis; Staging; Sterility; Structure; TEP1 gene; Threonine; Transcript; Transcriptional Activation; Translation Initiation; Translations; Trichostatin A; Variant; base; cell type; demethylation; derepression; dimer; disulfide bond; gonad function; helicase; inhibitor/antagonist; leydig interstitial cell; malignant breast neoplasm; men; messenger ribonucleoprotein; mutant; novel; particle; peptide hormone; prevent; promoter; protein kinase C zeta; receptor; second messenger; sperm cell; transition protein 1

The Luteinizing Hormone receptor (LHR): The LHR gene is subject to repression by histone deacetylation at its promoter region. An Sp1 site was shown to be an essential mediator of trichostatin A (TSA)-induced LHR gene activation. TSA-induced histone acetylation and gene activation 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. This indicated the participation of an additional mechanism in TSA-induced derepression. Phosphatidylinositol 3-kinase/protein kinase C zeta (PI3K/PKCzeta)-mediated Sp1 phosphorylation at ser 641 accounts for Sp1 site-dependent LHR activation by TSA. Sp1 phosphorylation was required for the release of inhibitor p107 from Sp1. These findings have revealed a novel mechanism of TSA-regulated gene expression through derecruitment of a repressor from the LHR gene promoter in a PI3K/PKC-induced Sp1 phosphorylation-dependent manner. They also, supported the participation of phosphatase(s) in the control of LHR gene transcription, where a coordinate balance between PI3K/PKC (constitutive and/or induced activity) and phosphatase(s) could be critical for up- or down-regulation of LHR gene expression by its effect on the Sp1 phosphorylation status. Our studies revealed an important contribution of phosphatases PP2A and PP1 to TSA-induced activation of LHR gene transcription in a cell type-specific manner. TSA caused dose-dependent release of PP2A and PP1 from the LHR promoter in JAR and MCF-7 cells, respectively. Blockade of phosphatase activities enhanced the TSA effect, while overexpression largely prevented both the promoter induction and Sp1 phosphorylation elicited by TSA. PP2A was shown to associate with Sp1 at both its N- and C-termini of Sp1, where the phosphorylation site (at 641), that is critical for the PI3K/PKC mediated Sp1 phosphorylation induced by TSA, is located. Our findings have demonstrated that TSA-induced changes in chromatin structure cause a cell-specific release of a phosphatase that is associated with Sp1 directly (PP2A)or through HDAC (PP1). This favors the phosphorylation of Sp1 mediated by the PI3K/ PKC pathway (constitutively active) which in turn causes the release of the p107 inhibitor from SP1 and the marked transcriptional activation of the hLHR. Gonadotropin-Regulated Genes: We have previously identified a novel Gonadotropin-Regulated Testicular Helicase (GRTH/Ddx25). This enzyme, which 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-mice are sterile due to spermatid arrest at step 8 of spermatogenesis, and failure to elongate. The transcription of messages in spermatids steps 1-8 of these mice was not altered, but their translation was abrogated in a selective manner. Our current studies are defining the function of this helicase as an RNA binding protein, and its storage and translational functions during sperm progression. We have demonstrated differential localization of two GRTH protein species in subcellular compartments of germ cells. These include the 56 kDa species and the phosphorylated 61 kDa form primarily localized in the nucleus and cytoplasm respectively of germ cells. The post-transcriptional modification appears to be related to cytoplasmic-related events induced by protein kinase A at threonine residue(s). We have recently provided evidence for dual functional roles of GRTH as a component of mRNP in RNA export from nucleus to cytoplasm and in the translation of specific RNA transcripts at specific stages in germ cell development. The 56 kDa nuclear species interacted with CRM1 and participated in mRNA transport. The phosphorylated cytoplasmic 61kDa was associated with polyribosomes and selectively regulates the translation of specific genes. Phosphorylation of GRTH, in addition to facilitating the initiation of translation of its target genes, might induce conformational changes to recruit protein(s) to gain access of mRNAs to chromatoid bodies for storage and/or degradation. The N-terminal leucine-rich region was identified as the nuclear export signal that participates in the CRM1-dependent nuclear export pathway. A 14 amino acid GRTH sequence at 100-114 residues was identified as the nuclear localization signal. GRTH selectively regulated the translation of specific genes including histone 4 H4 and high mobility group protein HMG2 in germ cells. In addition, GRTH participated in the nuclear export of RNA messages including phosphoglyrate kinase 2 PGK2, testicular angiotensin converting enzyme (tACE), transition protein 1 & 2 TP1 & TP2. The cytoplamic levels of these proteins are markedly reduced in GRTH null mice. Our studies have demonstrated that GRTH/Ddx25 is a multifunctional RNA helicase that is an essential regulator of sperm maturation. Two polymorphic forms of GRTH were identified by genetic screening of GRTH/DDX25 in fertile and infertile Japanese men with non-obstructive azoospermia. We found heterozygous mutations in exons 8 (missense) and exon 11 (sense). The missense mutation Arg242 His in exon 8 was identified in 5.8% of infertile patients and 1% of normal subjects, and the silent was observed in 2% of infertile patients. Although the mutant protein was efficiently expressed in COS-1 cells, only the 56 kDa nuclear/cytoplasmic non-phosphorylated species was present and the 61 kDa species was absent. This finding highlights the relevance of the R242 residue for the post-transcriptional modification leading to generation of this relevant species. The absence of the phospho-GRTH species could be of relevance to some of the functional aspects of the protein that impact on germ cell development and/or function. Prolactin Receptors Prolactin:Short forms of the prolactin receptor (PRLR) with abbreviated cytoplasmic sequences (S1 a and S1b) exert dominant-negative effects on prolactin (PRL)-induced activation of transcription by the long form (LF). The LF homodimer is the only PRLR form able to activate the JAK2/STAT5 pathway, which is essential for PRL-induced gene transcription. The inhibitory action of SFs results from their heterodimerization with the LF. The presence of preexisting homodimers of the LF receptor in the absence of prolactin indicated that PRL is a conformational modifier rather that a dimer-inducer. Bioluminescence resonance energy transfer analysis (BRET) demonstrated PRL-independent homo- and heterodimer association of hPRLR variants. However, PRL binding could alter dimerized PRLR conformation to facilitate signal transduction. Mutation of four conserved extracelular cys residues, which form intramolecular disulfide bonds (36-46, 75-86 aa) that are necessary for PRL binding to the PRLR, completely eliminated the PRL-stimulated JAK phosphorylation that is normally observed in the wild type LFs and SFs. The wild type SF (S1b), in contrast to the LF, displayed high constitutive JAK2 phosphorylation that was not observed in S1bX mutant. This indicates that intra-molecular Cys-bridge(s)are required for the correct conformation to allow JAK binding to box-1. Also, the inhibitory action of the wild type S1b on LF mediated -casein promoter activity was not observed in the S1bX. BRET analysis showed reduced heterodimeric association of LF with S1bX versus wild type and an increased affinity in homodimers of SFX. The increased association of S1bX homodimers leaves the LFs to form homodimers for ligand-induced signaling.

黄体生成素受体（LHR）： LHR基因在其启动子区域受到组蛋白去乙酰化的抑制。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。tsa诱导的组蛋白乙酰化和基因激活在Sp1/Sp3表达、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没有变化的情况下普遍存在。这表明在tsa诱导的抑郁中有一个额外的机制参与。磷脂酰肌醇3-激酶/蛋白激酶Czeta （PI3K/PKCzeta）介导的Sp1在ser 641位点的磷酸化解释了TSA对Sp1位点依赖性LHR的激活。Sp1磷酸化是Sp1释放抑制剂p107的必要条件。这些发现揭示了一种新的tsa调控基因表达的机制，通过PI3K/ pkc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。他们还支持磷酸酶参与控制LHR基因转录，其中PI3K/PKC（构成性和/或诱导活性）和磷酸酶之间的协调平衡可能通过其对Sp1磷酸化状态的影响对LHR基因表达的上调或下调至关重要。我们的研究揭示了磷酸酶PP2A和PP1以细胞类型特异性的方式对tsa诱导的LHR基因转录激活的重要贡献。TSA分别在JAR和MCF-7细胞中引起LHR启动子中PP2A和PP1的剂量依赖性释放。磷酸酶活性的阻断增强了TSA的作用，而过表达在很大程度上阻止了启动子的诱导和TSA引起的Sp1磷酸化。PP2A在Sp1的N端和c端与Sp1结合，其中磷酸化位点（641）对TSA诱导的PI3K/PKC介导的Sp1磷酸化至关重要。我们的研究结果表明，tsa诱导的染色质结构变化导致细胞特异性磷酸酶的释放，该磷酸酶与Sp1直接相关（PP2A）或通过HDAC （PP1）。这有利于由PI3K/ PKC通路介导的Sp1磷酸化（组成活性），这反过来导致Sp1释放p107抑制剂和hLHR的显著转录激活。