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: 9349279
• 负责人: MARIA DUFAU
• 金额: $ 82.23万
• 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/9349279
• 关键词: 5' Flanking Region; Acetylation; Adjuvant; Affect; Androgens; Binding; Binding Proteins; Binding Sites; Boxing; Breast; Breast Cancer Cell; Breeding; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Chromatin; Complex; DNA; DNA Methylation; DNA Polymerase II; Development; Distal; ERBB2 gene; Endocrine; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Epigenetic Process; Estradiol; Estrogens; Etiology; Failure; Family; Fibroblasts; Flutamide; Gametogenesis; Genes; Genetic Transcription; Germ; Germ Cells; Gonadal structure; Gonadotropins; Growth; Histone Deacetylase; Histones; Hormone Receptor; Human; Investigation; JAK2 gene; Knockout Mice; LH Receptors; Lactation; Ligands; Link; LoxP-flanked allele; MAPK3 gene; MCF7 cell; Mammary Neoplasms; Mammary gland; Mediating; Meiosis; Messenger RNA; Methylation; Mitogen-Activated Protein Kinases; Molecular; Mus; Neoplasm Metastasis; Nuclear Orphan Receptor; Ovarian; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Polyribosomes; Preparation; Process; Production; Prolactin; Prolactin Receptor; Protein Export Pathway; Proteins; Proto-Oncogene Proteins c-akt; RNA Helicase; Receptor Gene; Receptor Up-Regulation; Recruitment Activity; Refractory; Regulation; Resistance; Role; Route; SP1 gene; Second Messenger Systems; Signal Pathway; Signal Transduction; Site; Spermatids; Spermatocytes; Spermatogenesis; Staging; Stat5 protein; Sterility; Steroid biosynthesis; Structure; System; Testis; Tissues; Transcription Factor TFIIB; Transcriptional Activation; Transgenic Mice; Translating; Up-Regulation; autocrine; base; chromatin remodeling; cofactor; demethylation; derepression; dimer; gene product; gonad function; helicase; hormone therapy; in vitro Model; in vivo; inhibitor/antagonist; knock-down; leydig interstitial cell; mRNA Stability; male; malignant breast neoplasm; mammary epithelium; member; mouse model; novel; nucleocytoplasmic transport; overexpression; paracrine; peptide hormone; prevent; promoter; receptor; receptor expression; second messenger; sertoli cell; sperm cell; steroidogenic acute regulatory protein; transcription factor

The Luteinizing Hormone Receptor (LHR)is expressed primarily in the gonads where it mediates LH signals that regulate ovarian and testicular function. The LHR gene transcription is regulated by diverse networks, in which coordination and interactions between regulatory effectors are essential for silencing/activation of LHR expression. The proximal Sp1 site of the promoter recruits histone (H) deacetylases and the Sin3A corepressor complex that contributes to the silencing of LHR transcription. Site specific acetylation/methylation-induced phosphatase release serve as switch for Sp1 phosphorylation, recruitment of TFIIB and Pol II and transcriptional activation. Maximal derepression of the gene is dependent on DNA demethylation of the promoter, H3/H4 acetylation and HDAC/Sin3A release. Positive Cofactor 4 (PC4) has an important role in the formation/assembly of PIC in TSA-mediated LHR transcription. It is recruited by Sp1 following TSA treatment and acts as its coactivator. However, PC4 does not participate in TSA release of phosphatases, Sp1 phosphorylation or release repressor/complexes. TFIIB recruitment is dependent on PC4 and have ruled out TFIIB as its direct target. However, TSA induced acetylation of a PC4 interacting proteins, was identified as Acetyl H3 by MS, and its presence in the complex in association to chromatin at the promoter was demonstrated by ChiP/reChiP. The role of these interactions and their participation in the assembly of the PIC and transcriptional activation are under investigation. Immunoprecipitated flag-tagged PC4/H3-complexes in transfected MCF-7 cells revealed Acetyl H3 at various K pulled-down by Flag Ab. To elucidate the physiological impact of PC4 on Sp1 directed transcription in gonads, we generated a PC4-floxed mice by breeding with transgenic mice expressing tissue specific Cyp17Cre. We are now analyzing PC4 null-mice with specific deletion in testicular Leydig cells. Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25, is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and meiotic germ 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. Besides, to its intrinsic helicase activity, it is a shuttling protein that exports specific mRNAs from the nucleus to cytoplasmic sites. Our studies have demonstrated the essential participation of the GRTH export/transport of mRNAs, in the structural integrity of the Chromatoid Body (storage/processing of mRNAs) and their transit/association to actively translating polyribosomes where it may regulate translational initiation of genes. GRTH is regulated by LH through androgen (A)/A receptor (AR)at the transcriptional level in LCs (direct)with impact in steroidogenesis, and germ cells (indirect-AR in Sertoli cells) where its expression is both cell-and stage specific. It displays negative autocrine control of A production in LCs by preventing overstimulation of the LH-induced A pathway through enhanced degradation of StAR protein. Tg-mice models carrying GRTH 5' flanking regions-GFP provided in vivo systems that permit differential elucidation of regions in the GRTH gene that directs its expression (upstream;-6.4kb/-3.6kb) in germ cells and downstream in LCs and its regulation by A/AR in LC (directly) and indirectly in germ cells. Functional binding sites for germ cell specific transcription factor (GCNF)are present in round spermatids (RS) and spermatocytes(SP) and its regulation by A/AR identified in the distal region-5270-5252 nt of the GRTH gene was operative selectively in RS. GCNF knock-down in RS preparation from testis of TG mice decreased GFP/GRTH expression upon in vivo treatment of mice with flutamide (Flu)- an AR antagonist. Moreover, Flu treatment of WT mice caused selective reduction of GRTH in RS. The studies provided evidence for actions of A on GCNF cell specific regulation of GRTH expression in germ cells. Also, GRTH associates with GCNF mRNA and its absence causes increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. Our in vivo/in vitro models link A actions to germ cells through GCNF as an A regulated transfactor that controls transcription/expression of GRTH, provided a connection of A action to two relevant germ cell genes essential for the progress of spermatogenesis and established their regulatory interrelationship. The study provides valuable links and facilitate what could be a difficult search for gene products in Sertoli cells affecting germ cell function and spermatogenesis (1). Transition protein 2 (Tp2), a chromatin remodeling protein whose mRNA associates with GRTH-required for spermatid elongation, fail to express in GRTH KO with impaired nuclear transport. We have determined Tp2 mRNA and GRTH protein binding regions. Nt 1-47&78-127 downstream of TGA of Tp2 bind conserved GRTH motifs(Ia/V)(3). Prolactin receptor (PRLR)-mediates the diverse cellular actions of Prolactin (PRL). PRL is a major factor in the proliferation and differentiation of breast epithelium and essential for lactation. It has an important role in the etiology and progression of breast cancer, tumoral growth and chemoresistance. hPRLR expression is controlled at the transcriptional level by the preferentially utilized PIII promoter which lacks and ERE. Complex formation of ERa homodimer (non-DNA bound) with Sp1 and C/EBPb dimers bound to their sites at the PIII promoter is required for basal and E2-induced transcriptional activation of the PRLR gene. PRL in tumoral breast causes cell proliferation via activation of its cognate receptor. Exacerbation of PRLs actions resulting from increased receptor number can explain resistance to E2 inhibitors in breast cancer. Our recent studies demonstrated in MCF7 breast cancer ERa+ HER2+ cells, upregulation of PRLR transcription/expression induced by endogenous/exogenous PRL in absence or estrogen (E2) via the the PRLR with essential participation ERa and JAK2/STAT5, mitogen activated protein kinase (MAPK) and PI3K pathways. This occurs by interaction of phosphorylated ERa (pERa)-generated by PRL/PRLR/JAK2 actions to SP1 and CEBPb and of STA5a,b to a GAS site in PIII. We also found that ERBB2/HER2, which is overexpressed in 10 of breast cancers, phosphorylated and activated by JAK2 via PRL/PRLR induce ERa phosphorylation. Such cross-talk activation of ERBB2/HER2 signalling was identified as an alternate route in the increase of PRLR induced by PRL in breast cancer cells. Paracrine inputs have an active role in breast tumor development, progression and metastasis . Stromal fibroblasts secrete Epidermal growth factor (EGF) activates through its receptor EGFR/ERRB1 present in breast tumor cells (BTC) signaling pathways which in turn trigger requisite transcription factors and co-activators that can affect the proliferation of BTC. We have shown in MCF7 cells marked activation of PRLR gene transcription/expression by exogenous EGF independent of PRL/PRLR/JAK2 with the essential involvement of the MPK; ERK1/2 and PI3K-AKT pathways. These are mediated by EGFR Y1068,1086 and the c-SRC dependent EGFR Y845 for ERa and STAT5b phosphorylation, respectively -for their recruitment to the PRLR promoter. Aside from its independence in E2 and the activators requirements (PRL vs EGF ) there are important commonalities (requisite ERa,STAT5)in the mechanism of PRLR transcription /expression. Also,the studies revealed STAT5 interaction with ERa as essential for PRLR as essential for PRLR up-regulation. Our findings provide mechanistic avenues for the increase of PRLR that upon resistance to hormonal therapy could promote progression and metastasis in breast cancer.

黄体生成激素受体（LHR）主要在介导调节卵巢和睾丸功能的LH信号的性腺中表达。 LHR基因转录受到不同网络的调节，其中协调和调节效应子之间的相互作用对于LHR表达的沉默/激活至关重要。启动子的近端SP1位点募集组蛋白（H）脱乙酰基酶和SIN3A Corepressor复合物，有助于LHR转录的沉默。位点特异性乙酰化/甲基化诱导的磷酸酶释放是SP1磷酸化，TFIIB和POL II的募集以及转录激活的开关。该基因的最大压抑取决于启动子H3/H4乙酰化和HDAC/SIN3A释放的DNA脱甲基化。正辅因子4（PC4）在TSA介导的LHR转录中PIC的形成/组装中具有重要作用。它是通过TSA治疗后通过SP1招募的，并充当其共激活剂。 但是，PC4不参与TSA的磷酸酶，SP1磷酸化或释放抑制剂/复合物的释放。 TFIIB招聘取决于PC4，并排除了TFIIB为直接目标。然而，TSA诱导的PC4相互作用蛋白的乙酰化通过MS鉴定为乙酰H3，并且通过ChIP/Rechip证明了其在启动子处与染色质相关的复合物中的存在。这些相互作用的作用及其在PIC和转录激活组装中的参与正在研究中。转染的MCF-7细胞中的免疫沉淀标记的PC4/H3复合物显示在FLAG AB拉下的各种K下方乙酰基H3。为了阐明PC4对性腺中SP1定向转录的生理影响，我们通过用表达组织特异性CYP17CRE的转基因小鼠繁殖来产生PC4-串联小鼠。现在，我们正在分析睾丸leydig细胞中具有特定缺失的PC4无效小鼠。 Gonadotropin regulated Testicular RNA Helicase (GRTH/DDX25, is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig cells (LC) and meiotic germ 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. Besides, to its intrinsic旋转酶活性，这是一种从细胞核到细胞质部位的特定mRNA的蛋白质，我们的研究表明，mRNA的GRTH出口/运输的重要参与是在色物体的结构完整性中（MRNAS的存储/处理）及其通过统一性的统一性的， LCS（直接）的转录水平的雄激素（A）/A受体（AR）在类固醇生成中影响，生殖细胞（Sertoli细胞中的间接AR​​）其表达既是细胞和阶段的特异性）。它通过通过增强恒星蛋白的降解来防止LH诱导的途径过度刺激来显示LCS生产的负自分泌控制。 带有GRTH 5'侧面区域GFP的TG - 小组模型在体内系统中提供了允许对GRTH基因中区域的差异化，该区域指导其表达（上游； -6.4KB/-3.6KB）在LCS中的生殖细胞和下游的LC及其在LC中的AR（直接）（直接在LC（直接）和独立细胞中）中的TG-RICE。生殖细胞特异性转录因子（GCNF）的功能结合位点存在于圆形的精子（RS）和精子细胞（SP）中，并且在RS中选择性地在RS中选择性地操作了GRTH基因的远端区域-5270-5252 NT中鉴定出的A/AR调节。 TG小鼠睾丸制剂中的GCNF敲低敲低，在用氟他胺（Flu）（流感）（AR拮抗剂）体内治疗小鼠时，GFP/GRTH表达降低了GFP/GRTH。此外，WT小鼠的流感治疗导致Rs的GRTH选择性降低。研究提供了A对GCNF细胞特异性调节GRTH表达在生殖细胞中的作用的证据。同样，GRTH与GCNF mRNA及其缺失有关GCNF表达和mRNA稳定性的增加，表明GRTH对GCNF的负自分泌调节负。我们的体内/体外模型通过GCNF作为控制GRTH的转录/表达的一个调节转录器将A的作用与生殖细胞联系起来，它提供了与两个相关的生殖细胞基因有关对精子发生进展至关重要的相关生殖细胞基因的联系，并确定了其调节性相互关系。该研究提供了有价值的联系，并促进了影响生殖细胞功能和精子发生的Sertoli细胞中难以搜索的基因产物（1）。过渡蛋白2（TP2）是一种染色质重塑蛋白，其mRNA与GRTH需要用于精子伸长的蛋白，无法在核转运受损的Grth KO中表达。我们已经确定了TP2 mRNA和GRTH蛋白结合区域。 TP2 TGA下游的NT 1-47和78-127结合了保守的GRTH基序（IA/V）（3）。 催乳素受体（PRLR）降低了催乳素（PRL）的各种细胞作用。 PRL是乳腺上皮增殖和分化的主要因素，对于哺乳为必不可少的。 它在乳腺癌，肿瘤生长和化学抗性的病因和进展中具有重要作用。 HPRLR表达在转录水平上由缺乏和ERE的优先使用的PIII启动子控制。 ERA同二聚体（非DNA结合）与SP1和C/EBPB二聚体的复合形成是在PIII启动子处与其位点结合的，这是基础和E2诱导的PRLR基因的转录激活所必需的。 肿瘤乳房中的PRL通过激活其同源受体引起细胞增殖。受体数量增加引起的PRLS作用加剧可以解释乳腺癌中对E2抑制剂的抗性。 我们最近的研究证明了MCF7乳腺癌ERA+ HER2+细胞，在缺席或雌激素（E2）中通过具有必要的参与ERA和JAK2/STAT5的PRLR（E2）诱导的PRLR转录/表达上调，以及有丝分裂原激活的蛋白质激酶（MAPK）和PI3K PATH的PRLR。这是通过PRL/PRLR/JAK2对SP1和CEBPB以及STA5A的磷酸化ERA（PERA）的相互作用发生的，B，B，b与PIII的气体位点。 我们还发现，在10种乳腺癌中过表达的ERBB2/HER2通过JAK2通过PRL/PRLR诱导ERA磷酸化。 ERBB2/HER2信号传导的这种串扰激活被确定为PRL在乳腺癌细胞中诱导的PRLR增加的替代途径。旁分泌输入在乳腺肿瘤的发育，进展和转移中具有积极作用。 基质成纤维细胞分泌表皮生长因子（EGF）通过其受体EGFR/ERRB1激活乳腺肿瘤细胞（BTC）信号通路，从而触发必要的转录因子和可能影响BTC增殖的共同激活剂。我们已经在MCF7细胞中显示了通过prl/prlr/jak2独立于MPK的基本参与的外源EGF来标志着PRLR基因转录/表达的激活。 ERK1/2和PI3K-AKT途径。这些由EGFR Y1068,1086和ERA和STAT5B磷酸化的C -SRC依赖性EGFR Y845介导 - 将其招募到PRLR启动子。 除了其在E2中的独立性和激活剂的要求（PRL与EGF）外，PRLR转录 /表达的机理中还有重要的共同点（必要的ERA，STAT5）。此外，研究揭示了STAT5与ERA的相互作用对于PRLR至关重要，这对于PRLR上调至关重要。我们的发现为增加PRLR的增加提供了机械途径，抗激素疗法可以促进乳腺癌的进展和转移。