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.

黄体生成素受体（LHR）主要在性腺中表达，介导黄体生成素信号，调节卵巢和睾丸功能。LHR基因的转录受到多种网络的调控，其中调控效应物之间的协调和相互作用对于LHR表达的沉默/激活至关重要。启动子的近端Sp1位点招募组蛋白(H)去乙酰化酶和Sin3A共抑制因子复合物，这有助于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募集和转录激活的开关。该基因的最大抑制依赖于启动子的DNA去甲基化、H3/H4乙酰化和HDAC/Sin3A的释放。在tsa介导的LHR转录中，正辅因子4 （PC4）在PIC的形成/组装中起重要作用。它在TSA治疗后被Sp1招募并作为其辅激活剂。然而，PC4不参与TSA磷酸酶的释放、Sp1磷酸化或释放抑制物/复合物。TFIIB的招募依赖于PC4，并且已经排除了TFIIB作为其直接靶点的可能性。然而，TSA诱导PC4相互作用蛋白乙酰化，MS鉴定为乙酰H3， ChiP/reChiP证实其存在于启动子处与染色质相关的复合体中。这些相互作用及其在PIC组装和转录激活中的作用正在研究中。在转染的MCF-7细胞中，免疫沉淀标记的PC4/H3复合物在不同的K位点上被Flag Ab拉低，显示乙酰H3。为了阐明PC4对性腺Sp1定向转录的生理影响，我们通过与表达组织特异性Cyp17Cre的转基因小鼠杂交，产生了PC4-floxed小鼠。我们现在正在分析睾丸间质细胞特异性缺失的PC4缺失小鼠。