INHIBITION OF SECRETORY ACTIVATION BY PROGESTERON

黄体酮对分泌激活的抑制

• 批准号: 7634423
• 负责人: Dean P Edwards
• 金额: $ 19.49万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7634423
• 关键词: Ablation; Adrenalectomy; Alveolar; Binding; Biological Assay; Birth; Caseins; Cells; Chemosensitization; Closure; Code; Cultured Cells; Data; Development; Doctor of Philosophy; Dominant-Negative Mutation; Ductal; Endocrine; Epithelial Cell Proliferation; Epithelial Cells; Equilibrium; Gene Expression; Gene Expression Profile; Gene Targeting; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Hand; Hormones; Immunohistochemistry; In Situ; In Situ Hybridization; In Vitro; Knock-in Mouse; Laboratories; LacZ Genes; Lipids; Localized; Mammary gland; Mediating; Mediator of activation protein; Messenger RNA; Milk; Milk Proteins; Modeling; Molecular; Morphogenesis; Mouse Mammary Tumor Virus; Mus; Ovariectomy; Pattern; Play; Pregnancy; Production; Progesterone; Progesterone Receptors; Prolactin; Protein Biosynthesis; Protein Overexpression; Proteins; Protocols documentation; RNA Interference; Reflux; Regulation; Reporter; Reporter Genes; Research Personnel; Response Elements; Role; Serum; Side; Site; System; Testing; Tight Junctions; Transactivation; Transcription Coactivator; Transfection; Transforming Growth Factor beta; Transgenic Animals; Transgenic Mice; Work; beta-Casein; chromatin immunoprecipitation; falls; in vivo; interstitial; knock-down; mouse model; paracrine; prevent; progesterone receptor negative; programs; promoter; protein distribution; protein expression; receptor; receptor binding; research study; response; tissue culture

Progesterone plays a central role in the development and differentiation of the mammary gland during pregnancy. It stimulates extensive epithelial cell proliferation leading to ductal side branching and alveolar morphogenesis, and late in pregnancy, has the additional function of inhibiting secretory activation, defined as prolactin (PRL) and glucocortiocoid (GC) stimulation of milk protein synthesis and tight junction closure. The mechanism for this important inhibitory action of progesterone is not well defined and is the overall objective of this proposal. Our preliminary studies taken together with the fact that progesterone receptor (PR) is expressed in only a fraction of epithelial cells, indicates that both direct and indirect paracrine mechanisms mediate progesterone-dependent inhibition of secretory activation. Our Preliminary results show that PR can directly repress beta-casein gene transcription induced by PRL/GC by interfering with StatS transactivation at the level of the beta-casein promoter and they suggest that TGFbeta acts as a paracrine factor mediating progesterone-dependent inhibition of beta-casein synthesis and tight junction closure. In this proposal we seek to test and define these direct and indirect mechanisms with the following aims. AIM#1 will define the molecular mechanisms by which PR directly inhibits PRL/GC induction of beta-casein transcription in primary mouse mammary epithelial cells. We will determine the extent to which progesterone inhibits transcriptional vs mRNA translational control of beta-casein expression stimulated by PRL/GC, and explore the hypothesis that PR blocks the transcriptional activity of StatS by disrupting the normal balance of transcriptional coactivators and corepressors at the promoter. AIM#2 will test the hypothesis that TGFbeta is a paracrine mediator of progesterone-dependent inhibition of casein synthesis and tight junction closure. Mammary gland explants and cell cultures will be used for in vitro studies and transgenic mice expressing a dominant negative TGFbeta receptor under the control of MMTV will be used for in vivo analysis. AIM#3 will confirm that direct and indirect mechanisms established from Aims #1 and #2 occur in vivo by use of PR-LacZ and beta-casein-EGFP reporter gene mice and crosses of these transgenic animals. We will determine the spatial and temporal expression patterns of beta-casein and PR promoters, along with endogenous TGFbeta, in response to short term endocrine ablation/hormone replacement protocols.

孕酮在妊娠期间乳腺的发育和分化中起着核心作用。它刺激广泛的上皮细胞增殖，导致导管侧支和肺泡形态发生，在妊娠晚期，具有抑制分泌激活的附加功能，定义为催乳素(PRL)和糖皮质激素(GC)刺激乳蛋白合成和紧密连接关闭。黄体酮这种重要的抑制作用的机制还没有很好地确定，这是本提案的总体目标。我们的 初步研究表明，孕激素受体(PR)仅在一小部分上皮细胞中表达，表明直接和间接旁分泌机制都介导孕酮依赖的分泌激活抑制。我们的初步结果表明，PR可以直接抑制PRL/GC诱导的β-酪蛋白基因转录，在β-酪蛋白启动子水平上干扰STATS的反式激活，提示TGFβ作为旁分泌因子介导孕酮依赖的抑制β-酪蛋白合成和紧密连接关闭。在这项提案中，我们寻求测试和界定这些直接和间接机制，目的如下。目的#1将明确PR直接抑制PRL/GC诱导原代小鼠乳腺上皮细胞β-酪蛋白转录的分子机制。我们将确定黄体酮在多大程度上抑制PRL/GC刺激的β-酪蛋白表达的转录与mRNA翻译控制， 并探索PR通过破坏转录辅助激活因子和辅助抑制因子在启动子上的正常平衡来阻断STATS的转录活性的假设。目的#2将验证这样的假设，即转化生长因子β是孕酮依赖的抑制酪蛋白合成和紧密连接关闭的旁分泌介体。乳腺外植体和细胞培养将用于体外研究，在MMTV控制下表达显性负TGFbeta受体的转基因小鼠将用于体内分析。目标3将确认直接和 从AIMS#1和#2建立的间接机制通过使用PR-LacZ和β-酪蛋白-EGFP报告基因小鼠和这些转基因动物的杂交在体内发生。我们将确定β-酪蛋白和PR启动子以及内源性转化生长因子β在短期内分泌消融/激素作用下的时空表达模式。 替换协议。