DIRECT AND INDIRECT MECHANISM FOR THE INHIBITION OF SECRETORY ACTIVATION BY PROGE

PROGE 抑制分泌激活的直接和间接机制

• 批准号: 7018037
• 负责人: Dean P Edwards
• 金额: $ 18.46万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7018037
• 关键词: RNA interference; caseins; chromatin immunoprecipitation; epidermal growth factor; gene expression; genetic promoter element; genetic transcription; genetically modified animals; glucocorticoids; growth factor receptors; hormone regulation /control mechanism; laboratory mouse; mammary epithelium; paracrine; progesterone; progesterone receptors; prolactin; reporter genes; secretion; tight junctions; tissue /cell culture; transforming growth factors

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.

黄体酮在怀孕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，导致导管侧分支和肺泡形态发生，并且在妊娠晚期，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闭合。黄体酮这种重要抑制作用的机制尚未明确定义，但这是该提案的总体目标。我们的 初步研究与黄体酮受体（PR）仅在一小部分上皮细胞中表达的事实相结合，表明直接和间接旁分泌机制介导黄体酮依赖性分泌激活抑制。我们的初步结果表明，PR 可以通过干扰 β-酪蛋白启动子水平的 StatS 反式激活，直接抑制 PRL/GC 诱导的 β-酪蛋白基因转录，并​​且表明 TGFbeta 作为旁分泌因子介导黄体酮依赖性β-酪蛋白合成和紧密连接闭合的抑制。在本提案中，我们试图测试和定义这些直接和间接机制，其目标如下。 AIM#1 将定义 PR 直接抑制原代小鼠乳腺上皮细胞中 PRL/GC 诱导 β-酪蛋白转录的分子机制。我们将确定黄体酮抑制 PRL/GC 刺激的 β-酪蛋白表达的转录与 mRNA 翻译控制的程度， 并探讨 PR 通过破坏启动子处转录共激活子和辅阻遏子的正常平衡来阻断 StatS 转录活性的假设。 AIM#2 将检验以下假设：TGFbeta 是黄体酮依赖性酪蛋白合成和紧密连接闭合抑制的旁分泌介质。乳腺外植体和细胞培养物将用于体外研究，而在 MMTV 控制下表达显性失活 TGFbeta 受体的转基因小鼠将用于体内分析。 AIM#3 将确认直接和 通过使用 PR-LacZ 和 β-酪蛋白-EGFP 报告基因小鼠以及这些转基因动物的杂交，从目标 #1 和 #2 建立的间接机制发生在体内。我们将确定 β-酪蛋白和 PR 启动子以及内源性 TGFbeta 响应短期内分泌消融/激素的空间和时间表达模式 更换协议。