INHIBITION OF SECRETORY ACTIVATION BY PROGESTERON

孕激素在妊娠期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，并且在妊娠后期具有抑制分泌激活的额外功能，分泌激活定义为催乳素（PRL）和糖皮质激素（GC）刺激乳蛋白合成和紧密连接闭合。孕酮的这种重要抑制作用的机制尚未明确，这是本提案的总体目标。我们 结合孕酮受体（PR）仅在一部分上皮细胞中表达的事实进行的初步研究表明，直接和间接的旁分泌机制都介导了孕酮依赖性的分泌激活抑制。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，通过干扰β-酪蛋白启动子水平的StatS反式激活，他们建议TGF β作为一种旁分泌因子介导的β-酪蛋白合成和紧密连接关闭的甾体酮依赖性抑制。在本提案中，我们试图测试和界定这些直接和间接机制，目的如下。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。我们将确定孕酮抑制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通过破坏启动子转录辅激活子和辅抑制子的正常平衡来阻断StatS转录活性的假说。AIM#2将检验TGF β是酪蛋白合成和紧密连接闭合的胆固醇依赖性抑制的旁分泌介质的假设。乳腺外植体和细胞培养物将用于体外研究，在MMTV控制下表达显性阴性TGF β受体的转基因小鼠将用于体内分析。目标3将确认直接和 通过使用PR-LacZ和β-酪蛋白-EGFP报告基因小鼠和这些转基因动物的杂交，由目的#1和#2建立的间接机制在体内发生。我们将确定β-酪蛋白和PR启动子的空间和时间表达模式，沿着内源性TGF β，以响应短期内分泌消融/激素 替换协议。

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.