Progesterone Inhibition--Milk Protein Gene Transcription

孕激素在妊娠乳腺的结构和功能发育过程中具有两种相反的生物学作用。增生作用刺激导管侧分支和小叶肺泡的形成。同时黄体酮具有抑制乳蛋白基因表达直至分娩的抗分化作用。乳蛋白表达调控的主要激素是催乳素，其作用是通过激活Stat5转录因子与乳蛋白基因启动子的相互作用来介导的。糖皮质激素通过糖皮质激素受体（GR）与Stat5的积极合作相互作用增强催乳素的作用。我们初步研究了糖皮质激素受体（GR）与Stat5的体外正合作相互作用。我们的初步体外实验结果表明，孕激素通过糖皮质激素受体（GR）与Stat5的直接相互作用，在基因转录水平上抑制β -酪蛋白的表达。我们的初步体外实验结果表明，孕激素通过与β -酪蛋白启动子上的孕激素受体（PR）直接相互作用，干扰泌乳素/Stat5信号传导，在基因转录水平上抑制β -酪蛋白的表达。本提案的目的是确定体外和体内这种直接依赖pr的β -酪蛋白转录抑制的机制。在AIM #1中，生化方法将用于定义PR和Stat5之间的蛋白质-蛋白质和蛋白质- dna相互作用，这些相互作用有助于PR介导的Stat5活性抑制或GR的增强作用。在AIM #2中，将使用乳腺上皮细胞培养中的基于细胞的转录测定来定义PR抑制Stat5和GR依赖的β -酪蛋白报告基因转录的机制。目的3，我们将通过分析重组腺病毒表达的β -酪蛋白报告基因的调控元件，确定体外确定的PR依赖性β -酪蛋白基因转录抑制机制是否也发生在体内乳腺中。在AIM #4中，我们将确定直接和间接重组腺病毒的相对贡献。在AIM #4中，我们将确定PR在体外和体内抑制β -酪蛋白转录的直接和间接（旁分泌）机制的相对贡献。本研究的目的是确定一种乳蛋白基因所特有的PR负基因调控机制，从而实现孕激素在妊娠期乳腺中的特定生物学作用。

Progesterone has two opposing biological actions during the structural and functional development of the pregnant mammary gland. A proliferative action stimulates ductal side branching and formation of lobuloalveoli. At the same time progesterone has the anti-differentiative effect of repressing milk protein gene expression until parturition. The primary hormone responsible for regulation of milk protein expression is prolactin whose effect is mediated through activating interaction of the Stat5 transcription factor with the promoters of milk protein genes. Glucocorticoids potentiate the effect of prolactin through a positive cooperative interaction of the glucocorticoid receptor (GR) with Stat5. Our preliminary in vitro positive cooperative interaction of the glucocorticoid receptor (GR) with Stat5. Our preliminary in vitro results indicate that progesterone inhibits beta-casein expression at the level of gene transcription through a direct interaction of the glucocorticoid receptor (GR) with Stat5. Our preliminary in vitro results indicate that progesterone inhibits beta-casein expression at the level of gene transcription through a direct interaction of the progesterone receptor (PR) at the beta-casein promoter that interferes with prolactin/Stat5 signaling. The goal of this proposal is to define the mechanism of this direct PR-dependent inhibition of beta-casein transcription in vitro and in vivo. In AIM #1, biochemical approaches will be used to define cooperative protein-protein and protein-DNA interactions between PR and Stat5 that contribute to PR-mediated inhibition of Stat5 activity, or to the potentiating effect of GR. In AIM #2, cell-based transcription assays in mammary epithelial cell cultures will be used to define the mechanism by which PR inhibits Stat5 and GR-dependent transcription of beta-casein reporter genes. Aim #3, we will determine whether mechanisms of PR- dependent inhibition of beta-casein gene transcription defined in vitro also occur in the mammary gland in vivo by analysis of regulatory elements of beta-casein reporter genes expressed by recombinant adenovirus. In AIM #4, we will determine the relative contribution of direct and indirect recombinant adenovirus. In AIM #4, we will determine the relative contribution of direct and indirect (paracrine) mechanisms of repression of beta-casein transcription by PR in vitro and in vivo. The expectation of this research is to define a mechanism of negative gene regulation by PR that is unique to milk protein genes and thus fulfills a specific biological role of progesterone in the mammary gland during pregnancy.