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。增殖作用刺激导管侧分支和小叶肺泡的形成。同时，孕酮具有抑制乳蛋白基因表达的抗分化作用，直到分娩。负责调节乳蛋白表达的主要激素是催乳素，其作用通过激活Stat 5转录因子与乳蛋白基因启动子的相互作用来介导。糖皮质激素通过糖皮质激素受体（GR）与Stat 5的正协同相互作用增强催乳素的作用。我们的初步体外积极合作相互作用的糖皮质激素受体（GR）与Stat 5。我们的初步体外研究结果表明，孕酮抑制β-酪蛋白的表达在基因转录水平通过糖皮质激素受体（GR）与Stat 5的直接相互作用。我们的初步体外研究结果表明，孕酮抑制β-酪蛋白表达的基因转录水平，通过直接相互作用的孕酮受体（PR）在β-酪蛋白启动子，干扰催乳素/Stat 5信号。本提案的目的是确定这种直接PR依赖性抑制β-酪蛋白转录的体外和体内机制。在AIM#1中，生物化学方法将用于定义PR和Stat 5之间的协同蛋白质-蛋白质和蛋白质-DNA相互作用，其有助于PR介导的Stat 5活性抑制或GR的增强作用。在乳腺上皮细胞培养物中的基于细胞的转录测定将用于确定PR抑制Stat 5和GR依赖性β-酪蛋白报告基因。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节元件来确定体外定义的β-酪蛋白基因转录的PR依赖性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。这项研究的期望是定义一种由PR负基因调控的机制，这种机制是乳蛋白基因所特有的，从而实现孕激素在妊娠期间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.