Progesterone Inhibition--Milk Protein Gene Transcription

黄体酮抑制--乳蛋白基因转录

• 批准号: 6602427
• 负责人: Dean P Edwards
• 金额: $ 15万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6602427
• 关键词: Adenoviridae; beta galactosidase; caseins; female; gene induction /repression; hormone regulation /control mechanism; laboratory mouse; mammary epithelium; pregnancy; progesterone; progesterone receptors; protein structure function; reporter genes; reproductive development; tissue /cell culture; transcription factor; transfection /expression vector

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.

黄体酮在怀孕乳腺的结构和功能发育过程中具有两种相反的生物作用。增殖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。负责调节乳蛋白表达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 的积极协同相互作用来增强催乳素的作用。我们初步在体外观察到糖皮质激素受体 (GR) 与 Stat5 的积极协同相互作用。我们的初步体外结果表明，黄体酮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-酪蛋白表达。我们的初步体外结果表明，孕酮通过 β-酪蛋白启动子上孕酮受体 (PR) 的直接相互作用，干扰催乳素/Stat5 信号传导，从而在基因转录水平上抑制 β-酪蛋白表达。该提案的目的是确定这种直接 PR 依赖性抑制体外和体内 β-酪蛋白转录的机制。在 AIM #1 中，生化方法将用于定义 PR 和 Stat5 之间的协同蛋白质-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，或增强 GR 的作用。在 AIM #2 中，乳腺上皮细胞培养物中基于细胞的转录测定将用于定义 PR 抑制 Stat5 和 GR 依赖性 β-酪蛋白报告基因转录的机制。目标#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，确定体外定义的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。这项研究的期望是确定一种乳蛋白基因特有的 PR 负基因调控机制，从而实现妊娠期间孕激素在乳腺中的特定生物学作用。