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.

孕酮在妊娠乳腺的结构和功能发育过程中有两种截然相反的生物学作用。增殖作用刺激导管侧支和小叶肺泡的形成。同时，孕酮具有抑制乳蛋白基因表达直至分娩的抗分化作用。催乳素是调节乳蛋白表达的主要激素，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而实现的。糖皮质激素通过糖皮质激素受体(GR)与Stat5的正向协同作用来增强催乳素的作用。我们初步研究了糖皮质激素受体(GR)与Stat5的体外正协同作用。我们的初步体外结果表明，孕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-酪蛋白的表达。我们的初步体外实验结果表明，孕酮通过与β-酪蛋白启动子上的孕酮受体(PR)直接相互作用而在基因转录水平上抑制β-酪蛋白的表达，从而干扰催乳素/STAT5信号转导。这项建议的目的是明确这种依赖PR的直接抑制体外和体内β-酪蛋白转录的机制。在AIM#1中，将使用生化方法来确定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活性的抑制，或者有助于GR的增强效应。在AIM#2中，将使用乳腺上皮细胞培养中基于细胞的转录分析来确定PR抑制β-酪蛋白报告基因Stat5和GR依赖的转录的机制。目的#3，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，我们将确定体外确定的PR依赖的抑制β-酪蛋白基因转录的机制是否也存在于体内乳腺。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PR对基因负调控的机制，这种机制是牛奶蛋白基因所特有的，从而在怀孕期间发挥孕酮在乳腺中的特定生物学作用。