Obr Alison E, Grimm Sandra L, Bishop Kathleen A, Pike J Wesley, Lydon John P, Edwards Dean P
PhD, Department of Molecular & Cellular Biology, Baylor College of Medicine, BCM Box 130, One Baylor Plaza, Houston, Texas 77030.
Mol Endocrinol. 2013 Nov;27(11):1808-24. doi: 10.1210/me.2013-1077. Epub 2013 Sep 6.
Progesterone (P4) stimulates proliferation of the mammary epithelium by a mechanism that involves paracrine signaling mediated from progesterone receptor (PR)-positive to neighboring PR-negative cells. Here we used a primary mouse mammary epithelial cell (MEC) culture system to define the molecular mechanism by which P4 regulates the expression of target gene effectors of proliferation including the paracrine factor receptor and activator of nuclear factor κB ligand (RANKL). MECs from adult virgin mice grown and embedded in three-dimensional basement-membrane medium resemble mammary ducts in vivo structurally and with respect to other properties including a heterogeneous pattern of PR expression, P4 induction of RANKL and other target genes in a PR-dependent manner, and a proliferative response to progestin. RANKL was demonstrated to have multiple functional P4-responsive enhancers that bind PR in a hormone-dependent manner as detected by chromatin immunoprecipitation assay. P4 also stimulated recruitment of signal transducer and activator of transcription (Stat)5a to RANKL enhancers through an apparent tethering with PR. Analysis of primary MECs from Stat5a knockout mice revealed that P4 induction of RANKL and a broad range of other PR target genes required Stat5a, as did P4-stimulated cell proliferation. In the absence of Stat5a, PR binding was lost at selective RANKL enhancers but was retained with others, suggesting that Stat5a acts to facilitate PR DNA binding at selective sites and to function as a coactivator with DNA-bound PR at others. These results show that RANKL is a direct PR target gene and that Stat5a has a novel role as a cofactor in PR-mediated transcriptional signaling in the mammary gland.
孕酮（P4）通过一种涉及从孕酮受体（PR）阳性细胞向相邻PR阴性细胞介导的旁分泌信号传导的机制来刺激乳腺上皮细胞的增殖。在这里，我们使用原代小鼠乳腺上皮细胞（MEC）培养系统来确定P4调节增殖靶基因效应物表达的分子机制，这些效应物包括旁分泌因子受体和核因子κB配体激活剂（RANKL）。来自成年处女小鼠的MEC在三维基底膜培养基中生长并包埋，在结构上以及其他特性方面类似于体内的乳腺导管，包括PR表达的异质性模式、P4以PR依赖的方式诱导RANKL和其他靶基因，以及对孕激素的增殖反应。通过染色质免疫沉淀试验检测发现，RANKL具有多个功能性P4反应性增强子，它们以激素依赖的方式结合PR。P4还通过与PR的明显连接刺激信号转导和转录激活因子（Stat）5a募集到RANKL增强子。对来自Stat5a基因敲除小鼠的原代MEC的分析表明，P4诱导RANKL和一系列其他PR靶基因需要Stat5a，P4刺激的细胞增殖也需要Stat5a。在没有Stat5a的情况下，PR在选择性RANKL增强子处的结合丧失，但在其他增强子处保留，这表明Stat5a在选择性位点促进PR与DNA结合，并在其他位点作为与结合DNA的PR的共激活因子发挥作用。这些结果表明，RANKL是PR的直接靶基因，并且Stat5a在乳腺中PR介导的转录信号传导中作为辅助因子具有新的作用。
