Activin regulation of the follicle-stimulating hormone β-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1
Activin regulation of the follicle-stimulating hormone β-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1
复制标题
DOI：
10.1210/me.2003-0442
复制
发表时间：
2004-05-01
影响因子：
--
通讯作者：
Mellon, PL
中科院分区：
文献类型：
--
作者：
Bailey, JS;Rave-Harel, N;Mellon, PL
FSH对男性和女性的正常生殖功能至关重要。活化素是TGF β家族的一个成员，是FSH表达的重要调节因子，但对其作用的分子机制知之甚少。我们使用瞬时转染到永生化促性腺细胞系LbetaT 2中，以确定绵羊FSH β亚基基因的三个区域（-973/-962，-167和-134），这些区域是激活素完全应答所必需的。所有三个区域都含有与Smad蛋白的共有结合位点的同源性，Smad蛋白是TGF β家族信号传导的细胞内介质。远端位点的突变降低激活素的反应性，而近端位点的突变深刻地破坏激活素对FSH β基因的调节。这些位点特异性结合EMSA中的LbetaT 2核蛋白，并且-973/-962位点结合Smad 4蛋白。有趣的是，结合到-134位点的蛋白质复合物包含Smad 4与同源结构域蛋白Pbx 1和Prep 1。使用谷胱甘肽S-转移酶相互作用测定，我们表明，Pbx 1和Prep 1与Smads 2和3以及相互作用。两个近端激活素反应元件在物种间是保守的，Pbx 1和Prep 1蛋白在体内与小鼠基因结合。此外，任一近端位点的突变也消除了小鼠FSH β报告基因的激活素反应性，证实了它们的功能保守性。我们的研究为理解激活素对FSH β基因表达的调控提供了基础，并将Pbx 1和Prep 1鉴定为Smad伴侣和激活素作用的新型介质。
FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions ( at - 973/ - 962, - 167, and - 134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the - 973/ - 962 site binds Smad4 protein. Interestingly, the protein complex binding to the - 134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.