Safwat Nedal, Ninomiya-Tsuji Jun, Gore A Jesse, Miller William L
Department of Molecular and Structural Biochemistry, Box 7622, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
Endocrinology. 2005 Nov;146(11):4814-24. doi: 10.1210/en.2005-0457. Epub 2005 Aug 4.
FSH, a key regulator of gonadal function, contains a beta-subunit (FSHbeta) that is transcriptionally induced by activin, a member of the TGFbeta-superfamily. This study used 4.7 kb of the ovine FSHbeta-promoter linked to luciferase (oFSHbetaLuc) plus a well-characterized activin-responsive construct, p3TPLuc, to investigate the hypothesis that Smad3, TGFbeta-activated kinase 1 (TAK1), or both cause activin-mediated induction of FSH. Overexpression of either Smad3 or TAK1 induced oFSHbetaLuc in gonadotrope-derived LbetaT2 cells as much as activin itself. Induction of p3TPLuc by activin is known to require Smad3 activation in many cell types, and this was true in LbetaT2 cells, where 10-fold induction by activin (2-8 h after activin treatment) was blocked more than 90% by two dominant negative (DN) inhibitors of Smad3 [DN-Smad3 (3SA) and DN-Smad3 (D407E)]. By contrast, 6.5-fold induction of oFSHbetaLuc by activin (10-24 h after activin treatment) was not blocked by either DN-Smad inhibitor, suggesting that activation of Smad3 did not trigger induction of oFSHbetaLuc. By contrast, inhibition of TAK1 by a DN-TAK1 construct led to a 50% decrease in activin-mediated induction of oFSHbetaLuc, and a specific inhibitor of TAK1 (5Z-7-Oxozeanol) blocked induction by 100%, indicating that TAK1 is necessary for activin induction of oFSHbetaLuc. Finally, inhibiting p38-MAPK (often activated by TAK1) blocked induction of oFSHbetaLuc by 60%. In conclusion, the data presented here indicate that activation of TAK1 (and probably p38-MAPK), but not Smad3, is necessary for triggering induction of oFSHbeta by activin.
促卵泡激素（FSH）是性腺功能的关键调节因子，它包含一个β亚基（FSHβ），该亚基由激活素转录诱导产生，激活素是转化生长因子β（TGFβ）超家族的成员。本研究使用与荧光素酶相连的4.7 kb绵羊FSHβ启动子（oFSHβLuc）以及一个特征明确的激活素反应性构建体p3TPLuc，来研究以下假设：Smad3、TGFβ激活激酶1（TAK1）或两者共同导致激活素介导的FSH诱导。在促性腺激素细胞来源的LβT2细胞中，Smad3或TAK1的过表达诱导oFSHβLuc的程度与激活素本身相同。已知在许多细胞类型中，激活素诱导p3TPLuc需要Smad3激活，在LβT2细胞中也是如此，在LβT2细胞中，激活素（激活素处理后2 - 8小时）10倍的诱导作用被两种Smad3显性负性（DN）抑制剂[DN - Smad3（3SA）和DN - Smad3（D407E）]阻断了90%以上。相比之下，激活素（激活素处理后10 - 24小时）对oFSHβLuc 6.5倍的诱导作用未被任何一种DN - Smad抑制剂阻断，这表明Smad3的激活并未触发oFSHβLuc的诱导。相反，用DN - TAK1构建体抑制TAK1导致激活素介导的oFSHβLuc诱导降低50%，并且TAK1的特异性抑制剂（5Z - 7 - 氧代泽anol）将诱导作用100%阻断，表明TAK1是激活素诱导oFSHβLuc所必需的。最后，抑制p38 - MAPK（通常由TAK1激活）将oFSHβLuc的诱导作用阻断了60%。总之，此处呈现的数据表明，激活TAK1（可能还有p38 - MAPK）而非Smad3是激活素触发oFSHβ诱导所必需的。
