Park Youngkyu, Maizels Evelyn T, Feiger Zachary J, Alam Hena, Peters Carl A, Woodruff Teresa K, Unterman Terry G, Lee Eun Jig, Jameson J Larry, Hunzicker-Dunn Mary
Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.
J Biol Chem. 2005 Mar 11;280(10):9135-48. doi: 10.1074/jbc.M409486200. Epub 2004 Dec 21.
Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.
卵巢卵泡会因促卵泡激素（FSH）而呈指数增长，这主要是颗粒细胞（GCs）增殖的结果。在无血清条件下的体外实验中，大鼠颗粒细胞会对FSH产生分化反应，但除非同时存在激活素，否则不会增殖。在FSH加激活素存在的情况下，颗粒细胞中细胞周期蛋白D2以及抑制素α、芳香化酶、类固醇生成因子-1（SF-1）、胆固醇侧链（SCC）和表皮调节素的表达会增强。在本报告中，我们试图确定FSH和激活素促进颗粒细胞增殖和分化的信号通路。我们的结果表明，相对于时间匹配的对照组，这些反应与Akt磷酸化的延长有关，并且基于磷脂酰肌醇3激酶（PI 3激酶）抑制剂LY294002或腺病毒显性负性Smad3（DN-Smad3）突变体感染减弱细胞周期蛋白D2、抑制素α、芳香化酶、SCC、SF-1和表皮调节素诱导的能力，这些反应依赖于PI 3激酶和Smad2/3信号传导。DN-Smad3突变体在处理后24小时也消除了FSH加激活素刺激的Akt磷酸化延长。用腺病毒组成型活性含叉头框蛋白O亚家族（FOXO）1突变体感染可抑制细胞周期蛋白D2、芳香化酶、抑制素α、SF-1和表皮调节素的诱导。用组成型活性FOXO1突变体对颗粒细胞进行瞬时转染也可抑制细胞周期蛋白D2、抑制素α和表皮调节素启动子-报告基因活性。染色质免疫沉淀结果在体内证明了未处理的颗粒细胞中FOXO1与细胞周期蛋白D2启动子的关联，以及添加FSH加激活素后FOXO1从细胞周期蛋白D2启动子上的释放。这些结果表明，颗粒细胞对FSH加激活素的增殖和分化需要消除FOXO1依赖性抑制以及来自Smad2/3的正向信号传导。
