Law Nathan C, Weck Jennifer, Kyriss Brandon, Nilson John H, Hunzicker-Dunn Mary
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-7520, USA.
Mol Endocrinol. 2013 Aug;27(8):1295-310. doi: 10.1210/me.2013-1025. Epub 2013 Jun 10.
Ovarian follicles lacking FSH or FSH receptors fail to progress to a preovulatory stage, resulting in infertility. One hallmark of the preovulatory follicle is the presence of luteinizing hormone/choriogonadotropin receptors (LHCGR) on granulosa cells (GCs). However, the mechanisms by which FSH induces Lhcgr gene expression are poorly understood. Our results show that protein kinase A (PKA) and phosphoinositide 3-kinase (PI3K)/AKT pathways are required for FSH to activate both the murine Lhcgr-luciferase reporter and expression of Lhcgr mRNA in rat GCs. Based on results showing that an adenovirus (Ad) expressing a steroidogenic factor 1 (SF1) mutant that cannot bind β-catenin abolished FSH-induced Lhcgr mRNA, we evaluated the role of β-catenin in the regulation of Lhcgr gene expression. FSH promoted the PKA-dependent, PI3K-independent phosphorylation of β-catenin on Ser552 and Ser665. FSH activated the β-catenin/T-cell factor (TCF) artificial promoter-reporter TOPFlash via a PKA-dependent, PI3K-independent pathway, and dominant-negative (DN) TCF abolished FSH-activated Lhcgr-luciferase reporter and induction of Lhcgr mRNA. Microarray analysis of GCs treated with Ad-DN-TCF and FSH identified the Lhcgr as the most down-regulated gene. Chromatin immunoprecipitation results placed β-catenin phosphorylated on Ser552 and Ser675 and SF1 on the Lhcgr promoter in FSH-treated GCs; TCF3 was constitutively associated with the Lhcgr promoter. Transduction with an Ad-phospho-β-catenin mutant (Ser552/665/Asp) enhanced Lhcgr mRNA expression in FSH-treated cells greater than 3-fold. Finally, we identified a recognized PI3K/AKT target, forkhead box O1, as a negative regulator of Lhcgr mRNA expression. These results provide new understanding of the complex regulation of Lhcgr gene expression in GCs.
缺乏促卵泡激素（FSH）或FSH受体的卵巢卵泡无法发育到排卵前阶段，从而导致不孕。排卵前卵泡的一个标志是颗粒细胞（GCs）上存在促黄体生成素/绒毛膜促性腺激素受体（LHCGR）。然而，FSH诱导Lhcgr基因表达的机制尚不清楚。我们的结果表明，蛋白激酶A（PKA）和磷酸肌醇3激酶（PI3K）/AKT信号通路是FSH激活小鼠Lhcgr荧光素酶报告基因以及大鼠GCs中Lhcgr mRNA表达所必需的。基于表达不能结合β-连环蛋白的类固醇生成因子1（SF1）突变体的腺病毒（Ad）消除了FSH诱导的Lhcgr mRNA这一结果，我们评估了β-连环蛋白在Lhcgr基因表达调控中的作用。FSH促进了β-连环蛋白在Ser552和Ser665位点的PKA依赖性、PI3K非依赖性磷酸化。FSH通过PKA依赖性、PI3K非依赖性途径激活β-连环蛋白/T细胞因子（TCF）人工启动子报告基因TOPFlash，并且显性负性（DN）TCF消除了FSH激活的Lhcgr荧光素酶报告基因以及Lhcgr mRNA的诱导。对用Ad-DN-TCF和FSH处理的GCs进行微阵列分析，确定Lhcgr是下调最明显的基因。染色质免疫沉淀结果显示，在FSH处理的GCs中，Ser552和Ser675位点磷酸化的β-连环蛋白以及SF1位于Lhcgr启动子上；TCF3与Lhcgr启动子持续相关。用Ad-磷酸化β-连环蛋白突变体（Ser552/665/Asp）转导可使FSH处理细胞中的Lhcgr mRNA表达增强3倍以上。最后，我们确定了一个公认的PI3K/AKT靶点，即叉头框O1，作为Lhcgr mRNA表达的负调节因子。这些结果为GCs中Lhcgr基因表达的复杂调控提供了新的认识。
