Wayne Chad M, Fan Heng-Yu, Cheng Xiaodong, Richards Joanne S
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Endocrinol. 2007 Aug;21(8):1940-57. doi: 10.1210/me.2007-0020. Epub 2007 May 29.
FSH regulates ovarian granulosa cell differentiation not only by activating adenylyl cyclase and protein kinase A (PKA) but also by other complex mechanisms. Using primary rat granulosa cell cultures, we provide novel evidence that FSH rapidly activates two small GTP-binding proteins RAP1 and RAS. FSH activation of RAP1 requires cAMP-mediated activation of exchange factor activated by cAMP/RAPGEF3 whereas FSH activation of RAS and downstream signaling cascades involves multiple factors. Specifically, FSH activation of RAS required Rous sarcoma oncogene (SRC) family tyrosine kinase (SFK) and epidermal growth factor receptor (EGFR) tyrosine kinase activities but not PKA. FSH-induced phosphorylation of ERK1/2 was blocked by dominant-negative RAS as well as by inhibitors of EGFR tyrosine kinase, metalloproteinases involved in growth factor shedding, and SFKs. In contrast, FSH-induced phosphorylation of protein kinase B (PKB/AKT) and the Forkhead transcription factor, FOXO1a occurred by SFK-dependent but RAS-independent mechanisms. The SFKs, c-SRC and FYN, and the SRC-related tyrosine kinase ABL were present and phosphorylated rapidly in response to FSH. Lastly, the EGF-like factor amphiregulin (AREG) activated RAS and ERK1/2 phosphorylation in granulosa cells by mechanisms that were selectively blocked by an EGFR antagonist but not by an SFK antagonist. However, AREG-mediated phosphorylation of PKB and FOXO1a required both EGFR and SFK activation. Moreover, we show that FSH induces AREG and that activation of the EGFR impacts granulosa cell differentiation and the expression of genes characteristic of the luteal cell phenotype. Thus, FSH orchestrates the coordinate activation of three diverse membrane-associated signaling cascades (adenylyl cyclase, RAS, and SFKs) that converge downstream to activate specific kinases (PKA, ERK1/2, and PKB/FOXO1a) that control granulosa cell function and differentiation.
促卵泡生成素（FSH）不仅通过激活腺苷酸环化酶和蛋白激酶A（PKA）来调节卵巢颗粒细胞分化，还通过其他复杂机制发挥作用。利用原代大鼠颗粒细胞培养物，我们提供了新的证据表明，FSH能快速激活两种小GTP结合蛋白RAP1和RAS。FSH对RAP1的激活需要cAMP介导的由cAMP激活的交换因子（cAMP/RAPGEF3）的激活，而FSH对RAS及下游信号级联反应的激活涉及多种因素。具体而言，FSH对RAS的激活需要劳斯肉瘤癌基因（SRC）家族酪氨酸激酶（SFK）和表皮生长因子受体（EGFR）酪氨酸激酶的活性，但不需要PKA。FSH诱导的细胞外信号调节激酶1/2（ERK1/2）的磷酸化被显性负性RAS以及EGFR酪氨酸激酶抑制剂、参与生长因子释放的金属蛋白酶和SFK阻断。相反，FSH诱导的蛋白激酶B（PKB/AKT）和叉头转录因子FOXO1a的磷酸化是通过依赖SFK但不依赖RAS的机制发生的。SFK、c-SRC和FYN以及与SRC相关的酪氨酸激酶ABL在FSH刺激下迅速出现并被磷酸化。最后，表皮生长因子样因子双调蛋白（AREG）通过一种机制激活颗粒细胞中的RAS和ERK1/2磷酸化，该机制可被EGFR拮抗剂选择性阻断，但不能被SFK拮抗剂阻断。然而，AREG介导的PKB和FOXO1a的磷酸化需要EGFR和SFK的共同激活。此外，我们发现FSH能诱导AREG的产生，并且EGFR的激活会影响颗粒细胞的分化以及黄体细胞表型特征基因的表达。因此，FSH协调激活三种不同的膜相关信号级联反应（腺苷酸环化酶、RAS和SFK），这些反应在下游汇聚以激活特定的激酶（PKA、ERK1/2和PKB/FOXO1a），从而控制颗粒细胞的功能和分化。
