Mukherjee A, Park-Sarge O K, Mayo K E
Department of Biochemistry, Northwestern University, Evanston, Illinois 60208, USA.
Endocrinology. 1996 Aug;137(8):3234-45. doi: 10.1210/endo.137.8.8754745.
The pituitary gonadotropins FSH and LH exert their effects on gonadal target cells, at least in part, through the activation of adenylyl cyclase and the production of the second messenger cAMP. To elucidate further the signal transduction pathways regulating gonadotropin-responsive genes in ovarian granulosa cells, we have investigated the expression of the cAMP response element binding protein (CREB), which mediates many of the effects of cAMP by modulating the transcription of target genes in a cAMP-dependent fashion. In situ hybridization, RNA blot analysis and RT-PCR RNA quantification demonstrated that CREB messenger RNA (mRNA) is expressed at low levels throughout the ovary, and that CREB mRNA levels do not change appreciably after gonadotropin stimulation. Similar results were obtained using immunohistochemistry and Western protein blotting to examine CREB protein in ovaries isolated from immature animals treated with gonadotropins or immunocytochemistry and Western protein blotting to examine the CREB protein in cultured granulosa cells after gonadotropin treatment. In contrast, immunocytochemistry and Western protein blotting using an antipeptide antibody specific to CREB phosphorylated at serine 133 (P-CREB), which is the activated from of the CREB protein, revealed a dramatic increase in the phosphorylated form of CREB within 20 min of gonadotropin treatment of granulosa cells that was transient and was decreased by 60 min after gonadotropin treatment. Stimulation of P-CREB was observed using granulosa cells isolated from immature animals and treated with recombinant human FSH in vitro, or using granulosa cells isolated from immature animals primed with PMSG in vivo and treated with human CG (hCG) in vitro. Stimulation of P-CREB was also observed in ovarian granulosa cells isolated from animals treated with PMSG in vivo. These results indicate that both gonadotropins can induce a rapid and transient phosphorylation of the CREB protein in granulosa cells, leading to the activation of a factor likely to play an important role in the transcription of many gonadotropin-regulated ovarian genes.
垂体促性腺激素FSH和LH至少部分地通过激活腺苷酸环化酶和产生第二信使cAMP来对性腺靶细胞发挥作用。为了进一步阐明调节卵巢颗粒细胞中性腺促激素反应性基因的信号转导途径，我们研究了环磷酸腺苷反应元件结合蛋白（CREB）的表达，该蛋白通过以环磷酸腺苷依赖性方式调节靶基因的转录来介导环磷酸腺苷的许多作用。原位杂交、RNA印迹分析和逆转录-聚合酶链反应（RT-PCR）RNA定量表明，CREB信使核糖核酸（mRNA）在整个卵巢中低水平表达，并且在促性腺激素刺激后CREB mRNA水平没有明显变化。使用免疫组织化学和蛋白质免疫印迹法检测用促性腺激素处理的未成熟动物卵巢中的CREB蛋白，或者使用免疫细胞化学和蛋白质免疫印迹法检测促性腺激素处理后培养的颗粒细胞中的CREB蛋白，也得到了类似的结果。相比之下，使用对丝氨酸133磷酸化的CREB（P-CREB）具有特异性的抗肽抗体进行免疫细胞化学和蛋白质免疫印迹分析，结果显示，在促性腺激素处理颗粒细胞20分钟内，CREB的磷酸化形式急剧增加，这种增加是短暂的，在促性腺激素处理60分钟后下降。在用重组人FSH体外处理从未成熟动物分离的颗粒细胞时，或者在用孕马血清促性腺激素（PMSG）体内预处理未成熟动物并在体外用人绒毛膜促性腺激素（hCG）处理后分离的颗粒细胞中，均观察到了P-CREB的激活。在用PMSG体内处理的动物分离的卵巢颗粒细胞中也观察到了P-CREB的激活。这些结果表明，两种促性腺激素均可诱导颗粒细胞中CREB蛋白快速且短暂的磷酸化，从而导致一种可能在许多促性腺激素调节的卵巢基因转录中起重要作用的因子被激活。
