Kurten R C, Richards J S
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Endocrinology. 1989 Sep;125(3):1345-57. doi: 10.1210/endo-125-3-1345.
The following studies were conducted with the goal of understanding some of the molecular mechanisms by which cAMP alters granulosa cell function. In this regard, we characterized the expression of messages for the cholesterol side-chain cleavage cytochrome P450 (P450scc) enzyme and a type II cAMP-dependent protein kinase subunit (RII beta) in granulosa cells isolated from small antral follicles and cultured in 1% fetal bovine serum-containing medium. Forskolin (FSK) stimulated cAMP production followed by accumulation of RII beta mRNAs, induction of P450scc mRNA, and, finally, progesterone biosynthesis. The regulation of each mRNA displayed a different sensitivity to actinomycin-D treatment. To determine if the modulation of RII beta or the induction of P450scc could be mediated by the enhancer activity of a cAMP-responsive DNA element (CRE), plasmid DNA containing the CRE and TATA box of the human glycoprotein alpha-subunit (alpha G) gene fused to the chloramphenicol acetyl transferase (CAT) gene was introduced into cultured granulosa or interstitial cells, and the ability of FSK to stimulate the expression of the CAT enzyme was measured. When granulosa cells were isolated from preantral/small antral follicles and maintained for at least 5 days in culture, 5 or 10 microM FSK reversibly stimulated expression of the CAT enzyme within 18 h posttransfection. Under similar conditions, interstitial ovarian cells (prepared from the residual ovarian tissue remaining after granulosa cell isolation) were unable to express the template unless the cells were pretreated for 24 h with FSK before transfection. Thereafter, CAT gene expression by interstitial cells was maintained in a FSK-insensitive manner. To determine if cAMP-dependent transcription of the reporter gene required the same sequences that had been characterized for placenta-derived cells, a truncated plasmid lacking the CRE of the alpha G gene was transfected. Under no condition was expression of the CAT gene observed from a CRE-deficient template. The acute transcriptional activation of a CRE/TATA box-containing gene by cAMP in granulosa cells indicated that transcription-activating proteins interacting with the CRE were either present in these cells or were rapidly synthesized after stimulation. To distinguish between these two possibilities, protein synthesis was transiently inhibited after transfection, before the addition of FSK, by incubating the cells for 2 h with 25 micrograms/ml cycloheximide. Cycloheximide treatment alone did not stimulate transcription from the CRE-containing molecule. Incubation with cycloheximide, followed by treatment with FSK, increased CAT activity 2-fold compared to t
进行了以下研究，目的是了解环磷酸腺苷（cAMP）改变颗粒细胞功能的一些分子机制。在这方面，我们对从小窦状卵泡分离并在含1%胎牛血清的培养基中培养的颗粒细胞中胆固醇侧链裂解细胞色素P450（P450scc）酶和II型cAMP依赖性蛋白激酶亚基（RIIβ）的信使表达进行了表征。福斯高林（FSK）刺激cAMP产生，随后RIIβ信使核糖核酸（mRNAs）积累，P450scc信使核糖核酸诱导，最终是孕酮生物合成。每种信使核糖核酸的调节对放线菌素-D处理表现出不同的敏感性。为了确定RIIβ的调节或P450scc的诱导是否可由cAMP反应性DNA元件（CRE）的增强子活性介导，将含有人类糖蛋白α亚基（αG）基因的CRE和TATA盒并与氯霉素乙酰转移酶（CAT）基因融合的质粒DNA导入培养的颗粒细胞或间质细胞，并测量FSK刺激CAT酶表达的能力。当从窦前/小窦状卵泡分离颗粒细胞并在培养中维持至少5天时，5或10微摩尔FSK在转染后18小时内可逆地刺激CAT酶表达。在类似条件下，间质卵巢细胞（由颗粒细胞分离后剩余的残余卵巢组织制备）无法表达模板，除非细胞在转染前用FSK预处理24小时。此后，间质细胞的CAT基因表达以FSK不敏感的方式维持。为了确定报告基因的cAMP依赖性转录是否需要与胎盘来源细胞相同的序列，转染了一个缺少αG基因CRE的截短质粒。在任何条件下，均未观察到来自CRE缺陷模板的CAT基因表达。颗粒细胞中cAMP对含CRE/TATA盒基因的急性转录激活表明，与CRE相互作用的转录激活蛋白要么存在于这些细胞中，要么在刺激后迅速合成。为了区分这两种可能性，在转染后、添加FSK之前，通过用25微克/毫升环己酰亚胺孵育细胞2小时来短暂抑制蛋白质合成。单独用环己酰亚胺处理不会刺激含CRE分子的转录。与单独用FSK处理相比，先用环己酰亚胺孵育，然后用FSK处理，CAT活性增加了2倍。
