Clemens J W, Robker R L, Kraus W L, Katzenellenbogen B S, Richards J S
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Endocrinol. 1998 Aug;12(8):1201-14. doi: 10.1210/mend.12.8.0157.
Expression of progesterone receptor (PR) mRNA in granulosa cells of ovarian preovulatory follicles is induced by LH (1, 2) and is essential for ovulation (3). Although 17beta-estradiol (E) can induce PR mRNA and activate PR promoter-reporter constructs in other cell types, the effects of E in granulosa cells appear to be indirect. We show herein that E alone does not induce the expression of PR mRNA in preovulatory granulosa cells. Rather, induction of PR mRNA depends on the differentiation of granulosa cells in response to E and a physiological amount of FSH followed by exposure to agonists (elevated levels of LH, FSH, and forskolin) that markedly increase cAMP. Induction of PR mRNA by forskolin is blocked by the A-kinase inhibitor H89 and cycloheximide but not by the E antagonist, ICI 164,384. These results indicate that phosphorylation and synthesis of some regulatory factor(s) other than or in addition to the estrogen receptor (ER) are essential for transactivation of the PR gene. When distal and proximal PR promoter-reporter constructs that are responsive to E in other cell types were transiently transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Likewise, when a vector containing the consensus vitellogenin B1 gene estrogen response element (ERE) was transfected into differentiated granulosa cells, forskolin, but not E, induced activity. Using electrophoretic mobility shift assays, the consensus ERE was shown to bind ERbeta, the predominant subtype present in rat granulosa cells, and ERalpha, the predominant subtype present in luteal cells, whereas the putative ERE-like region (ERE3) of the proximal PR promoter did not bind either ER subtype. Although the identity of the specific factors binding to the ERE3 site remain to be determined, mutation of this region abolished forskolin-induced activity of ERE3-PR-CAT constructs. The GC-rich region of the distal PR promoter bound Sp1 and Sp3 but not C/EBPalpha/beta, indicating that factors binding to ERE3 interact synergistically with Sp1/Sp3 to confer increased responsiveness of the distal promoter to forskolin. Taken together, these results indicate that activation of the A-kinase pathway leads to the phosphorylation of some transcription factor(s) other than or in addition to ER that is (are) critical for the transactivation of the PR gene and that this mechanism is selectively activated in differentiated granulosa cells possessing a preovulatory phenotype.
促黄体生成素（LH）可诱导卵巢排卵前卵泡颗粒细胞中孕激素受体（PR）mRNA的表达（1,2），且该表达对排卵至关重要（3）。尽管17β-雌二醇（E）可在其他细胞类型中诱导PR mRNA表达并激活PR启动子-报告基因构建体，但E对颗粒细胞的作用似乎是间接的。我们在此表明，单独的E不会诱导排卵前颗粒细胞中PR mRNA的表达。相反，PR mRNA的诱导取决于颗粒细胞对E和生理量促卵泡激素（FSH）的反应而发生的分化，随后暴露于能显著增加环磷酸腺苷（cAMP）的激动剂（升高水平的LH、FSH和福斯可林）。福斯可林对PR mRNA的诱导被A激酶抑制剂H89和放线菌酮阻断，但未被E拮抗剂ICI 164,384阻断。这些结果表明，除雌激素受体（ER）之外或与之一起的某些调节因子的磷酸化和合成对于PR基因的反式激活至关重要。当将在其他细胞类型中对E有反应的远端和近端PR启动子-报告基因构建体瞬时转染到分化的颗粒细胞中时，福斯可林而非E诱导了活性。同样，当将含有共有的卵黄蛋白原B1基因雌激素反应元件（ERE）的载体转染到分化的颗粒细胞中时，福斯可林而非E诱导了活性。使用电泳迁移率变动分析表明，共有ERE可结合大鼠颗粒细胞中主要的亚型ERβ以及黄体细胞中主要的亚型ERα，而近端PR启动子的假定ERE样区域（ERE3）不结合任何一种ER亚型。尽管结合ERE3位点的特定因子的身份仍有待确定，但该区域的突变消除了福斯可林诱导的ERE3-PR-CAT构建体的活性。远端PR启动子富含鸟嘌呤-胞嘧啶的区域结合Sp1和Sp3，但不结合C/EBPα/β，这表明结合ERE3的因子与Sp1/Sp3协同作用，赋予远端启动子对福斯可林更高的反应性。综上所述，这些结果表明，A激酶途径的激活导致除ER之外或与之一起的某些转录因子的磷酸化，这些转录因子对PR基因的反式激活至关重要，并且这种机制在具有排卵前表型的分化颗粒细胞中被选择性激活。
