An E-box region within the prostaglandin endoperoxide synthase-2 (PGS-2) promoter is required for transcription in rat ovarian granulosa cells
An E-box region within the prostaglandin endoperoxide synthase-2 (PGS-2) promoter is required for transcription in rat ovarian granulosa cells
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DOI：
10.1074/jbc.271.28.16633
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发表时间：
1996-07-12
影响因子：
4.8
通讯作者：
Richards, JS
中科院分区：
文献类型：
--
作者：
Morris, JK;Richards, JS
前列腺素内过氧化物合酶 2 (PGS-2) 基因编码前列腺素合酶同种型，该同工型由排卵卵泡颗粒细胞中的蛋白激酶 A（黄体生成素/cAMP）和蛋白激酶 C（促性腺激素释放激素）激动剂瞬时诱导。大鼠 PGS-2 基因的启动子含有一个 CAAT 增强子结合蛋白共有位点（CAAT 盒），它可以赋予 PGS-2-CAT 报告基因激素诱导性，以及一个推定的 E-盒区域。为了确定 E-box 区域是否参与激素诱导的大鼠 PGS-2 基因反式激活，将具有 CAAT 盒和 E-box 区域 (-192 PGS-2-CAT) 的构建体、仅假定的 E-box (-110 PGS-2 CAT) 或两个区域 (-52 PGS-2-CAT) 瞬时转染至大鼠颗粒细胞培养物中。 -192 和 -110 PGS-S-CAT 载体中的 CAT 活性均由黄体生成素（10 倍）和促性腺激素释放激素（6 倍）诱导，而 -52 PGS-S-CAT 构建体的 CAT 活性与无启动子载体 (pCAT-Basic) 没有差异。从 -110 PGS-S CAT 构建体内的 E-box 删除 1 个碱基对，以及 -192 PGS-S CAT 构建体的 CAAT 盒、E-box 或两个区域内的点突变，证明 E-box 对于基础转录至关重要，并且除 CAAT 盒外，该区域也参与 PGS-S 基因的激素诱导。跨越大鼠 PGS-2 E-box 的寡核苷酸结合了两种特定的蛋白质复合物，这些蛋白质复合物在上游刺激因子特异性抗体存在的情况下发生超位移。因此，在大鼠颗粒细胞中，PGS-2 E-盒区域似乎与除 CAAT 盒之外的上游顺式作用元件相互作用，以赋予基因激素调节作用。大鼠 PGS-2 启动子的 E-box 区域不包含人类和小鼠 PGS-2 启动子中发现的 ATF/CRE 活性，但对于大鼠颗粒细胞中 PGS-2 基因的基础转录至关重要，并结合上游刺激因子（卵巢中调节的其他基因的 E-box 区域也是如此）。
The prostaglandin endoperoxide synthase-2 (PGS-2) gene encodes an isoform of prostaglandin synthase that is transiently induced by protein kinase A (luteinizing hormone/cAMP) and protein kinase C (gonadotropin-releasing hormone) agonists in granulosa cells of ovulating follicles. The promoter of the rat PGS-2 gene contains a CAAT enhancer-binding protein consensus site (CAAT box) which can confer hormone inducibility to a PGS-2-CAT reporter gene, as well as a putative E-box region. To determine if the E-box region was involved in hormone induced trans-activation of the rat PGS-2 gene, constructs with the CAAT box and E-box regions (-192 PGS-2-CAT), only the putative E-box (-110 PGS-2 CAT), or neither region (-52 PGS-2-CAT) were transiently transfected into rat granulosa cell cultures. CAT activity was induced in both the -192 and -110 PGS-S-CAT vectors by luteinizing hormone (10-fold) and gonadotropin releasing hormone (6-fold), whereas CAT activity of the -52 PGS-S-CAT construct did not differ from the promoterless vector (pCAT-Basic). Deletion of 1 base pair from the E-box within the -110 PGS-S CAT construct, as well as point mutations within the CAAT box, E-box, or both regions of the -192 PGS-S CAT construct, demonstrated that the E-box is critical for basal transcription, and that regions, in addition to the CAAT box, are involved in hormone induction of the PGS-S gene. An oligonucleotide spanning the rat PGS-2 E-box bound two specific protein complexes which were supershifted in the presence of antibody specific for the upstream stimulatory factor. Thus, in rat granulosa cells, the PGS-2 E-box region appears to interact with upstream cis-acting elements other than the CAAT box to confer hormonal regulation of the gene. The E-box region of the rat PGS-2 promoter does not contain ATF/CRE activity found in the human and mouse PGS-2 promoters, but is critical for basal transcription of the PGS-2 gene in rat granulosa cells and binds the upstream stimulatory factor (as do E-box regions of other genes regulated in the ovary).