Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBP beta promoter element.
Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBP beta promoter element.
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发表时间：
1993-10
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通讯作者：
J. Sirois;J. Richards
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作者：
J. Sirois;J. Richards
大鼠前列腺素内过氧化物合成酶2 （PGS-2）基因的启动子最近被证明，当连接到CAT（氯霉素乙酰转移酶）报告基因并转染到分化颗粒细胞的原代培养物中时，可诱导促性腺激素（促卵泡激素（FSH），促黄体生成素（LH）， cAMP）。为了描述介导这种反应的顺式作用元件和反式激活因子，我们测试了活性启动子区域（转录起始位点上游-192/-53碱基对）的缺失，以检测它们结合颗粒细胞核提取物蛋白和激活报告基因活性的能力。电泳迁移位移分析显示，-142/-120亚区抑制颗粒细胞核提取物与标记的PGS-2片段-192/-53之间的蛋白/DNA结合。子区域-142/-120作用元素C/EBP β， 5‘-TTATGCAAT-3’。C/EBP β元件内的点突变破坏了蛋白质/DNA结合，导致报告基因活性的forskolin/LH/FSH诱导性丧失50%。人绒毛膜促性腺激素（hCG）在体内快速诱导颗粒细胞中C/EBP β mRNA和蛋白表达。总之，这些结果表明，C/EPB β似乎在排卵前颗粒细胞中调控PGS-2基因的诱导中起关键作用。
The promoter of the rat prostaglandin endoperoxide synthase 2 (PGS-2) gene has recently been shown to confer gonadotropic hormone (follicle-stimulating hormone (FSH), luteinizing hormone (LH), cAMP) inducibility when ligated to a CAT (chloramphenicol acetyltransferase) reporter gene and transfected into primary cultures of differentiated granulosa cells. To delineate cis-acting elements and trans-activating factors mediating this response, deletions of the active promoter region (-192/-53 base pairs upstream of the transcriptional start site) were tested for their ability to bind protein of granulosa cell nuclear extracts and activate reporter gene activity. Electrophoretic mobility shift assays revealed that the DNA subregion -142/-120 inhibited protein/DNA binding observed between granulosa cell nuclear extracts and the labeled PGS-2 fragment -192/-53. The subregion -142/-120 acting element C/EBP beta, 5'-TTATGCAAT-3'. Point mutations within the C/EBP beta element abolished protein/DNA binding and resulted in a 50% loss of forskolin/LH/FSH inducibility of reporter gene activity. C/EBP beta mRNA and protein were induced rapidly in granulosa cells in vivo by an ovulatory dose of human chorionic gonadotropin (hCG). Collectively, these results indicate that C/EPB beta appears to play a key role in regulating induction of the PGS-2 gene in granulosa cells prior to ovulation.