Effects of hormones and protein kinase inhibitors on expression of steroidogenic enzyme promoters in electroporated primary rat granulosa cells.
Effects of hormones and protein kinase inhibitors on expression of steroidogenic enzyme promoters in electroporated primary rat granulosa cells.
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激素和蛋白激酶抑制剂对电穿孔原代大鼠颗粒细胞中类固醇生成酶启动子表达的影响。
DOI：
10.1095/biolreprod54.1.208
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发表时间：
1996
影响因子：
3.6
通讯作者：
Richards,JS
中科院分区：
文献类型：
--
作者：
Orly,J;Clemens,JW;Singer,O;Richards,JS
以往的研究表明，蛋白酪氨酸激酶抑制剂，tyrphostins，可以显着减弱卵巢细胞中表达的蛋白诱导基因的mRNA的稳态水平。为了进一步阐明酪氨酸磷酸化抑制剂的作用机制，用嵌合表达载体电穿孔大鼠颗粒细胞，所述嵌合表达载体含有两个关键类固醇生成基因的启动子，胆固醇侧链裂解细胞色素P450（CYP 11 A; P450 SCC）和芳香化酶细胞色素P450（CYP 19; P450 arom），连接到CAT报告基因。转染的电穿孔方法证明，相应的启动子-报告基因构建体，-379sccCAT和-534aromCAT，可以赋予大于10倍的FSH/cAMP对幼稚颗粒细胞中表达的报告基因的反应性。此外，电穿孔方法允许将DNA转染到少量细胞中，并有助于测定从处于晚期分化阶段的卵泡分离的细胞中的表达。在幼稚颗粒细胞中，-379sccCAT、-534aromCAT和-169α CGCAT的功能活性被A激酶特异性抑制剂H89消除，支持蛋白激酶A的激活是这些基因启动子区域转录激活的必要条件。对酪氨酸磷酸化抑制素AG 18也观察到类似的抑制作用，从而暗示酪氨酸激酶参与类固醇生成基因的调节。作为eCG/hCG处理的结果，在分化的颗粒体-叶黄素细胞中观察到转染效率的逐渐丧失，伴随着毛喉素对CAT表达的诱导降低。尽管激素调节嵌合基因表达的能力明显丧失的原因仍有待确定，但细胞和启动子对激素处理的不敏感性似乎反映了与幼稚颗粒细胞相比，分化细胞中启动子激活机制的根本变化。
Previous studies have shown that inhibitors of protein tyrosine kinases, tyrphostins, can markedly attenuate the steady-state levels of mRNAs of hormone-induced genes expressed in ovarian cells. To further elucidate the mechanism of tyrphostin action, rat granulosa cells were electroporated with chimeric expression vectors containing the promoters of two key steroidogenic genes, cholesterol side chain cleavage cytochrome P450 (CYP11A; P450scc) and aromatase cytochrome P450 (CYP19; P450arom), ligated to the CAT reporter gene. The electroporation method of transfection documents that the respective promoter-reporter constructs, –379sccCAT and –534aromCAT, can confer greater than 10-fold FSH/cAMP responsiveness to the reporter genes expressed in naive granulosa cells. Furthermore, the electroporation approach allows transfection of DNA into small numbers of cells and facilitates the assay of expression in cells isolated from follicles at advanced stages of differentiation. In naive granulosa cells, the functional activities of –379sccCAT, –534aromCAT, and –169α CGCAT were abolished by the A-kinase specific inhibitor, H89, supporting the notion that activation of protein kinase A is obligatory for transcriptional activation of the promoter regions within these genes. Similar inhibitory effects were also observed for tyrphostin AG18, thus implicating a tyrosine kinase in the regulation of the steroidogenic genes. As a result of eCG/hCG treatments, a gradual loss of transfection efficiency accompanied by decreasing forskolin induction of CAT expression was observed in the differentiating granulosa-lutein cells. Although the reason(s) for the apparent loss in the ability of hormones to regulate chimeric gene expression remains to be determined, cell and promoter refractoriness to hormone treatment appears to reflect a fundamental change in the mechanism of promoter activation in the differentiated cells compared to the naive granulosa cells.