EFFECTS OF HORMONES AND PROTEIN-KINASE INHIBITORS ON EXPRESSION OF STEROIDOGENIC ENZYME PROMOTERS IN ELECTROPORATED PRIMARY RAT GRANULOSA-CELLS

Previous studies have shown that inhibitors of protein tyrosine kinase s, tyrphostins, can markedly attenuate the steady-state levels of mRNA s of hormone-induced genes expressed in ovarian cells. To further eluc idate the mechanism of tyrphostin action, rat granulosa cells were ele ctroporated with chimeric expression vectors containing the promoters of two key steroidogenic genes, cholesterol side chain cleavage cytoch rome P450 (CYP11A; P450(scc)) and aromatase cytochrome P450 (CYP19; P4 50(arom)), ligated to the CAT reporter gene. The electroporation metho d of transfection documents that the respective promoter-reporter cons tructs, -379sccCAT and -534aromCAT, can confer greater than 10-fold FS H/cAMP responsiveness to the reporter genes expressed in naive granulo sa cells, Furthermore, the electroporation approach allows transfectio n of DNA into small numbers of cells and facilitates the assay of expr ession in cells isolated from follicles at advanced stages of differen tiation, In naive granulosa cells, the functional activities of -379sc cCAT, -534aromCAT, and -169uCGCAT were abolished by the A-kinase speci fic inhibitor, H89, supporting the notion that activation of protein k inase A is obligatory for transcriptional activation of the pro meter regions within these genes. Similar inhibitory effects were also obser ved for tyrphostin AG18, thus implicating a tyrosine kinase in the reg ulation of the steroidogenic genes. As a result of eCG/hCG treatments, a gradual loss of transfection efficiency accompanied by decreasing f orskolin induction of CAT expression was observed in the differentiati ng granulosa-lutein cells. Although the reason(s) for the apparent los s in the ability of hormones to regulate chimeric gene expression rema ins to be determined, cell and promoter refractoriness to hormone trea tment appears to reflect a fundamental change in the mechanism of prom oter activation in the differentiated cells compared to the naive gran ulosa cells.