Effects of hormones and protein kinase inhibitors on expression of steroidogenic enzyme promoters in electroporated primary rat granulosa cells

Abstract

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 -169 alpha 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.

Transactivation of the Progesterone Receptor Gene in Granulosa Cells: Evidence that Sp1/Sp3 Binding Sites in the Proximal Promoter Play a Key Role in Luteinizing Hormone Inducibility

“…Specifically, we have shown that LH rapidly, but transiently, induces PR mRNA and protein in FSH/Tdifferentiated (preovulatory) granulosa cells in culture (10). Although these differentiated cells provide the most physiological model in which to conduct transfection assays with PR promoter-reporter constructs, they do not transfect easily or efficiently (21). Therefore, to determine the efficacy of using immature granulosa cells as a model for analyzing PR promoterreporter constructs, it was necessary to determine whether the endogenous PR gene could be activated in these cells, if given the appropriate stimulus.…”

Transcriptional Regulation of <i>CYP11A1</i>

“…To understand the mechanism of the cAMP-PKA signal on the promoter activity of CYP11A1, the regulatory elements in its promoter have been analyzed in bovine CYP11A [1,23], rat CYP11A [28], mouse Cyp11a [2,21], and human CYP11A1 [3,16,39,40]. These investigations have identified certain cisacting elements conferring cAMP responsiveness via interactions with their specific transcription factors, using standard methods for the analysis of regulatory interacting proteins [29].…”

Protein Kinase A-Independent cAMP Stimulation of Progesterone in a Luteal Cell Model Is Tyrosine Kinase Dependent but Phosphatidylinositol-3-Kinase and Mitogen-Activated Protein Kinase Independent1

“…Various reports have directly implicated tyrosine kinase pathways in ovarian steroidogenesis. Addition of specific tyrosine kinase inhibitors to cultured rat granulosa cells results in inhibition of LH-stimulated progesterone production and Cyp11a1 mRNA expression [57][58][59]. Other studies have shown synergistic effects of insulin or IGF1 on gonadotropin-stimulated progesterone production and expression of Cyp11a1 and Hsd3b mRNAs by rat and human granulosa cells [3,4,8].…”