PROTEIN-KINASE-C IS A TONIC NEGATIVE REGULATOR OF STEROIDOGENESIS AND STEROID HYDROXYLASE GENE-EXPRESSION IN Y1 ADRENAL-CELLS AND FUNCTIONS INDEPENDENTLY OF PROTEIN KINASE-A

The role of protein kinase C (PKC) in the regulation of basal steroidogenesis and steroid hydroxylase gene expression in Y1 adrenocortical cells was investigated. Treatment of Y1 cells with either staurosporine or calphostin C, inhibitors of PKC, increases steroid hormone production up to 7-fold. Induction of P450-cholesterol side chain cleavage enzyme (SCC) mRNA expression parallels induction of steroidogenesis by the PKC inhibitors. Staurosporine increases expression of a transiently transfected SCC promoter-human growth hormone construct in Y1 cells, indicating that PKC regulates expression of SCC mRNA at the level of transcription. Treatment with staurosporine increases expression of mRNA for two additional steroid synthetic enzymes, P450-11beta-hydroxylase and 3beta-hydroxysteroid dehydrogenase. These data indicate that PKC acts as a tonic negative regulator of basal steroidogenesis in Y1 cells by suppressing expression of mRNA encoding the steroid synthetic enzymes. Protein kinase A (PKA) and PKC have reciprocal effects on steroidogenesis and expression of the steroid synthetic enzymes in Y1 cells. However, the results of this study demonstrate that these signaling pathways are not interdependent. Steroid production by Y1 cells treated with (Bu)2cAMP and calphostin C together is equal to the sum of steroid production after treatment with either agent alone. Pretreatment of Y1 cells with Rp-8-Bromo-cAMP, a specific inhibitor of PKA, prevents induction of steroidogenesis by (Bu)2CAMP, but not by staurosporine, indicating that PKC is not dependent on PKA activity. In addition, induction of SCC mRNA expression by staurosporine, in Y1 cells which are defective in activation of PKA (Y1 kin-8), is equivalent to induction in Y1 cells. These data indicate that PKA and PKC regulate basal steroidogenesis through independent effects on expression of the steroid synthetic enzymes.