Protein kinase A and protein kinase C differentially regulate steroidogenesis in human ovarian thecal tumor cells

The stimulatory role of protein kinase A in thecal cell steroidogenesis is well documented, whereas the role of protein kinase C is not well defined. In this study, using monolayer cultures of human ovarian tumor cells that are steroidogenically similar to thecal cells, we examined the effects of the protein kinase C activator tetradecanoylphorbol-13-acetate (TPA) on steroidogenesis and the expression of 17α-hydroxylase cytochrome P450 (P450c17), 3β-hydroxysteroid dehydrogenase (3βHSD), and cholesterol side-chain cleavage cytochrome P450 (P450scc). Cells were uniformly plated and grown to confluence prior to experimental treatment in serum-free medium. Treatments were control, forskolin (10 μM), TPA (0.01-1000 nM), and TPA with forskolin. Treatment with TPA alone for 24 h had little effect on basal steroid production, enzyme activities, or mRNA levels. However, when added with forskolin, TPA augmented progesterone production in a concentration-dependent manner. In contrast, TPA inhibited forskolin stimulation of androstenedione production and P450c17 activity. To define better the mechanism of TPA action, Northern analysis of P450c17, P450scc, and 3βHSD mRNA was accomplished using total RNA isolated from cells treated for 24 h. 3βHSD mRNA was increased by forskolin and was not significantly inhibited by treatment with TPA. P450c17 mRNA, however, was suppressed to near undetectable levels by TPA at doses as low as 1 nM. In addition, P450scc mRNA expression was inhibited in a manner similar to that seen for P450c17 mRNA. In summary, activation of the protein kinase A pathway increases expression of3βHSD, P450c17, and P450scc in this thecal cell model. Simultaneous activation of protein kinase A and protein kinase C enhances progesterone production while decreasing androstenedione production and the levels of mRNA encoding P450c17 and P450scc. This differential regulation of steroidogenesis suggests that protein kinase C may play a role in decreased androstenedione production during thecal cell luteinization.