Stimulation of progesterone production by phorbol-12-myristate-13-acetate in MA-10 Leydig tumor cells

Abstract

The tumor-promoting phorbol ester, phorbol-12-myristate-13-acetate (PMA) markedly stimulated progesterone production in MA-10 Leydig tumor cells. A slight but significant increase (35%) in the activity of the cholesterol side-chain cleavage (CSCC) enzyme was observed in mitochondria isolated from the PMA-treated MA-10 Leydig cells when compared to mitochondria isolated from non-treated cells. However, this stimulation of CSCC activity appears to be of limited importance when compared to the 240-fold increase observed in progesterone production following PMA stimulation. In contrast, the inactive phorbol ester 4α-phorbol-12,13-didecanoate (α-PD) had no effect on either progesterone production or CSCC activity. PMA had no effect on the conversion of 25-hydroxycholesterol and 22 R-hydroxycholesterol into progesterone suggesting that one of the mechanism(s) of PMA action may involve the delivery of cholesterol to the mitochondria and/or the affinity of cholesterol with cytochrome P-450 scc. Stimulation of steroidogenesis by PMA was also shown to be inhibited by cycloheximide. When PMA was added together with a submaximal dose of hCG, hCG-stimulated steroidogenesis was inhibited. However, at a maximal dose of human chorionic gonadotropin (hCG), PMA inhibited steroid synthesis at 1 and 2 h but had no significant effect at 3 h. Conversely, PMA had an additive effect on cAMP induced steroidogenesis. It was further demonstrated that PMA resulted in a decrease in the hCG-induced accumulation of cAMP. Our results suggest that PMA is able to stimulate progesterone production by MA-10 Leydig cells and that this stimulation of steroid synthesis by PMA is not mediated through an increase in cAMP production as seen with luteinizing hormone/hCG. Since the action of PMA is mediated through the activation of protein kinase C, we conclude, therefore, that in addition to cAMP-dependent protein kinase A, another protein phosphorylating enzyme, protein kinase C plays an important role in the regulation of steroidogenesis in MA-10 Leydig tumor cells.