Protein kinase C increases 11β-hydroxysteroid dehydrogenase oxidation and inhibits reduction in rat Leydig cells

Glucocorticoid hormone controls Leydig cell steroidogenic function through a receptor-mediated mechanism. The enzyme 11 beta -hydroxysterold dehydrogenase (11 beta HSD) plays an important role in Leydig cells by metabolizing glucocorticoids, and catalyzing the interconversion of corticosterone (the active form in rodents) and 11-dehydrocorticosterone (the biologically inert form). The net direction of this interconversion determines the amount of biologically active ligand, corticosterone, available for glucocorticoid receptor binding. We hypothesize that 11 beta HSD oxidative and reductive activities are controlled separately in Leydig cells, and that shifts in the favored direction of 11 beta HSD catalysis provide a mechanism for the control of intracellular corticosterone levels. Therefore, in the present study, we tested the dependency of 11 beta HSD oxidative and reductive activities on protein kinase C (PKC) and calcium-dependent signaling pathways, 11 beta HSD oxidative and reductive activities were measured in freshly isolated intact rat Leydig cells using 25 nM radiolabeled substrates after treatment with protein kinase modulators. We found that PKC and calcium-dependent signaling had opposing effects on 11 beta HSD oxidative and reductive activities. Stimulation of PKC using the PKC activator, 6-[N-decylamino]-4-hydroxymethylinole (DHI), increased 11 beta HSD oxidative activity from a conversion rate of 5.08% to 48.23% with an EC50 of 1.70 +/- 0.44 muM (mean +/- SEM), and inhibited reductive activity from 26.90% to 3.66% conversion with an IC50 of 0.22 +/- 0.05 muM. This indicated that PKC activation in Leydig cells favors 11 beta HSD oxidation and lower levels of corticosterone. The action of DHI was abolished by the PKC inhibitor bisindolylmaleimide I. In contrast, addition of calcium to Leydig cells increased 11 beta HSD reductive activity while decreasing oxidative activity, thereby favoring reduction and conversion of inert 11-dehydrocorticosterone into active corticosterone. The opposite effect was seen after elimination of calcium-dependent signaling, including removal of calcium by EGTA or addition of the calmodulin (calcium binding protein) inhibitor SKF7171A, or the calcium/calmodulin-dependent protein kinase I (CaMK II) inhibitor, KN62. We conclude that 11 beta HSD oxidative and reductive activities are separately regulated and that, in contrast to calcium-dependent signaling, PKC stimulates 11 beta HSD oxidation while inhibiting 11 beta HSD reduction. Maintenance of a predominantly oxidative 11 beta HSD could serve to eliminate adverse glucocorticoid-induced action in Leydig cells.