Evidence for Protein Kinase C Regulation of Ovarian Theca-Interstitial Cell Androgen Biosynthesis1

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Abstract
The hypothesis that protein kinase C may be an important regulator of ovarian theca-interstitial cell steroidogenesis was tested by using phorbol-12-myristate-13-acetate (PMA) and phorobol-12,13-dibutyrate (PDB) to directly stimulate protein kinase C activity. Collagenase-dispersed cells (4 .times. 105 viable cells/dish) from ovaries of hypophysectomized immature rats were cultured in serum-free medium in the presence and absence of 0-100 ng/ml of luteinizing hormone (LH), PMA (0-100 nM), and/or PDB (0-100 nM). Treatment with 100 ng/ml LH stimulated androsterone production 100-fold at Day 4 of culture. The presence of 100 nM PMA or PDB had no effect on basal androsterone production; however, treatment with increasing concentrations of PMA or PDB (0-100 nM) caused a dose-related inhibition (maximum 70%) of LH-stimulated androsterone synthesis (ID50 = 1.8 nM and 2.4 nM, respectively). PMA and PDB did not significantly alter DNA, protein, or cell viability, indicating that their inhibitory effects were not due to changes in cell number or viability. Cells treated with LH and 100 nM 4.alpha.-phorbol didecanoate (4.alpha.-PDD; a phorbol ester that does not activate protein kinase C) failed to show significant decreases in androsterone production. Time-course studies revealed that when PMA treatment was delayed until Day 2 or 4 of culture, dramatic inhibitory effects on LH-stimulated androsterone production were still observed. These results suggest that the biological activity of protein kinase C is retained after the cells have expressed their differentiated state. In reversibility studies, the PMA effect persisted; however, the inhibitory effect of the more hydrophilic PDB was reversed when PDB was washed from the culture medium. Dose-response studies showed that although PMA inhibited the magnitude of LH-stimulated androsterone production, the concentrations of LH that stimulated minimum, maximum, and ED50 responses were not affected. To examine the intracellular site of PMA action, androsterone synthesis was stimulated with cholera toxin, prostaglandin E2, and 8-bromo cAMP. In each case, concomitant treatment with PMA (100 nM) inhibited androsterone synthesis 81-96%. Metabolic studies revealed that PMA decreased the levels of pregnenolone, progesterone, 17.alpha.-hydroxyprogesterone, androstenedione, testosterone, androsterone, and 5.alpha.-androstan-3.alpha., 17.beta.-diol by equal amounts (70%). These results suggest that PMA blocked steroid biosynthesis at or prior to the cholesterol side chain cleavage step. These results provide the first evidence to support the concept that protein kinase C may play an important role in the mechanisms regulating androgen biosynthesis by ovarian theca-interstitial cells.

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