Mechanisms of action for an androgen-mediated autoregulatory process in rat thecal-interstitial cells

Abstract

In rat thecal-interstitial cells (TIC), treatment with the synthetic androgen mibolerone has led to the documentation of an autoregulatory process for androgen production. In the present study, accumulated evidence has provided insight into the mechanisms of mibolerone action that control this process. Investigations using the nonsteroidal antiandrogen hydroxyflutamide were conducted to characterize mibolerone's mode of action. Hydroxyflutamide had differential effects on hCG action, the 1-μM dose stimulating hCG- induced androsterone synthesis by 27% and the 10-μM concentration decreasing the androgen levels by 84%. In addition, treatment with 1 μM hydroxyflutamide was effective in partially reversing the inhibitory action of mibolerone on hCG-stimulated androsterone production. Thus, the data indicated that mibolerone's mode of action may be mediated, at least in part, via the androgen receptor. The possibility that mibolerone had multiple sites of action prompted studies on the effectiveness of this androgen to alter various signaling pathways. Treatment with increasing concentrations (0.01- 100 nM) of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C, resulted in a 75% decrease in hCG- stimulated androgen production at a dose of 100 nM TPA. Treatment with mibolerone (100 nM) was unable to alter the action of TPA on androgen synthesis when doses of 1 and 10 nM TPA were employed. It was also found that Ca2+ can serve as a mediator of mibolerone action. Treatment with a 0.01- μM dose of A23187, a Ca2+ ionophore known to increase intracellular Ca2+ was ineffective in altering hCG-stimulated androsterone synthesis. The concurrent treatment of mibolerone (100 nM) and A23187 (0.01 μM) resulted in the potentiation of mibolerone's inhibitory effects on hCG-stimulated androgen production, thereby suggesting that mibolerone can stimulate Ca2+ influx. Additional studies revealed that the administration of a 1-μM dose of the L-type Ca2+ channel blocker verapamil to TIC cultures was able to partially block the inhibitory effect of mibolerone on androgen synthesis. Evidence for an additional site of mibolerone action was revealed through an analysis of the mRNA levels of P450(scc), and P450(17α) enzymes. Although hCG and insulin-like growth factor I treatment resulted in 20- and 32-fold increases in the amount of P450(scc) and P450(17α) mRNA, respectively, the addition of mibolerone (100 nM) reduced only P450(17α) mRNA levels by 91%. Overall, the evidence indicates that mibolerone has multiple sites of action in exerting its regulatory effect on androgen synthesis.