Differential control of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells.

Abstract

Previous studies of human adrenocortical cells have given inconsistent findings concerning the effects of angiotensin II (AII) alone or in combination with activators of the protein kinase A-signaling pathway on expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450c17), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as well as the corresponding effects on adrenocortical cell steroid secretory products. We have used the human adrenocortical carcinoma H295R cell to evaluate further this question and determine the role of protein kinase C in each of these responses to AII. Treatment with AII alone (10 nmol/L, 48 h) resulted in a significant increase in cortisol production (1.8-fold), as well as a much greater effect on aldosterone production. This increased formation of 17 alpha-hydroxysteroids was accompanied by increased expression of P450c17 as determined at the level of messenger RNA (mRNA) and enzyme activity. Similar increases in expression of P450scc were observed at the level of mRNA. Increases in 3 beta-HSD expression were also seen at the level of mRNA and, to a lesser extent, at the level of enzyme activity. Because of the comparatively low basal 17 alpha-hydroxylase and high basal 3 beta-HSD activity of H295R cells, however, the overall effect of AII treatment was actually a rise in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, resulting in increased formation of 17 alpha-hydroxysteroids such as cortisol. Whereas treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) reproduced the effect of AII on 3 beta-HSD expression, TPA failed to reproduce the effects of AII on P450c17 and P450scc and even resulted in a marked decrease in expression of P450c17. Thus, the stimulatory effect of AII alone on P450c17 expression was not mediated via protein kinase C but, like the action of K+, was probably mediated via the Ca(2+)-signaling pathway. Treatment with forskolin (10 mumol/L, 48 h) resulted in a dramatic increase in both cortisol and dehydroepiandrosterone production together with increases in expression of P450c17, P450scc, and 3 beta-HSD as measured at the level of mRNA and activity. Consistent with the increase in 17 alpha-hydroxysteroid formation, the effect on 17 alpha-hydroxylase expression was greater than that on 3 beta-HSD at the level of enzyme activity, so a larger 17 alpha-hydroxylase/3 beta-HSD activity ratio was achieved. Cotreatment with forskolin and AII, however, resulted in a dose-dependent reduction in cortisol and DHEA production concomitant with a marked attenuation of P450scc and P450c17 expression. Although forskolin-induced expression of 3 beta-HSD was not further increased at the level of mRNA by cotreatment with AII, additivity was observed at the level of changes in enzyme activity. Thus, AII cotreatment resulted in a marked reduction of the forskolin-induced increase in 17 alpha-hydroxylase/3 beta-HSD activity ratio, and so, 17 alpha-hydroxysteroid synthesis was attenuated. These effects of AII cotreatment on expression of P450c17 and P450scc were reproduced by cotreatment with TPA (10 nmol/L), suggesting the involvement of protein kinase C in these attenuative responses. Furthermore, the effect of AII cotreatment on changes in forskolin-induced 17 alpha-hydroxylase and 3 beta-HSD activities were blocked by the AII Type 1 (AT1) receptor antagonist DuP753 (Losartan), confirming the involvement of an AT1 receptor-linked phospholipase C in activating protein kinase C.