Regulation of 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells.

Previous studies of the effects of angiotensin II (All), alone or in combination with activators of the protein kinase. A signalling pathway, have yielded inconsistent findings on the expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD and 17 alpha-hydroxylase cytochrome P450 (P450c17) as well as the corresponding responses on steroid secretory products in human adrenocortical cells. We have used the human adrenocortical carcinoma H295R cell further to evaluate this question, as well as to determine the role of protein kinase C in each of these responses to All. Treatment with All alone resulted in a marked increase in aldosterone secretion and a significant increase in cortisol secretion (1-8-fold). The increased formation of 17-hydroxysteroids was accompanied by an increased level of P450c17 mRNA and activity. Increases in 3 beta-HSD expression were also seen at the level of mRNA and to a lesser extent, at the level of activity. Because of the comparatively low basal 17 alpha-hydroxylase and high basal 3 beta-HSD activities of H295R cells, however, the overall effect of All treatment was actually a rise in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, so resulting in increased formation of 17 alpha-hydroxysteroids such as cortisol. While treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) reproduced the effect of All on 3 beta-HSD expression, TPA failed to reproduce the effects of All on P450c17 because it caused a marked decrease in P450c17 expression. Thus the stimulatory effect of All on P450c17 expression, unlike that on 3 beta-HSD expression, was not mediated by protein kinase C but, like the action of K, was probably mediated via the Ca2+ signalling pathway. Treatment with forskolin resulted in a dramatic increase in both cortisol and dehydroepiandrosterone (DHEA) secretion together with increases in expression of 3 beta-HSD and P450c17 as measured at the level of mRNA and activity. Consistent with the increase in 17 alpha-hydroxysteroid formation, the effect on P450c17 expression was greater than that on 3 beta-HSD at the level of activity, so a larger 17 alpha-hydroxylase/3 beta-HSD activity ratio was achieved. Cotreatment with forskolin and All, however, resulted in a dose-dependent reduction in cortisol and DHEA secretion concomitant with a marked attenuation of 3 beta-HSD and P450c17 expression. While forskolin-induced expression of 3 beta-HSD was not further increased at the level of mRNA by cotreatment with All, additivity was observed as the level of activity changed. Thus All cotreatment resulted in a marked reduction in the forskolin-induced increase in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, and so 17 alpha-hydroxysteroid synthesis was attenuated. The effect of All cotreatment on changes in forskolin-induced 3 beta-HSD activity was blocked by the All type 1 (AT1) antagonist DuP753 (Losartan), confirming the involvement of the AT1 receptor-linked phospholipase C in activating protein kinase C.