Regulation of 11β- and 17α-hydroxylases in cultured bovine adrenocortical cells: 3’, 5’-cyclic adenosine monophosphate, insulin-like growth factor-i, and activators of protein kinase C

The induction of steroid 11β-hydroxylase and 17α-hydroxylase was studied in bovine adrenocortical cell cultures in serum-free medium. In the absence of insulin-like growth factor (IGF)-I or insulin, cholera toxin failed to increase 11β -hydroxylase enzyme activity or messenger RNA (mRNA) levels; cholera toxin increased 11β -hydroxylase activity and mRNA only in the presence of 10 nM IGF-I or of higher concentrations of insulin. 17α -Hydroxylase enzyme activity and mRNA, in contrast, were increased maximally by cholera toxin in the absence of insulin or IGF. We also compared the induction of 11β -hydroxylase and 17α -hydroxylase by intracellular second messengers. When cultures were incubated with cholera toxin, cAMP analogs, forskolin, ACTH, or prostaglandin E₁ in defined medium with insulin, all agents increased the mRNA levels for 11β -hydroxylase and 17α -hydroxylase. 11β -Hydroxylase enzyme activity was detectable in control (insulin only) cultures and was increased to varying extents by the different agents. 17α -Hydroxylase enzyme activity was undetectable in control cultures and was increased more than 50-fold by all agents. We compared the sensitivity of induction of 11β -hydroxylase and 17α -hydroxylase enzyme activities by cAMP using serial dilutions of an equimolar mixture of N⁶-monobutyryl cAMP and 8-bromo cAMP. For both enzymes, the response curve was biphasic, with a maximal response in the range of 20 to 100 μM each analog, but the decline in response at higher cAMP concentrations was much more marked for 11β -hydroxylase than for 17 α -hydroxylase. The effects of activation of protein kinase C were studied in cultures incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA) together with a cAMP analog mixture. TPA decreased cAMP-induced 11β -hydroxylase mRNA; TPA also decreased the induction of 17α -hydroxylase mRNA, as previously reported. TPA caused a dose-dependent decrease in cAMPinduced 11β -hydroxylase enzyme activity. Angiotensin II at 0.1 to 10 μM also decreased induction of 11β -hydroxylase. Induction of 11β -hydroxylase and 17α -hydroxylase is coordinately regulated by cAMP, protein kinase C, and IGF-I/insulin, but responses to these regulators differ in various respects between these two cytochrome P450 enzymes.