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Protein Kinase A, Protein Kinase C, and Ca²⁺-Regulated Expression of 21-Hydroxylase Cytochrome P450 in H295R Human Adrenocortical Cells¹

Department of Obstetrics and Gynecology, University of Wisconsin (I.M.B.), Madison, Wisconsin 53715; the Department of Clinical Biochemistry, University of Edinburgh, Royal Infirmary of Edinburgh (J.I.M.), Edinburgh, Scotland EH3 9YW; and the Department of Obstetrics and Gynecology and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center (W.E.R.), Dallas, Texas 75235

Address all correspondence and requests for reprints to: Dr. Ian M. Bird, 7E Meriter Hospital Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: imbird{at}facstaff.wisc.edu

The physiological importance of adrenal 21-hydroxylase cytochrome P450 (CYP21) expression is clearly demonstrated by 21-hydroxylase deficiency, which results in adrenal hyperplasia and overproduction of C19 steroids, leading to virilization. The mechanisms regulating normal expression of this key enzyme in human adrenocortical cells are ill defined. Herein we examine the role of the calcium, protein kinase C, and protein kinase A signaling pathways in the expression of CYP21 messenger ribonucleic acid (mRNA) using the H295R human adrenocortical cell model. Forskolin (10 µmol/L) treatment caused a progressive increase in CYP21 mRNA levels (maximum, 4-fold; P < 0.05) over 36 h of treatment, whereas angiotensin II (AII; 10 nmol/L) produced a smaller, biphasic rise (maximum, 1.8-fold at 12 h; P < 0.05). K⁺ (14 mmol/L) also induced a time-dependent (maximal, 1.5-fold at 12 h; P < 0.05) and dose-dependent (P < 0.05 12 mmol/L or above at 20 h) rise in CYP21 mRNA levels. The action of forskolin was reproduced by dibutyryl cAMP, confirming the involvement of cAMP in this response. The action of AII was greater than that of K⁺ or the calcium channel agonist BAYK8644, suggesting that AII action was not solely through the Ca²⁺ signaling pathway. The action of AII was reproduced and indeed exceeded by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nmol/L; 5.5-fold increase; P < 0.05). The actions of forskolin alone were not significantly increased by combined treatment with AII, suggesting neither synergy nor attenuation of the effects of protein kinase A activation. This was further demonstrated at the level of mRNA and 21-hydroxylase activity by the observation that the effect of forskolin and TPA in combination did not exceed that of TPA alone. Inhibition of protein synthesis with cycloheximide blocked induction of CYP21 as well as type II 3ß-hydroxysteroid dehydrogenase (3ßHSDII) mRNA expression in response to AII, forskolin, and dibutyryl cAMP, but had no effect on 17-hydroxylase cytochrome P450 (CYP17) or cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA. Together, these findings were remarkably similar to those of our previous studies regarding mechanisms regulating 3ßHSDII expression and underline the existence of a subset of steroidogenic enzymes regulated positively (CYP21 and 3ßHSDII) as opposed to negatively (CYP17 and CYP11A) by the protein kinase C signaling pathway. The additional finding of a small induction of CYP21 expression in response to increased Ca²⁺, as previously reported for CYP17, but not 3ßHSDII, expression, also demonstrates that the mechanisms of control of CYP21 and 3ßHSDII are not identical. This latter finding may also relate to how CYP21 as well as CYP17 expression continues in the zona reticularis after adrenarche, whereas 3ßHSD expression declines.

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