title: Ca(2+)-regulated expression of steroid hydroxylases in H295R human adrenocortical cells.
abstract: Although changes in the expression of key steroidogenic enzymes such as cytochrome P450 cholesterol side-chain cleavage, 17 alpha-hydroxylase (P450c17), aldosterone synthase, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the human adrenal cortex are known to be controlled by factors activating the protein kinase A or protein kinase C signaling pathways, little is known concerning the effects of increased intracellular Ca2+. In this study we describe the effects of K+, an agent known to increase intracellular Ca2+ through the opening of voltage-sensitive Ca2+ channels, on steroidogenesis in H295R human adrenocortical cells and corresponding changes in expression of these vital steroidogenic enzymes. Treatment of cells for 48 h with K+ (14 mM) resulted in an increase in aldosterone (3.5-fold) as well as the 17 alpha-hydroxylated steroids cortisol (2.9-fold) and dehydroepiandrosterone (DHEA; 3.7-fold). This action of K+ was accompanied by a dose-dependent (P < 0.05 at 6 mM K+ or above) and time-dependent (P < 0.05 at 24 h and beyond) increase in expression of P450c17 and, to a lesser extent, cytochrome P450 cholesterol side-chain cleavage messenger RNA (mRNA). Treatment with K+ also caused a time-dependent increase in aldosterone synthase mRNA levels, which were detectable by 12 h. Treatment with K+, however, was without effect on 3 beta HSD expression. These effects contrast with those of (Bu)2cAMP, which stimulated a greater increase in cortisol and DHEA secretion as well as P450c17 expression. The effects of K+ treatment also differ from those of AII, which promoted a greater aldosterone secretory response (5.7-fold), but a lesser effect on DHEA secretion (2.2-fold) and P450c17 expression. Although AII and TPA (known activators of protein kinase C) as well as forskolin and (Bu)2cAMP (known activators of protein kinase A) increased the expression of 3 beta HSD mRNA, K+ treatment was without effect, suggesting that elevation of [Ca2+]i in response to K+ did not activate the protein kinase C or protein kinase A signaling pathways. Furthermore, the effects of K+ on steroid secretion and 17 alpha-hydroxylase activity were reproduced by the voltage-sensitive Ca2+ channel activator BAYK 8644, and increases in P450c17 mRNA in response to K+ were reversed by the Ca2+ channel antagonist, nifedipine. We conclude that K+ can modulate the expression of key steroidogenic enzymes in H295R cells through the Ca2+ signaling pathway without involvement of the protein kinase A or protein kinase C pathways.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
authors.forename: I M
authors.initials: IM
authors.lastname: Bird
authors.forename: J M
authors.initials: JM
authors.lastname: Mathis
authors.forename: J I
authors.initials: JI
authors.lastname: Mason
authors.forename: W E
authors.initials: WE
authors.lastname: Rainey
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000096:biosynthesis; D000302:Adrenal Cortex / Q000201:enzymology*; D001483:Base Sequence; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000096:biosynthesis; D019405:Cytochrome P-450 CYP11B2; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D011188:Potassium / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D013252:Steroid 11-beta-Hydroxylase / Q000096:biosynthesis; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D015096:3-Hydroxysteroid Dehydrogenases; D013250:Steroid Hydroxylases; D013254:Steroid 17-alpha-Hydroxylase; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D011188:Potassium; D002118:Calcium