The response of 21-hydroxylase messenger ribonucleic acid levels to adenosine 3',5'-monophosphate and 12-O-tetradecanoylphorbol-13-acetate in bovine adrenocortical cells is dependent on culture conditions.
The regulation of 21-hydroxylase mRNA and enzyme activity by cAMP and 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied as a function of different culture conditions in bovine adrenocortical cells. Induction of 21-hydroxylase mRNA and enzyme activity was assessed after cultures were incubated with cAMP analogs. Basal 21-hydroxylase mRNA and enzyme activities were high; cAMP addition resulted in some increase in the level of mRNA, but little increase in enzyme activity. When cultures were made mitotically quiescent by using a higher starting cell density or by allowing cells to grow for a longer period before incubation with cAMP, basal mRNA and enzyme activity were low and increased greatly in response to cAMP. Other agents that raise intracellular cAMP (ACTH, prostaglandin E1, cholera toxin, and forskolin) increased 21-hydroxylase mRNA in quiescent cultures. As previously demonstrated for 11 beta-hydroxylase,21-hydroxylase mRNA induction by cholera toxin or cAMP analogs was dependent on the presence of insulin-like growth factor-I or insulin in the culture medium. When cultures were not quiescent, 21-hydroxylase mRNA and enzyme activity were highly variable, but characterized by 1) high basal levels, 2) small increases or actual decreases in response to cAMP, and 3) variable increases in response to TPA, not observed in quiescent cultures. This last effect was observed within a very narrow range of TPA concentrations (0.3-3 nM); at higher concentrations 21-hydroxylase activity decreased to values below those in the control culture. The protein kinase inhibitor staurosporine at 1-5 nM inhibited basal 21-hydroxylase enzyme activity in nonquiescent cultures by up to 80%. The probable existence of controlling factors for 21-hydroxylase expression other than cAMP suggests that the regulation of this gene differs substantially from that of the other steroid hydroxylases.
Chang
C W
CW
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912.
Naseeruddin
S A
SA
Hornsby
P J
PJ
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000302:Adrenal Cortex / Q000187:drug effects / Q000201:enzymology*; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000031:analogs & derivatives / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D000242:Cyclic AMP / Q000494:pharmacology* / Q000502:physiology; D004790:Enzyme Induction; D007700:Kinetics; D008297:Male; D009919:Orchiectomy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics*; D019311:Staurosporine; D013255:Steroid 21-Hydroxylase / Q000096:biosynthesis / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000470:Alkaloids; D003470:Culture Media; D012333:RNA, Messenger; C002373:monobutyryl cyclic AMP; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003994:Bucladesine; D000242:Cyclic AMP; D013255:Steroid 21-Hydroxylase; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate