Multiple Mechanisms for Regulation of 3<i>β</i>-Hydroxysteroid Dehydrogenase/Δ⁵→Δ⁴-Isomerase, 17<i>α</i>-Hydroxylase/C_17–20Lyase Cytochrome P450, and Cholesterol Side-Chain Cleavage Cytochrome P450 Messenger Ribonucleic Acid Levels in Primary Cultures of Mouse Leydig Cells^*

Abstract

The regulation of mRNA levels for Δ5–3β-hy-droxysteroid dehydrogenase/Δ5→Δ4-isomerase (3βHSD), 17α-hydroxylase/C17–20 lyase cytochrome P450 (P45017α) and cholesterol side-chain cleavage cytochrome P450 (P45scc) was studied in primary cultures of mouse Leydig cells. Treatment of Leydig cells with 8-bromo-cAMP (cAMP) was essential for expression of P45017α mRNA, but not for 3βHSD. Treatment with cAMP caused a decrease in basal levels of 3βHSD mRNA. The addition of aminoglutethimide (AG), an inhibitor of cholesterol metabolism, to the cAMP-treated cultures resulted in increased expression of both 3βHSD and P45017α mRNA levels. The addition of testosterone or the androgen agonist mibolerone to cAMP- plus AG-treated cultures reduced 3βHSD and P45017α mRNA to levels comparable to those observed when cells were treated with cAMP only. The glucocorticoid dexamethasone reduced both basal and cAMP- plus AG-induced increases in 3βHSD mRNA, but not in P45017α mRNA. Estradiol at a concentration of 1 μM had no effect on cAMP- plus AG-induced 3βHSD or P45017α mRNA levels. The role of protein synthesis in mediating the cAMP induction of 3αHSD, P45017α, and P450scc was investigated. The addition of cycloheximide (10 μg/ml) to cAMP-treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3βHSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of P45017α to 12% of levels observed in the absence of cycloheximide. In sharp contrast, 24-h treatment with cycloheximide did not suppress cAMP induction of P450scc mRNA, but reduced basal levels by approximately 50%. A time course of induction by cAMP (50 μM) of P45017α and P450scc mRNA showed very similar rates of increase in P45017α and P450scc mRNA, with the greatest increase occurring between 12 and 24 h of treatment. The results of the study demonstrate that in normal mouse Leydig cells steady state levels of mRNA for 3βHSD, P45017α, and P450scc are differentially regulated. cAMP is required for maximal levels of all three mRNAs. There is high constitutive expression of 3βHSD and P450scc mRNA, while expression of P45017α mRNA is absolutely dependent on cAMP stimulation. Endogenously produced testosterone negatively regulates the expression of cAMP-induced P45017α and 3βHSD, while the glucocorticoid dexamethasone negatively regulates 3βHSD and P450scc. Newly synthesized protein(s) is required for cAMP induction of P45017α and 3βHSD mRNA levels, but not for P450scc mRNA. (Endocrinology129: 1429–1435, 1991)