REGULATION OF 3-BETA-HYDROXYSTEROID DEHYDROGENASE/DELTA-5-]4-ISOMERASE EXPRESSION BY ADRENOCORTICOTROPIN IN BOVINE ADRENOCORTICAL-CELLS

In the steroidogenic pathway, 3-beta-hydroxysteroid dehydrogenase-isomerase (3-beta-HSD) catalyzes the formation of hormonally active DELTA-4-3-ketosteroids from DELTA-5-3-beta-hydroxysteroids. In the present study the regulation of 3-beta-HSD by ACTH action on bovine adrenocortical (BAC) cells in primary culture was evaluated. Western blot analysis was accomplished using an antibody against human placental 3-beta-HSD. The relative molecular mass of 3-beta-HSD in these cells was 45K, which was similar to that in human placenta. A significant effect of ACTH was not detected until day 6 of culture due to the high basal levels of the enzyme in BAC cells. Treatment of cells with ACTH on day 8 of culture resulted in a marked increase in the amount of 3-beta-HSD protein, and this effect was correlated directly with enzymatic activity. The effects of ACTH were time and dose dependent, with an increase detectable only after 48 h of treatment; the maximal response was obtained with 10(-9) M ACTH. As demonstrated by Northern analysis, ACTH action was manifested by increasing the steady state level of 3-beta-HSD mRNA. A human 3-beta-HSD cDNA probe, which was used in this study, hybridized to a 1.7-kilobase species of BAC RNA. The effects of ACTH on 3-beta-HSD activity and increases in 3-beta-HSD protein and mRNA in BAC cells were mimicked by treatment with (Bu)2cAMP. The findings of this study suggest that ACTH controls 3-beta-HSD gene expression in BAC cells by a cAMP-dependent mechanism similar to that involved in the expression of steroid hydroxylase genes. However, because the different stabilities of 3-beta-HSD and hydroxylase proteins and/or mRNAs may play a critical role in determining the zone-specific steroids secreted from the adrenal cortex, other cAMP-dependent or independent regulatory mechanisms may also be important in regulating the expression of adrenal 3-beta-HSD.