Regulation of Human CYP11B2 and CYP11B1: Comparing the Role of the Common CRE/Ad1 Element

Abstract

In humans, the final steps in corticosteroid production results from the activity of aldosterone synthase in the glomerulosa and 11β-hydroxylase in the fasciculata. The regional expression of these isozymes is believed to result from transcriptional regulation of the aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) genes. Previous studies suggest that the primary cis-element needed for agonist enhanced transcription of the CYP11B genes shares high sequence similarity to a consensus cAMP Response Element (CRE). Here the role of the CRE/Adl was studied. Reporter constructs prepared with the 5′flanking DNA of hCYP11B2 and hCYP11B1 were transfected into NCI-H295R (H295R) adrenocortical tumor cells. Both hCYP11B2 and hCYP11B1 driven reporter constructs responded in a similar manner to treatment with angiotensin II, potassium, dbcAMP, or forskolin. Mutation of the hCYP11B1 CRE/Adl element decreased basal reporter expression and decreased response to agonist. Mutation of the hCYP11B2 CRE/Adl element caused a loss of basal expression but retained response to agonist suggesting a role for other cis-elements in hormonal regulation of hCYP11B2 In addition, both cis-elements were able to form complexes with in vitro prepared CRE binding (CREB) protein, activating transcription factor (ATF)-1 and ATF-2 in mobility shift assays. However, only the ATF-2 complex migrated similarly to a complex seen using H295R nuclear extract. Taken together these data suggest that the CRE/Adl element plays an important role in the transcriptional regulation of both hCYP11B genes but does not play an important role in the regional distribution of the two isozymes within the adrenal.