Angiotensin II and Potassium Regulate Human CYP11B2 Transcription through Common cis-Elements

Abstract

Aldosterone synthase is a mitochondrial enzyme that catalyzes the conversion of 11-deoxycorticosterone to the potent mineralocorticoid aldosterone. The gene encoding aldosterone synthase, CYP11B2, is expressed in the zona glomerulosa of the adrenal cortex. Although the major physiological regulators of aldosterone production are angiotensin II (ANG II) and potassium (K+), the mechanisms by which these compounds regulate CYP11B2 transcription are unknown. Therefore we analyzed the human CYP11B2 5'-flanking region using a transient transfection expression system in the H295R human adrenocortical cell line. ANG II and K+ increased expression of a luciferase reporter construct containing 2015 bp of human CYP11B2 5'-flanking DNA. This response was mimicked by treatment with the calcium channel activator BAYK8644, whereas activation of the protein kinase C pathway with 12-o-tetradecanoylphorbol-13-acetate had no effect. Reporter gene activity was also increased after activation of cAMP-dependent pathways by (Bu)2cAMP. Deletion, mutation, and deoxyribonuclease I footprinting analyses of the CYP11B2 5'-flanking region identified two distinct elements at positions -71/-64 (TGACGTGA) and -129/-114 (CTCCAGCCTTGACCTT) that were both required for full basal reporter gene activity and for maximal induction by either cAMP or calcium-signaling pathways. The -71/-64 element, which resembles a consensus cAMP response element (CRE), bound CRE-binding proteins from H295R cell nuclear extracts as determined by electrophoretic mobility shift analysis. Analysis of the -129/-114 element using electrophoretic mobility shift analysis demonstrated binding of the orphan nuclear receptors steroidogenic factor 1 and chicken ovalbumin upstream promoter transcription factor. These data demonstrate that ANG II, K+, and cAMP-signaling pathways utilize the same SF-1 and CRE-like cis-elements to regulate human CYP11B2 expression.

Janus Kinase 2 and Calcium Are Required for Angiotensin II-dependent Activation of Steroidogenic Acute Regulatory Protein Transcription in H295R Human Adrenocortical Cells

“…Similar results for a lack of an effect of phorbol ester treatment on CYP11B2 expression in H295R cells have been reported (38). We now have addressed the question of whether Ang II-dependent PKC activation contributes to StAR expression.…”

“…1C) levels in the cell medium, was correspondingly increased by agonist treatment as well. Aldosterone was increased to a greater degree than was progesterone by Ang II treatment, and this is likely the result of the stimulatory actions of Ang II on aldosterone synthase expression (37,38). For the remainder of the studies, progesterone served as the indicator of increased (or decreased) steroidogenic activity.…”

Genetic variation in aldosterone synthase predicts plasma glucose levels

“…Deletion of this region, however, did not affect transcription of the aldosterone synthase Table 5. Aldosterone synthase genotypes and fasting glucose levels by ethnicity, geographical origin, medication status, and within siblingships gene in in vitro experiments (25). This SNP was associated also with plasma and urinary aldosterone levels (26)(27)(28)(29)(30).…”

H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

“…Although the H295R cells increase aldosterone production in response to Ang II and potassium treatment , Clyne et al 1997, the cells have only moderate responsiveness to ACTH. Therefore, most experiments designed to examine the cAMP-dependent pathway require the addition of either forskolin or cAMP analogues (Rainey et al , 2004.…”