Angiotensin II-regulated transcription regulatory genes in adrenal steroidogenesis

Abstract

Transcription regulatory genes are crucial modulators of cell physiology and metabolism whose intracellular levels are tightly controlled in response to extracellular stimuli. We previously reported a set of 29 transcription regulatory genes modulated by angiotensin II in H295R human adrenocortical cells and their roles in regulating the expression of the last and unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways, 11β-hydroxylase and aldosterone synthase, respectively, using gene expression reporter assays. To study the effect of this set of transcription regulatory genes on adrenal steroidogenesis, H295R cells were transfected by high-efficiency nucleofection and aldosterone and cortisol were measured in cell culture supernatants under basal and angiotensin II-stimulated conditions. BCL11B, BHLHB2, CITED2, ELL2, HMGA1, MAFF, NFIL3, PER1, SERTAD1, and VDR significantly stimulated aldosterone secretion, while EGR1, FOSB, and ZFP295 decreased aldosterone secretion. BTG2, HMGA1, MITF, NR4A1, and ZFP295 significantly increased cortisol secretion, while BCL11B, NFIL3, PER1, and SIX2 decreased cortisol secretion. We also report the effect of some of these regulators on the expression of endogenous aldosterone synthase and 11β-hydroxylase under basal and angiotensin II-stimulated conditions. In summary, this study reports for the first time the effects of a set of angiotensin II-modulated transcription regulatory genes on aldosterone and cortisol secretion and the expression levels of the last and unique enzymes of the mineralocorticoid and glucocorticoid biosynthetic pathways. Abnormal regulation of mineralocorticoid or glucocorticoid secretion is involved in several pathophysiological conditions. These transcription regulatory genes may be involved in adrenal steroidogenesis pathologies; thus they merit additional study as potential candidates for therapeutic intervention.

Fig. 1.

Effect of transcription regulatory genes on aldosterone (A) and cortisol (B) secretion. H295R cells were transfected with transcription regulatory gene-expressing plasmids or control plasmid. Cells were incubated under basal or angiotensin II (ANG II; 10 nM)-stimulated conditions for 24 h. Aldosterone and cortisol were measured by ELISA in cell culture supernatants. Data are expressed as fold induction compared with control plasmid. Transfection experiments were performed in duplicate with 3 independent plasmid DNA preparations at least 3 times. *P < 0.05 vs. Control.

Fig. 2.

Effect of transcription regulatory genes on endogenous aldosterone synthase expression. H295R cells were transfected with transcription regulatory gene-expressing plasmids or control plasmid and stimulated with ANG II (10 nM) for 3 or 12 h, and then aldosterone synthase expression was quantified by real-time RT-PCR. Transfection experiments were performed in triplicate with 3 independent plasmid DNA preparations at least 3 times. *P < 0.05 vs. Control. AU, arbitrary unit.

Fig. 3.

Effect of transcription regulatory genes on endogenous 11β-hydroxylase expression. H295R cells were transfected with transcription regulatory gene-expressing plasmids or control plasmid and stimulated with ANG II (10 nM) for 3 or 12 h, and then 11β-hydroxylase expression was quantified by real-time RT-PCR. Transfection experiments were performed in triplicate with 3 independent plasmid DNA preparations at least 3 times. *P < 0.05 vs. Control.