Regulation of Aldosterone Synthase by Activator Transcription Factor/cAMP Response Element-Binding Protein Family Members

Abstract

Aldosterone synthesis is regulated by angiotensin II (Ang II) and K(+) acting in the adrenal zona glomerulosa, in part through the regulation of aldosterone synthase (CYP11B2). Here, we analyzed the role of cAMP response element (CRE)-binding proteins (CREBs) in the regulation of CYP11B2. Expression analysis of activator transcription factor (ATF)/CREB family members, namely the ATF1 and ATF2, the CREB, and the CRE modulator, in H295R cells and normal human adrenal tissue was performed using quantitative real-time PCR. Ang II-induced phosphorylation of ATF/CREB members was analyzed by Western blot analysis, and their subsequent binding to the CYP11B2 promoter using chromatin immunoprecipitation assay. Aldosterone production and CYP11B2 expression were measured in small interfering RNA-transfected cells to knockdown the expression of ATF/CREB members. CYP11B2 promoter activity was measured in H295R cells cotransfected with NURR1 (NR4A2) alone or with constitutively active vectors for ATF/CREB members. Ang II induced phosphorylation of ATF1, ATF2, and CRE modulator in a time-dependent manner. Based on chromatin immunoprecipitation analysis, there was an increased association of these proteins with the CYP11B2 promoter after Ang II and K(+) treatment. Phosphorylated ATF/CREB members also bound the CYP11B2 promoter. Knockdown of ATF/CREB members reduced Ang II and K(+) induction of adrenal cell CYP11B2 mRNA expression and aldosterone production. The constitutively active ATF/CREB vectors increased the promoter activity of CYP11B2 and had a synergistic effect with NURR1. In summary, these results suggest that ATF/CREB and NGFI-B family members play a crucial role in the transcriptional regulation of CYP11B2 and adrenal cell capacity to produce aldosterone.

Stimulus-selective induction of the orphan nuclear receptor NGFIB underlies different influences of angiotensin II and potassium on the human adrenal gland zona glomerulosa-specific 3β-HSD isoform gene expression in adrenocortical H295R cells

“…7C). As previously shown by Nogueira & Rainey [8], both AngII and K + significantly increased the levels of ATF2 recruitment to the promoter region of CYP11B2 (~3.2-and ~2.7-fold over vehicle control, respectively), confirming that the CYP11B2 CRE sequence analysis (Fig. 5B) and found that K + treatment failed to induce detectable protein expression of NGFIB, NURR1, and NOR1.…”

“…It has been well established that AngII and K + both activate transcription of CYP11B2 through a common cis-regulatory element [7]. The CYP11B2 promoter contains a Ca 2+ /cAMP-response element (CRE) to which the CREB/ATF family proteins bind in response to AngII and K + [8]. Thus, AngII and K + are thought to share, at least in part, the CREB/ ATF-mediated transcription pathway.…”

“…6). ATF2 was analyzed here because it has been reported [8] that in H295R cells mRNA levels of ATF2 are relatively high (7.8 nmol/mol) compared to those of the other members, such as CREB (3.2 × 10 signals from AngII and K + are not identical; a different set of genes was reported to be induced in response to AngII and K + in H295R cells [21], although the underlying mechanism(s) remains unknown. In the present study, we showed that the NGFIB/NURR1 pathway leading to HSD3B1 expression is only operative for AngII.…”

Angiotensin II Triggers Expression of the Adrenal Gland Zona Glomerulosa-Specific 3β-Hydroxysteroid Dehydrogenase Isoenzyme through De Novo Protein Synthesis of the Orphan Nuclear Receptors NGFIB and NURR1

“…Because H295R is a very difficult cell line to transfect (41,42), electroporation was performed with the Nucleofector system (Amaxa Biosystems) for efficient introduction of siRNAs (43,44). We observed that electroporation of siRNA mixtures, each directed against NGFIB or NURR1, led to a specific knockdown of the target gene without any apparent off-target cross-reactions between the members of the NGFIB family (Fig.…”

Antiaging Gene Klotho Regulates Adrenal CYP11B2 Expression and Aldosterone Synthesis

“…However, ATF2 serves as a positive transcription factor that increases CYP11B2 expression. 20 Activation of ATF2 requires phosphorylation. Silencing of KL increased phosphorylation of ATF2 ( Figure 7B), which also could contribute to upregulation of CYP11B2 expression (Figure 6, D-F).…”