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.

Figure 1

Relative role of NBRE and CRE DNA sequences in the regulation of CYP11B2 promoter activity. Analysis of the role of cis-elements present in the human CYP11B2 promoter. H295R transfected with vectors for WT CYP11B2 promoter linked to a luciferase reporter (−1521 wt), CRE mutated sequence (−1521 CREm), NBRE mutated sequence (−1521 NBREm), or CRE + NBRE mutated sequence (−1521 CREm/NBREm). Luciferase activity was compared in transfected cells treated with Ang II or K⁺. Data are expressed as a percentage of the basal for each individual vector. *, P < 0.05 (mutant vs. WT). §, P < 0.05 (basal vs. treatment with Ang II or K⁺). Results represent the mean ± sem from three independent experiments. P < 0.05 was considered significant.

Figure 2

Ang II-induced phosphorylation of ATF/CREB family members in human adrenocortical cells. H295R cells were treated for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 9 h, 12 h, and 24 h with 10 nm Ang II. Western blot analysis revealed time-dependent phosphorylation of ATF1 (panel A), ATF2 (panel B), and CREM (panel C). Western blot analysis of total protein for each individual transcription factor was used to normalize our results. Results represent the mean ± sem for five independent experiments.

Figure 3

Analysis of ATF/CREB protein recruitment to the human CYP11B2 promoter. EMSA was performed using ³²P-labeled oligonucleotide probes containing the CRE site from sequence of human CYP11B2 gene (panel A). Lane 1, Radiolabeled probe alone (FP, free probe); lane 2, labeled probe incubated with nuclear extract from H295R cells; lane 3, nonradiolabeled self-competitor oligonucleotides; lanes 4–6, labeled probe incubated with in vitro prepared CREB, ATF1, and ATF2. ChIP was used to investigate the induction of binding of ATF/CREB members to the segment of human CYP11B2 containing the CRE site (panels B–D). Incubation of H295R cells for 1 h with Ang II and K⁺ induced binding of ATF1, ATF2, and CREM to the CYP11B2 promoter. Recruitment of phosphorylated ATF2 and ATF1/CREM (cross-reacting antibody against serine 133) to the promoter region of CYP11B2 is shown in panels E and F. Antiacetylated histone antibody was used for the positive controls, antirabbit IgG was added to negative controls, and 1% of the supernatant (chromatin in dilution buffer) was used for the input. *, P < 0.05 (compared with basal). Results represent the mean ± sem for three or more independent experiments. P < 0.05 was considered significant.

Figure 4

Effects of the knockdown of ATF/CREB transcription factors on CYP11B2 mRNA expression and aldosterone production. H295R cells transfected with siRNA for ATF1, ATF2, and CREM, were incubated with Ang II or K⁺. Western blot analysis was used to confirm the knockdown of these proteins in transfected cells (panels A–C). Real-time qPCR was used to measure expression of CYP11B2 (panel D). Aldosterone levels in the cell culture medium were determined by RIA, and values were normalized to the amount of protein in each well (panel E). *, P < 0.05 in siATFs/CREM vs. control (panels A–C) and siATF/CREB in Control vs. Ang II-treated conditions (panels D and E). §, P < 0.05 in siATF/CREM vs. control in Control and K⁺-treated cells (panels D and E). Results represent the mean ± sem for three or more independent experiments. P < 0.05 was considered significant.

Figure 5

Effects of the knockdown of NURR1 alone and in combination with ATF/CREM transcription factors on CYP11B2 expression. H295R cells transfected with siRNA for NURR1, ATF1, ATF2, and CREM, were incubated with Ang II or K⁺. Real-time PCR analysis was used to confirm the knockdown of NURR1 (panel A) and to measure its effect on CYP11B2 mRNA expression in transfected cells (panel B–D). *, P < 0.05 (siRNA transfected cells vs. control); §, P < 0.05 (combined siNURR1 + siATF/CREB vs. control siNURR1 or siATF/CREB alone). Results represent the mean ± sem for three independent experiments. P < 0.05 was considered significant.

Figure 6

Effects of constitutively active ATF/CREB family members and NURR1 on the promoter activity of CYP11B2. Results are expressed as fold increase over control. In panel A, gray bars represent CYP11B2 cotransfected with empty vector or with each ATF/CREB family member. Black bars represent CYP11B2 cotransfected with empty vector or each ATF/CREB family member in the presence of NURR1. *, P < 0.05 (ATF/CREB members vs. empty vector); §, P < 0.05 (ATF/CREB member + NURR1 vs. NURR1 alone). In panel B, white bars represent the basal promoter activity of intact and mutant CYP11B2, black bars represent such promoter activity in the presence of constitutively active ATF2, and the gray bars represent CYP11B2 (intact and mutant) promoter activity in the presence of NURR1. *, P < 0.05 (basal vs. ATF2 or NURR1); §, P < 0.05 (mutant vs. intact vector). The results represent the mean ± sem for four independent experiments. P < 0.05 was considered significant.