Calcineurin regulates aldosterone production via dephosphorylation of NFATC4
Mesut Berber, Sining Leng, Agnieszka Wengi, Denise V. Winter, Alex Odermatt, Felix Beuschlein, Johannes Loffing, David T. Breault, David Penton
Mesut Berber, Sining Leng, Agnieszka Wengi, Denise V. Winter, Alex Odermatt, Felix Beuschlein, Johannes Loffing, David T. Breault, David Penton
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Research Article Endocrinology Metabolism

Calcineurin regulates aldosterone production via dephosphorylation of NFATC4

Abstract

The mineralocorticoid aldosterone, secreted by the adrenal zona glomerulosa (ZG), is critical for life, maintaining ion homeostasis and blood pressure. Therapeutic inhibition of protein phosphatase 3 (calcineurin, Cn) results in inappropriately low plasma aldosterone levels despite concomitant hyperkalemia and hyperreninemia. We tested the hypothesis that Cn participates in the signal transduction pathway regulating aldosterone synthesis. Inhibition of Cn with tacrolimus abolished the potassium-stimulated (K+-stimulated) expression of aldosterone synthase, encoded by CYP11B2, in the NCI-H295R human adrenocortical cell line as well as ex vivo in mouse and human adrenal tissue. ZG-specific deletion of the regulatory Cn subunit CnB1 diminished Cyp11b2 expression in vivo and disrupted K+-mediated aldosterone synthesis. Phosphoproteomics analysis identified nuclear factor of activated T cells, cytoplasmic 4 (NFATC4), as a target for Cn-mediated dephosphorylation. Deletion of NFATC4 impaired K+-dependent stimulation of CYP11B2 expression and aldosterone production while expression of a constitutively active form of NFATC4 increased expression of CYP11B2 in NCI-H295R cells. Chromatin immunoprecipitation revealed NFATC4 directly regulated CYP11B2 expression. Thus, Cn controls aldosterone production via the Cn/NFATC4 pathway. Inhibition of Cn/NFATC4 signaling may explain low plasma aldosterone levels and hyperkalemia in patients treated with tacrolimus, and the Cn/NFATC4 pathway may provide novel molecular targets to treat primary aldosteronism.

Authors

Mesut Berber, Sining Leng, Agnieszka Wengi, Denise V. Winter, Alex Odermatt, Felix Beuschlein, Johannes Loffing, David T. Breault, David Penton

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Figure 5

Active NFATC4 is crucial to drive CYP11B2 expression and aldosterone production in NCI-H295R cells.

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Active NFATC4 is crucial to drive CYP11B2 expression and aldosterone pro...
(A) Representative immunoblots (n = 3) showing NFATC4 in K+-stimulated control and NFATC4-KO NCI-H295R cells. (B) CYP11B2 expression (qPCR) in control and NFATC4-KO NCI-295R cells stimulated with 15 mM K+ in the presence or absence of tacrolimus. Statistical differences assessed by 2-way ANOVA with Tukey’s multiple-comparison posttest. (*P < 0.05, ****P < 0.0001; n = 3–6.) (C) Aldosterone concentration in the supernatant of control and NFATC4-KO NCI-295R cells stimulated with 15 mM K+ and 100 nM AngII for 48 hours assessed by ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS). The concentration of aldosterone in vehicle-treated cells was below the detection limit. Statistical differences assessed by 2-way ANOVA with Tukey’s multiple-comparison posttest. (****P < 0.0001; n = 4–5.) (D) CYP11B2 expression (qPCR) in NCI-H295R cells transiently overexpressing empty (mock), full-length NFATC4, and constitutively active NFATC4 (ΔNFATC4) plasmids, stimulated with 15 mM K+ in the presence or absence of tacrolimus (10 nM). Statistical differences assessed by 2-way ANOVA with Tukey’s multiple-comparison posttest. (*P < 0.05, **P < 0.01; n = 7–9.) (E) Heatmap of the most significantly changed transcripts due to deletion of NFATC4 or overexpression of ΔNFATC4 in NCI-H295R cells as assessed by RNA-Seq. (F) Representative gel electrophoresis showing CYP11B2 promoter enrichment (qPCR) assessed in sheared chromatin of ΔNFATC4-overexpressing cells pulled down with anti-T7 tag antibody or control IgG. In graph, ratio of anti-T7 ab to control IgG. Statistical differences assessed by Mann-Whitney test. (*P < 0.05; n = 6.) (G) Control and NFATC4-KO NCI-H295R cells were stimulated with 15 mM K+ in the presence or absence of tacrolimus (10 nM). NR4A2 expression levels were determined by qPCR. Statistical differences assessed by 2-way ANOVA with Tukey’s multiple-comparison posttest. (*P < 0.05; ***P < 0.001; ****P < 0.0001; n = 3–4.)