Calcineurin-NFATc4 Pathway Is Activated Upon K(+)-stimulation of Adrenal Aldosterone Production

The mineralocorticoid aldosterone secreted by the adrenal zona glomerulosa (ZG) cells promote renal K(+) secretion and Na(+) reabsorption; thereby it is critical for the control of ion homeostasis and blood pressure. While the Ca(2+)/calmodulin-dependent protein kinase (CAMK) pathway regulating K(+) stimulated aldosterone production is well studied, little is known about the potentially involved phosphatases. Interestingly, immunosuppression therapy of transplanted patients with protein phosphatase 3 (calcineurin) inhibitors often results in rather low plasma aldosterone levels despite a concomitant hyperkalemia and hyperreninemia. Calcineurin (Cn) is a calcium and calmodulin-dependent protein phosphatase expressed in the adrenal cortex. We tested the hypothesis that Cn participates in the signal transduction pathway mediating the K(+)-dependent stimulation of aldosterone production. To address this question, we used the adrenocortical cell model NCI-H295R, mouse and human ex vivo adrenal preparations and a ZG-specific and inducible Cn knockout mouse model (ZG-CnB1-KO). Inhibition of Cn with tacrolimus abolished the K(+)-stimulated expression of CYP11B2 in NCI-H295R cell line as well as mouse and human adrenal pieces, ex vivo. Using a phosphoproteomics analysis, we identified nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) as a critical downstream factor mediating Cn function. In support of this result, genetic deletion of NFATc4 reduced the basal expression of CYP11B2 and impaired the K(+)-stimulated expression of this gene. Conversely, the expression of a constitutively active form of NFATc4 drastically increased the expression of CYP11B2 in NCI-H295R cells which remained unaltered upon treatment with K(+) or tacrolimus. Finally, preliminary experiments using ZG-CnB1-KO mice suggest that Cn deletion in the ZG blunts the increase in aldosterone excretion triggered by high K(+) diet. Altogether, our data indicate that Cn function is indispensable for the physiological regulation of aldosterone production. Moreover, Cn may represent a novel molecular target for the pharmacological treatment of primary aldosteronism.