Inflammasomes are multiprotein complexes that sense pathogen-associated and danger-associated molecular patterns and induce inflammation in cells. The NALP3 inflammasome is tightly regulated by recently discovered control mechanisms, but other modulators still remain to be characterized. NLR family CARD-containing 3 (NLRC3) protein, a caspase recruitment domain (CARD)-containing member of the nucleotide oligomerization domain-like receptor (NLR) family, was found to down-regulate the NF-κB pathway and stimulator of interferon genes (STING)-dependent cytokine secretion. However, the effect of NLRC3 on the NALP3 inflammasome or other inflammasomes is still unknown. We hypothesized that NLRC3 might inhibit NALP3 inflammasome complex assembly. Toward this end, we tested whether NLRC3 overexpression or knockdown influences NALP3 activity in human monocyte and HEK293FT cells when the complex is ectopically reconstituted. We found that NLRC3 indeed decreases NALP3-induced IL-1β maturation and secretion, pro-caspase-1 cleavage, and speck formation by apoptosis-associated speck-like protein containing a CARD (ASC) protein in response to NALP3 activators. We also show that endogenous NLRC3 interacts with both ASC and pro-caspase-1 but not with NALP3, disrupts ASC speck formation through its CARD, and impairs the ASC and pro-caspase-1 interaction. Moreover, the NLRC3 CARD alone could dampen IL-1β secretion and ASC speck formation induced by NALP3 mutants associated with autoinflammatory diseases. In conclusion, we show here that, besides its role in the inhibition of the NF-κB pathway, NLRC3 interferes with the assembly and activity of the NALP3 inflammasome complex by competing with ASC for pro-caspase-1 binding.

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.

The mineralocorticoid aldosterone promotes renal K+ excretion and Na+ reabsorption; thereby it is critical for the regulation of ion homeostasis and blood pressure. Interestingly, immunosuppression therapy 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‐beta subunit 1 (CnB1) 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 in mouse and human adrenal pieces, ex vivo. Moreover, high K+ diet‐dependent increase in aldosterone production was blunted in wild type mice treated with tacrolimus. Furthermore, male ZG‐CnB1‐KO mice fed a high K+ diet exhibited a decreased aldosterone excretion compared to control animals while ZG‐CnB1‐KO females became hyperkalemic. Using a phosphoproteomics analysis, we identified the nuclear factor of activated T‐cells, cytoplasmic 4 (NFATc4) as a downstream factor regulated by Cn. The genetic deletion of NFATc4 in NCI‐H295R cells reduced the basal expression of CYP11B2 and blunted the K+‐stimulated expression of this gene. Conversely, the expression of a constitutively active form of NFATc4 in NCI‐H295R cells drastically increased the expression of CYP11B2 which remained unaltered upon treatment with K+ or tacrolimus. Altogether, our data indicate that the calcineurin‐NFATc4 pathway is activated by K+ to promote adrenal aldosterone production. Moreover, our data indicate that insufficient aldosterone production contributes to Cn inhibitor‐induced hyperkalemia.

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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 Ca2+/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.