RESUMO
The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl- ) accumulation. The anion Cl- , acting as a second messenger, stimulates the secretion of interleukin-1ß (IL-1ß), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl- concentration, showing maximal expression and activity at 75 mM Cl- , in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl- . The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl- -stimulated IL-1ß mRNA expression and partially the IL-1ß secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl- , a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1ß mRNA response to Cl- and the IL-1ß secretion, interrupting the autocrine IL-1ß loop. The results suggest that Cl- effects are mediated by SGK1, in which under Cl- modulation stimulates the secretion of mature IL-1ß, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1ß itself, through autocrine signalling.
Assuntos
Caspase 1/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Interleucina-1beta/metabolismo , Espaço Intracelular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Dipeptídeos/farmacologia , Retroalimentação Fisiológica , Furanos/farmacologia , Humanos , Proteínas Imediatamente Precoces/genética , Indenos/farmacologia , Interleucina-1beta/genética , Mutação/genética , Nigericina/farmacologia , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Sulfonamidas/farmacologia , para-Aminobenzoatos/farmacologiaRESUMO
Cystic fibrosis (CF) is caused by mutations in the CFTR gene, which encodes a cAMP-regulated chloride channel. Several cellular functions are altered in CF cells. However, it is not clear how the CFTR failure induces those alterations. We have found previously several genes differentially expressed in CF cells, including c-Src, MUC1, MTND4, and CISD1 (CFTR-dependent genes). Recently, we also reported the existence of several chloride-dependent genes, among them GLRX5 and RPS27. Here, varying the intracellular chloride concentration [Cl- ]i of IB3-1 CF bronchial epithelial cells, we show that IL-1ß mRNA expression and secretion are also under Cl- modulation. The response to Cl- is biphasic, with maximal effects at 75 mM Cl- . The regulation of the IL-1ß mRNA expression involves an IL-1ß autocrine effect, since in the presence of the IL-1ß receptor antagonist IL1RN or anti-IL-1ß blocking antibody, the mRNA response to Cl- disappeared. Similar effects were obtained with the JNK inhibitor SP600125, the c-Src inhibitor PP2 and the IKK inhibitor III (BMS-345541). On the other hand, the IL-1ß secretion is still modulated by Cl- in the presence of IL-1RN, IL-1ß blocking antibody, or cycloheximide, suggesting that Cl- is affecting the IL-1ß maturation/secretion, which in turn starts an autocrine positive feedback loop. In conclusion, the Cl- anion acts as a second messenger for CFTR, modulating the IL-1ß maturation/secretion. The results also imply that, depending on its intracellular concentration, Cl- could be a pro-inflammatory mediator. J. Cell. Biochem. 118: 2131-2140, 2017. © 2016 Wiley Periodicals, Inc.