Intracellular Chloride Concentration Changes Modulate IL-1β Expression and Secretion in Human Bronchial Epithelial Cultured Cells

Abstract

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 ] of IB3-1 CF bronchial epithelial cells, we show that IL-1β 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-1β mRNA expression involves an IL-1β autocrine effect, since in the presence of the IL-1β receptor antagonist IL1RN or anti-IL-1β 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-1β secretion is still modulated by Cl in the presence of IL-1RN, IL-1β blocking antibody, or cycloheximide, suggesting that Cl is affecting the IL-1β 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-1β 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.

The chloride anion as a signalling effector

“…These studies clearly demonstrate a proinflammatory effect of NaCl although the specific effects of Cl − or Na + were not addressed. However, they agree with recent observations that Cl − by itself can induce IL‐1β secretion and autocrine signalling (Clauzure et al ., ), and with previous studies showing that mutant CFTR accumulates in the ER, leading to ER stress and Ca 2+ release, with deletion of phenylalanine 508 (ΔF508)‐CFTR as a Cl − counter‐ion channel, culminating in IL‐8 release (Barro Soria et al ., ; Martins et al ., ). Counter‐ion effects of Cl − were originally observed for ER Ca 2+ release from skeletal muscle (Fink & Veigel, ), and more recently in endosomes during the activation of NOX1 through the chloride channel CLC‐3 (Miller Jr. et al ., ), and in the activation of the TRPC6 Ca 2+ channel (Riazanski et al ., ), where Cl − , through the CFTR channel, shunts the transmembrane potential generated by movement of protons.…”

“…It was not clear how reduced [Cl − ] i could have the same effect as increased [Cl − ] i . It is possible that JNK has a biphasic response to Cl − , with maximal activity at intermediate [Cl − ] – a pattern known for interleukin‐1β (IL‐1β), which also has a biphasic response to Cl − (Clauzure et al ., ).…”

“…Zhang et al . () recently showed that serum glucocorticoid regulated kinase 1 (SGK1) activity is under Cl − modulation and that this kinase might be involved in the mechanism by which Cl − acts as a proinflammatory signal (Clauzure et al ., ; Zhang et al ., ). Intriguingly, increasing Cl − concentration has opposing effects on WNK1 and SGK1 activities, decreasing the activity of WNK1 and increasing that of SGK1.…”

“…Using this strategy, we found several differentially expressed mRNAs and characterized two of these, corresponding to RPS27 and GLRX5 (Valdivieso et al ., ). We also demonstrated that IL‐1β is a Cl − ‐dependent gene (Clauzure et al ., ; Massip‐Copiz et al ., ). Our results demonstrated the existence of numerous Cl − ‐dependent genes, and that changes in [Cl − ] i could, directly or indirectly, modulate the expression of specific genes in mammalian cells.…”

“…We have recently identified additional steps in CFTR signalling mechanisms that involve Cl − (Valdivieso et al ., , ), IL‐1β (Clauzure et al ., ; Massip‐Copiz et al ., , ) and c‐Src (Massip‐Copiz et al ., , ), which may explain the reduced mitochondrial Complex I activity (Valdivieso, ; Valdivieso et al ., ; Clauzure et al ., ) and increased ROS levels found in cultured CF cells (Clauzure et al ., , ; Massip Copiz & Santa Coloma, ; Massip‐Copiz et al ., ). Cl − modulates IL‐1β mRNA and protein expression in a biphasic way, with maximal expression at 75 mM [Cl − ] i in the presence of nigericin and tributyltin (Clauzure et al ., ). We postulate that this is the result of two steps: ( i ) Δ[Cl − ] i modulates IL‐1β secretion; and ( ii ) the secreted IL‐1β initiates an autocrine positive‐feedback loop, increasing its own mRNA levels and the synthesis of immature pro‐IL‐1β, which in turn is cleaved and secreted to start a new cycle of IL‐1R stimulation, resulting in signal amplification.…”