THE CFTR CHLORIDE CHANNEL SIGNALING

TOMÁS A. SANTA-COLOMA; ÁNGEL G. VALDIVIESO; MARÍA M. MASSIP COPIZ; CONSUELO MORI; CRISTIAN ASENSIO; VERÓNICA SOTOMAYOR; MARIÁNGELES CLAUZURE; AGUSTINA VIDAL; IGNACIO VERGARA

Rio de Janeiro

Congreso; 38th World Congress of the International Union of Physiological Sciences (IUPS) ? Rhythms of Life; 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for the cystic fibrosis (CF) disease. The channel activity is up­regulated by cAMP, phosphorylation (PKA, PKC, c­Src) and ATP binding, and downregulated by WNK kinases. To explain the CF phenotype, after the CFTR cloning, initial studies focused in the non­genomic and extracellular effects of the CFTR failure. However, the phenotype was too complex to be explained only by the failure in the Cl­ transport and the accompanying Na+ and water permeation. Thus, two decades ago, we hypothesized that the complex CF phenotype might be the result of the expression of a net of CFTR­dependent genes, modulated by the activity of the CFTR channel (Cl­ transport). Applying differential display to CF cells, we found several CFTR­dependent genes, and characterized some of them, including c­Src, MUC1, MTND4 and CISD1. We then hypothesized that changes in the [Cl­]i could be the first step in the CFTR ?signaling mechanism?, with a role for Cl­ as a signaling effector or ?second messenger? in the modulation of CFTR and Cl­ specific genes. To demonstrate this hypothesis, we used again differential display and analyzed the mRNA expression pattern of IB3­1 cells, an immortalized human tracheobronchial epithelial cell line derived from a CF patient. The cells were incubated at different Cl­ concentrations, using nigericin and tributyltin to equilibrate the intracellular and extracellular Cl­ concentrations ([Cl­]i = [Cl­]e). We found several differentially expressed mRNAs and characterized two of them, the ribosomal protein S27 (RPS27) and glutaredoxin 5 (GLRX5). RPS27 was further analyzed. It responded to increased concentrations of CFTR inhibitors, which also produced a progressive Cl- accumulation. In parallel, we also found that IL­1β responded to changes in Cl­. Thus, the hypothesis of Cl­ acting as a second messenger for CFTR appear to be correct. We then identified additional steps in the CFTR signaling mechanisms that involve Cl­, IL­1β and c­Src that may explain the reduced mitochondrial Complex I activity and the increased reactive oxygen species (ROS) previously found in CF cells. In conclusion, the results suggest that Cl­ may act as a second messenger for CFTR, and that Cl- constitutes a proinflammatory stimulus, inducing IL­1β secretion and starting an autocrine, positive feedback loop, which in turn upregulates its own mRNA.