K Ca 3.1 mediates αSMA expression in human lung myofibroblast via the Smad2/3 and TGFβ1 signalling pathway

Introduction Idiopathic pulmonary fibrosis (IPF) is a common and invariably lethal interstitial lung disease with no effective therapy. K Ca 3.1 ion channel blockade inhibits several TGFβ1-dependent cell processes in human lung myofibroblasts (HLMFs), including contraction and collagen secretion. We hypothesised that K Ca 3.1 channel-dependent cell processes regulate HLMF αSMA expression via TGFβ1 signalling pathways. Methods HLMFs grown in vitro were examined for αSMA expression by immunofluorescence (IF), RT-PCR and flow cytommetry. Two specific and distinct K Ca 3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100nM) were used to determine their effects on TGFβ1-dependent processes. Expression of phosphorylated and total Smad 2/3 following TGFβ1 stimulation were examined by western blot and Smad 2/3 nuclear translocation examined by immunofluorescence. Results Constitutive αSMA expression in HLMFs was inhibited by the TRAM-34 and ICA-17043 (P<0.0001). TGFβ1-dependent increases in αSMA protein and mRNA expression in HLMFs were also inhibited by K Ca 3.1 channel blockers . TGFβ1- stimulated cells also showed increased mRNA expression of collagen type I (P=0.0378), and K Ca 3.1 (P=0.0313), which were inhibited by TRAM-34 and ICA-17043. After 1 hour of TGFβ1 stimulation, both phosphorylation of Smad 2/3 and Smad 2/3 nuclear translocation were inhibited by K Ca 3.1 channel block. Conclusions K Ca 3.1 activity promotes αSMA expression and myofibroblast differentiation through the TGFβ1/Smad signaling pathway. K Ca 3.1 blockers may offer a novel approach to treating IPF with the potential for rapid translation to the clinic. This work was funded by the The Dunhill Medical Trust.