Human lung myofibroblast TGF beta 1-dependent Smad2/3 signalling is Ca2+-dependent and regulated by K(Ca)3.1 K+ channels

Background: Idiopathic pulmonary fibrosis (IPF) is a common and invariably lethal interstitial lung disease with poorly effective therapy. Blockade of the K+ channel K(Ca)3.1 reduces constitutive alpha-SMA and Smad2/3 nuclear translocation in IPF-derived human lung myofibroblasts (HLMFs), and inhibits several transforming growth factor beta 1 (TGF beta 1)-dependent cell processes. We hypothesized that K(Ca)3.1-dependent cell processes also regulate the TGF beta 1-dependent Smad2/3 signalling pathway in HLMFs. HLMFs obtained from non-fibrotic controls (NFC) and IPF lungs were grown in vitro and examined for alpha SMA expression by immunofluorescence, RT-PCR, and flow cytometry. Two specific and distinct K(Ca)3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on TGF beta 1-dependent signalling. Expression of phosphorylated and total Smad2/3 following TGF beta 1 stimulation was determined by Western blot and Smad2/3 nuclear translocation by immunofluorescence. Results: K(Ca)3.1 block attenuated TGF beta 1-dependent Smad2/3 phosphorylation and nuclear translocation, and this was mimicked by lowering the extracellular Ca2+ concentration. K(Ca)3.1 block also inhibited Smad2/3-dependent gene transcription (alpha SMA, collagen type I), inhibited K(Ca)3.1 mRNA expression, and attenuated TGF beta 1-dependent alpha SMA protein expression. Conclusions: K(Ca)3.1 activity regulates TGF beta 1-dependent effects in NFC-and IPF-derived primary HLMFs through the regulation of the TGF beta 1/Smad signalling pathway, with promotion of downstream gene transcription and protein expression. K(Ca)3.1 blockers may offer a novel approach to treating IPF.