Human lung myofibroblast TGFβ1-dependent Smad2/3 signalling is Ca(2+)-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 α-SMA and Smad2/3 nuclear translocation in IPF-derived human lung myofibroblasts (HLMFs), and inhibits several transforming growth factor beta 1 (TGFβ1)-dependent cell processes. We hypothesized that K(Ca)3.1-dependent cell processes also regulate the TGFβ1-dependent Smad2/3 signalling pathway in HLMFs. HLMFs obtained from non-fibrotic controls (NFC) and IPF lungs were grown in vitro and examined for αSMA 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 TGFβ1-dependent signalling. Expression of phosphorylated and total Smad2/3 following TGFβ1 stimulation was determined by Western blot and Smad2/3 nuclear translocation by immunofluorescence. RESULTS: K(Ca)3.1 block attenuated TGFβ1-dependent Smad2/3 phosphorylation and nuclear translocation, and this was mimicked by lowering the extracellular Ca(2+) concentration. K(Ca)3.1 block also inhibited Smad2/3-dependent gene transcription (αSMA, collagen type I), inhibited K(Ca)3.1 mRNA expression, and attenuated TGFβ1-dependent αSMA protein expression. CONCLUSIONS: K(Ca)3.1 activity regulates TGFβ1-dependent effects in NFC- and IPF-derived primary HLMFs through the regulation of the TGFβ1/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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13069-015-0022-0) contains supplementary material, which is available to authorized users.