Abstract
BackgroundIdiopathic pulmonary fibrosis (IPF) is a common and invariably lethal interstitial lung disease with poorly effective therapy. Blockade of the K+ channel KCa3.1 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 KCa3.1-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 KCa3.1 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.ResultsKCa3.1 block attenuated TGFβ1-dependent Smad2/3 phosphorylation and nuclear translocation, and this was mimicked by lowering the extracellular Ca2+ concentration. KCa3.1 block also inhibited Smad2/3-dependent gene transcription (αSMA, collagen type I), inhibited KCa3.1 mRNA expression, and attenuated TGFβ1-dependent αSMA protein expression.ConclusionsKCa3.1 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. KCa3.1 blockers may offer a novel approach to treating IPF.Electronic supplementary materialThe online version of this article (doi:10.1186/s13069-015-0022-0) contains supplementary material, which is available to authorized users.
Highlights
Idiopathic pulmonary fibrosis (IPF) is a common and invariably lethal interstitial lung disease with poorly effective therapy
Transforming growth factor beta 1 (TGFβ1)-dependent Smad2/3 phosphorylation is attenuated by KCa3.1 blockade The ability of KCa3.1 blockers to attenuate several TGFβ1-dependent cell processes in Human lung myofibroblast (HLMF) [29] suggests that KCa3.1 potentially regulates the activity of TGFβ1dependent transcription factors
No significant differences in Smad2/3 phosphorylation were seen between non-fibrotic controls (NFC) and IPF data, and data were pooled for statistical analysis here and elsewhere where no disease-related differences were evident
Summary
Idiopathic pulmonary fibrosis (IPF) is a common and invariably lethal interstitial lung disease with poorly effective therapy. Blockade of the K+ channel KCa3.1 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. Phosphorylated Smad and Smad form hetero-oligomeric complexes with Smad and translocate to the nucleus to regulate gene expression through transcription and mediate the biological effects of TGFβ1 such as cell growth, differentiation and contraction [20,21,22]. This TGFβ1/Smad pathway contributes to myofibroblast differentiation by increasing α-smooth muscle actin expression [20,21]
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