title: Human lung myofibroblast TGFβ1-dependent Smad2/3 signalling is Ca(2+)-dependent and regulated by KCa3.1 K(+) channels.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  KCa3.1 block attenuated TGFβ1-dependent Smad2/3 phosphorylation and nuclear translocation, and this was mimicked by lowering the extracellular Ca(2+) 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.
  
  
  CONCLUSIONS
  KCa3.1 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.
authors.affiliation: Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Glenfield Hospital, Groby Road, Leicester, LE3 9QP UK.
authors.forename: Katy M
authors.initials: KM
authors.lastname: Roach
authors.affiliation: Department of Medicine, Division of Rheumatology and Immunology, University of South Carolina, Columbia, SC 29208 USA.
authors.forename: Carol
authors.initials: C
authors.lastname: Feghali-Bostwick
authors.affiliation: Department of Pharmacology, University of California, 451 Health Sciences Drive, Davis, CA 95616 USA.
authors.forename: Heike
authors.initials: H
authors.lastname: Wulff
authors.affiliation: Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Glenfield Hospital, Groby Road, Leicester, LE3 9QP UK.
authors.forename: Yassine
authors.initials: Y
authors.lastname: Amrani
authors.affiliation: Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Glenfield Hospital, Groby Road, Leicester, LE3 9QP UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Bradding
keywords: Human lung myofibroblast; Idiopathic pulmonary fibrosis; Potassium channel KCa3.1
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