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Title: Increased constitutive αSMA and Smad2/3 expression in idiopathic pulmonary fibrosis myofibroblasts is KCa3.1-dependent.
Authors: Roach, Katy M.
Wulff, H.
Feghali-Bostwick, C.
Amrani, Yassine
Bradding, Peter
First Published: 5-Dec-2014
Publisher: BioMed Central
Citation: Respir Res, 2014, 15:155
Abstract: Background Idiopathic pulmonary fibrosis is a common and invariably fatal disease with limited therapeutic options. Ca2+-activated KCa3.1 potassium channels play a key role in promoting TGFβ1 and bFGF-dependent profibrotic responses in human lung myofibroblasts (HLMFs). We hypothesised that KCa3.1 channel-dependent cell processes regulate HLMF αSMA expression via Smad2/3 signalling pathways. Methods In this study we have compared the phenotype of HLMFs derived from non-fibrotic healthy control lungs (NFC) with cells derived from IPF lungs. HLMFs grown in vitro were examined for αSMA expression by immunofluorescence (IF), RT-PCR and flow cytommetry. Basal Smad2/3 signalling was examined by RT-PCR, western blot and immunofluorescence. Two specific and distinct KCa3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on HLMF differentiation and the Smad2/3 signalling pathways. Results IPF-derived HLMFs demonstrated increased constitutive expression of both α-smooth muscle actin (αSMA) and actin stress fibres, indicative of greater myofibroblast differentiation. This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation. The increased Smad2/3 nuclear localisation was inhibited by removing extracellular Ca2+ or blocking KCa3.1 ion channels with selective KCa3.1 blockers (TRAM-34, ICA-17043). This was accompanied by de-differentiation of IPF-derived HLMFs towards a quiescent fibroblast phenotype as demonstrated by reduced αSMA expression and reduced actin stress fibre formation. Conclusions Taken together, these data suggest that Ca2+- and KCa3.1-dependent processes facilitate “constitutive” Smad2/3 signalling in IPF-derived fibroblasts, and thus promote fibroblast to myofibroblast differentiation. Importantly, inhibiting KCa3.1 channels reverses this process. Targeting KCa3.1 may therefore provide a novel and effective approach for the treatment of IPF and there is the potential for the rapid translation of KCa3.1-directed therapy to the clinic.
DOI Link: 10.1186/s12931-014-0155-5
ISSN: 1465-9921
eISSN: 1465-993X
Version: Publisher Version
Status: Peer-reviewed
Type: Journal Article
Rights: © 2014 Roach et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.
Description: PMCID: PMC4263015
Appears in Collections:Published Articles, Dept. of Infection, Immunity and Inflammation

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