title: Increased constitutive αSMA and Smad2/3 expression in idiopathic pulmonary fibrosis myofibroblasts is KCa3.1-dependent.
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.
authors.forename: Katy M
authors.initials: KM
authors.lastname: Roach
authors.forename: Heike
authors.initials: H
authors.lastname: Wulff
authors.forename: Carol
authors.initials: C
authors.lastname: Feghali-Bostwick
authors.forename: Yassine
authors.initials: Y
authors.lastname: Amrani
authors.forename: Peter
authors.initials: P
authors.lastname: Bradding
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism*; D016022:Case-Control Studies; D002454:Cell Differentiation; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D058628:Myofibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010641:Phenotype; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D026902:Potassium Channel Blockers; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051901:Smad4 Protein