Increased constitutive αSMA and Smad2/3 expression in idiopathic pulmonary fibrosis myofibroblasts is KCa3.1-dependent

Respir Res. 2014 Dec 5;15(1):155. doi: 10.1186/s12931-014-0155-5.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics
  • Actins / metabolism*
  • Case-Control Studies
  • Cell Differentiation
  • Cells, Cultured
  • Gene Expression Regulation
  • Humans
  • Idiopathic Pulmonary Fibrosis / genetics
  • Idiopathic Pulmonary Fibrosis / metabolism*
  • Idiopathic Pulmonary Fibrosis / pathology
  • Intermediate-Conductance Calcium-Activated Potassium Channels / antagonists & inhibitors
  • Intermediate-Conductance Calcium-Activated Potassium Channels / metabolism*
  • Lung / drug effects
  • Lung / metabolism*
  • Lung / pathology
  • Myofibroblasts / drug effects
  • Myofibroblasts / metabolism*
  • Myofibroblasts / pathology
  • Phenotype
  • Potassium Channel Blockers / pharmacology
  • Signal Transduction
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism*
  • Smad4 Protein / genetics
  • Smad4 Protein / metabolism*

Substances

  • ACTA2 protein, human
  • Actins
  • Intermediate-Conductance Calcium-Activated Potassium Channels
  • KCNN4 protein, human
  • Potassium Channel Blockers
  • SMAD2 protein, human
  • SMAD4 protein, human
  • Smad2 Protein
  • Smad4 Protein