Increased constitutive [alpha]SMA and Smad2/3 expression in idiopathic pulmonary fibrosis myofibroblasts is KCa 3.1-dependent

Abstract

Doc number: 155

Abstract

Background: Idiopathic pulmonary fibrosis is a common and invariably fatal disease with limited therapeutic options. Ca²⁺ -activated K_Ca 3.1 potassium channels play a key role in promoting TGFβ1 and bFGF-dependent profibrotic responses in human lung myofibroblasts (HLMFs). We hypothesised that K_Ca 3.1 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 K_Ca 3.1 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 K_Ca 3.1 ion channels with selective K_Ca 3.1 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 K_Ca 3.1-dependent processes facilitate "constitutive" Smad2/3 signalling in IPF-derived fibroblasts, and thus promote fibroblast to myofibroblast differentiation. Importantly, inhibiting K_Ca 3.1 channels reverses this process. Targeting K_Ca 3.1 may therefore provide a novel and effective approach for the treatment of IPF and there is the potential for the rapid translation of K_Ca 3.1-directed therapy to the clinic.