Abstract
BackgroundIdiopathic 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.MethodsIn 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.ResultsIPF-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.ConclusionsTaken 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.
Highlights
Idiopathic pulmonary fibrosis (IPF) has an unknown etiology [1] and is marked by progressive lung fibrosis leading to respiratory failure
IPF myofibroblasts have increased basal KCa3.1 expression Functional KCa3.1 channels are increased in IPF-derived human lung myofibroblast (HLMF) [22]
Smad2/3 expression is greater in IPF-derived HLMFs To elucidate the molecular mechanisms underlying the observed phenoytypic differences between non-fibrotic healthy control lungs (NFC)- and IPF-derived HLMFs we investigated the basal Smad2/3 content as Smad2/3 signalling is key for myofibroblast differentiation and α-smooth muscle actin (αSMA) gene transcription RT-PCR results confirmed that both Smad2 and Smad3 mRNA were significantly upregulated in IPF-derived HLMFs compared to NFC-derived cells, P = 0.0286 and P = 0.0286 respectively, Mann Whitney (Figure 3A and B)
Summary
Idiopathic pulmonary fibrosis (IPF) has an unknown etiology [1] and is marked by progressive lung fibrosis leading to respiratory failure. IPF evolves from dysfunctional interactions between the injured epithelium and fibroblasts which lead to pathologic lesions called fibroblast foci, which are comprised of activated myofibroblasts [13]. In their activated state, myofibroblasts are the primary cell responsible for the synthesis, secretion and remodelling of the extracellular matrix in IPF [14]. 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
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