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

Roach KM, Wulff H, Feghali-Bostwick C, Amrani Y, Bradding P - Respir. Res. (2014)

Bottom Line: 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.This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation.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.

View Article: PubMed Central - PubMed

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.

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HLMF αSMA stress fibres are significantly attenuated by KCa3.1 blockers. (A) TRAM-34 dose-dependently decreased the number of actin stress fibres (NFC n = 3 and IPF n = 3, data pooled for statistical analysis). (B) ICA-17043 dose-dependently decreased the number of actin stress fibres (NFC n = 5 and IPF n = 3, data pooled for statistical analysis). No difference in response to the KCa3.1 blockers were seen between NFC and IPF donors. Results are represented as mean ± SEM *P < 0.05, **P < 0.01 (2 Way ANOVA corrected by Sidaks multiple comparisons test).
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Fig7: HLMF αSMA stress fibres are significantly attenuated by KCa3.1 blockers. (A) TRAM-34 dose-dependently decreased the number of actin stress fibres (NFC n = 3 and IPF n = 3, data pooled for statistical analysis). (B) ICA-17043 dose-dependently decreased the number of actin stress fibres (NFC n = 5 and IPF n = 3, data pooled for statistical analysis). No difference in response to the KCa3.1 blockers were seen between NFC and IPF donors. Results are represented as mean ± SEM *P < 0.05, **P < 0.01 (2 Way ANOVA corrected by Sidaks multiple comparisons test).

Mentions: Following KCa3.1 inhibition the number of actin stress fibres in both NFC and IPF-derived HLMFs were dose-dependently inhibited by TRAM-34 (20 nM and 200 nM), P = 0.0235 and ICA-17043 (10 nM and 100 nM), P = 0.0095, (Figure 7A and B). Thus not only is cytosolic αSMA staining inhibited by KCa3.1 channel blockers but also αSMA stress fibres, suggesting a phenotypic transition from a myofibroblast into a more fibroblast-like phenotype.Figure 7


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

Roach KM, Wulff H, Feghali-Bostwick C, Amrani Y, Bradding P - Respir. Res. (2014)

HLMF αSMA stress fibres are significantly attenuated by KCa3.1 blockers. (A) TRAM-34 dose-dependently decreased the number of actin stress fibres (NFC n = 3 and IPF n = 3, data pooled for statistical analysis). (B) ICA-17043 dose-dependently decreased the number of actin stress fibres (NFC n = 5 and IPF n = 3, data pooled for statistical analysis). No difference in response to the KCa3.1 blockers were seen between NFC and IPF donors. Results are represented as mean ± SEM *P < 0.05, **P < 0.01 (2 Way ANOVA corrected by Sidaks multiple comparisons test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4263015&req=5

Fig7: HLMF αSMA stress fibres are significantly attenuated by KCa3.1 blockers. (A) TRAM-34 dose-dependently decreased the number of actin stress fibres (NFC n = 3 and IPF n = 3, data pooled for statistical analysis). (B) ICA-17043 dose-dependently decreased the number of actin stress fibres (NFC n = 5 and IPF n = 3, data pooled for statistical analysis). No difference in response to the KCa3.1 blockers were seen between NFC and IPF donors. Results are represented as mean ± SEM *P < 0.05, **P < 0.01 (2 Way ANOVA corrected by Sidaks multiple comparisons test).
Mentions: Following KCa3.1 inhibition the number of actin stress fibres in both NFC and IPF-derived HLMFs were dose-dependently inhibited by TRAM-34 (20 nM and 200 nM), P = 0.0235 and ICA-17043 (10 nM and 100 nM), P = 0.0095, (Figure 7A and B). Thus not only is cytosolic αSMA staining inhibited by KCa3.1 channel blockers but also αSMA stress fibres, suggesting a phenotypic transition from a myofibroblast into a more fibroblast-like phenotype.Figure 7

Bottom Line: 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.This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation.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.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus