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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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IPF myofibroblasts have increased basal αSMA expression and stress fibre formation. (A) HLMF αSMA expression was measured by grey scale intensity in n = 7 NFC and n = 7 IPF donors; a minimum of 10 random cells were measured in one field for each donor. IPF HLMF’s had significantly higher intensity of αSMA in comparison to NFC donors P = 0.0111, Mann–Whitney. (B) Immunofluorescent images displaying αSMA staining and actin stress fibers in the cytoplasmic matrix. (C and D) Western blot analysis of αSMA expression in NFC (n = 3) and IPF-derived (n = 3) HLMFs, **P = 0.0026. The detected band was at the correct molecular weight of 42 kDa for αSMA (E and F) the mean fluorescent intensity (MFI) of αSMA expression assessed by flow cytometry. IPF (n = 4) donors showed significantly higher expression than NFC (n = 4), P = 0.0159. (G) Illustration of how the αSMA stress fibres were assessed. A macro recorded the number of fibres per individual cell. Each fibre represents a peak in grey value and the number of peaks equating to the number of stress fibres was then counted per cell. A minimum of 10 cells were measured per donor. (H) The results show that the number of actin stress fibres were significantly higher in IPF (n = 8) donors in comparison to NFC (n = 8) donors P = 0.0437 (Un-paired t-test). Results are presented as median ± IQR, #P < 0.05 (Mann–Whitney), or mean ± SEM *P < 0.05, **P < 0.01 (Unpaired t-test).
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Fig1: IPF myofibroblasts have increased basal αSMA expression and stress fibre formation. (A) HLMF αSMA expression was measured by grey scale intensity in n = 7 NFC and n = 7 IPF donors; a minimum of 10 random cells were measured in one field for each donor. IPF HLMF’s had significantly higher intensity of αSMA in comparison to NFC donors P = 0.0111, Mann–Whitney. (B) Immunofluorescent images displaying αSMA staining and actin stress fibers in the cytoplasmic matrix. (C and D) Western blot analysis of αSMA expression in NFC (n = 3) and IPF-derived (n = 3) HLMFs, **P = 0.0026. The detected band was at the correct molecular weight of 42 kDa for αSMA (E and F) the mean fluorescent intensity (MFI) of αSMA expression assessed by flow cytometry. IPF (n = 4) donors showed significantly higher expression than NFC (n = 4), P = 0.0159. (G) Illustration of how the αSMA stress fibres were assessed. A macro recorded the number of fibres per individual cell. Each fibre represents a peak in grey value and the number of peaks equating to the number of stress fibres was then counted per cell. A minimum of 10 cells were measured per donor. (H) The results show that the number of actin stress fibres were significantly higher in IPF (n = 8) donors in comparison to NFC (n = 8) donors P = 0.0437 (Un-paired t-test). Results are presented as median ± IQR, #P < 0.05 (Mann–Whitney), or mean ± SEM *P < 0.05, **P < 0.01 (Unpaired t-test).

Mentions: We and others have shown previously that fibroblasts grown from lung parenchyma express relatively high levels of αSMA and are contractile [22,29], in keeping with a myofibroblast phenotype. Here both NFC and IPF-derived HLMFs expressed αSMA protein, but this was significantly increased in IPF derived cells when assessed by immunofluorescent staining (P = 0.0111, Figure 1A and B), western blot analysis (P = 0.0026, Figure 1C and D) and flow cytometry, P = 0.0159 (Figures 1E and F).Figure 1


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)

IPF myofibroblasts have increased basal αSMA expression and stress fibre formation. (A) HLMF αSMA expression was measured by grey scale intensity in n = 7 NFC and n = 7 IPF donors; a minimum of 10 random cells were measured in one field for each donor. IPF HLMF’s had significantly higher intensity of αSMA in comparison to NFC donors P = 0.0111, Mann–Whitney. (B) Immunofluorescent images displaying αSMA staining and actin stress fibers in the cytoplasmic matrix. (C and D) Western blot analysis of αSMA expression in NFC (n = 3) and IPF-derived (n = 3) HLMFs, **P = 0.0026. The detected band was at the correct molecular weight of 42 kDa for αSMA (E and F) the mean fluorescent intensity (MFI) of αSMA expression assessed by flow cytometry. IPF (n = 4) donors showed significantly higher expression than NFC (n = 4), P = 0.0159. (G) Illustration of how the αSMA stress fibres were assessed. A macro recorded the number of fibres per individual cell. Each fibre represents a peak in grey value and the number of peaks equating to the number of stress fibres was then counted per cell. A minimum of 10 cells were measured per donor. (H) The results show that the number of actin stress fibres were significantly higher in IPF (n = 8) donors in comparison to NFC (n = 8) donors P = 0.0437 (Un-paired t-test). Results are presented as median ± IQR, #P < 0.05 (Mann–Whitney), or mean ± SEM *P < 0.05, **P < 0.01 (Unpaired t-test).
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Related In: Results  -  Collection

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Fig1: IPF myofibroblasts have increased basal αSMA expression and stress fibre formation. (A) HLMF αSMA expression was measured by grey scale intensity in n = 7 NFC and n = 7 IPF donors; a minimum of 10 random cells were measured in one field for each donor. IPF HLMF’s had significantly higher intensity of αSMA in comparison to NFC donors P = 0.0111, Mann–Whitney. (B) Immunofluorescent images displaying αSMA staining and actin stress fibers in the cytoplasmic matrix. (C and D) Western blot analysis of αSMA expression in NFC (n = 3) and IPF-derived (n = 3) HLMFs, **P = 0.0026. The detected band was at the correct molecular weight of 42 kDa for αSMA (E and F) the mean fluorescent intensity (MFI) of αSMA expression assessed by flow cytometry. IPF (n = 4) donors showed significantly higher expression than NFC (n = 4), P = 0.0159. (G) Illustration of how the αSMA stress fibres were assessed. A macro recorded the number of fibres per individual cell. Each fibre represents a peak in grey value and the number of peaks equating to the number of stress fibres was then counted per cell. A minimum of 10 cells were measured per donor. (H) The results show that the number of actin stress fibres were significantly higher in IPF (n = 8) donors in comparison to NFC (n = 8) donors P = 0.0437 (Un-paired t-test). Results are presented as median ± IQR, #P < 0.05 (Mann–Whitney), or mean ± SEM *P < 0.05, **P < 0.01 (Unpaired t-test).
Mentions: We and others have shown previously that fibroblasts grown from lung parenchyma express relatively high levels of αSMA and are contractile [22,29], in keeping with a myofibroblast phenotype. Here both NFC and IPF-derived HLMFs expressed αSMA protein, but this was significantly increased in IPF derived cells when assessed by immunofluorescent staining (P = 0.0111, Figure 1A and B), western blot analysis (P = 0.0026, Figure 1C and D) and flow cytometry, P = 0.0159 (Figures 1E and F).Figure 1

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