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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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Smad2/3 nuclear translocation is Ca2+dependent. (A) When HLMFs were incubated with Ca2+-free medium for 1 hour there was a significant reduction in the amount of Smad2/3 located in the nucleus, NFC n = 3 and IPF n = 3 (a minimum of 10 random cells were measured in one field for each donor, data pooled for NFC and IPF). (B) Representative immunofluorescent staining demonstrating the attenuated nuclear expression of Smad2/3 in Ca2+-free medium. Results are represented as mean ± SEM, *P < 0.05 (Paired t-test).
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Fig5: Smad2/3 nuclear translocation is Ca2+dependent. (A) When HLMFs were incubated with Ca2+-free medium for 1 hour there was a significant reduction in the amount of Smad2/3 located in the nucleus, NFC n = 3 and IPF n = 3 (a minimum of 10 random cells were measured in one field for each donor, data pooled for NFC and IPF). (B) Representative immunofluorescent staining demonstrating the attenuated nuclear expression of Smad2/3 in Ca2+-free medium. Results are represented as mean ± SEM, *P < 0.05 (Paired t-test).

Mentions: KCa3.1 channel blockade depolarises the plasma membrane and thereby reduces receptor-dependent rises in intracellular Ca2+ concentrations in many cell types including HLMFs [22,33-36]. If Ca2+ signalling is mechanistically important for the effects of KCa3.1 blockers on constitutive Smad2/3 nuclear translocation, lowering the extracellular Ca2+ concentration should also inhibit nuclear translocation. Incubating HLMFs for 1 hour in Ca2+-free media significantly reduced the amount of Smad2/3 in the nuclei compared to cells incubated in media containing normal external Ca2+ (1.8 mM), P = 0.0114 (Figure 5A and B). This suggests that the enhancement of Ca2+-influx by KCa3.1 channels is an essential requirement for the efficient nuclear translocation of Smad2/3 and subsequent transcription and expression of αSMA.Figure 5


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)

Smad2/3 nuclear translocation is Ca2+dependent. (A) When HLMFs were incubated with Ca2+-free medium for 1 hour there was a significant reduction in the amount of Smad2/3 located in the nucleus, NFC n = 3 and IPF n = 3 (a minimum of 10 random cells were measured in one field for each donor, data pooled for NFC and IPF). (B) Representative immunofluorescent staining demonstrating the attenuated nuclear expression of Smad2/3 in Ca2+-free medium. Results are represented as mean ± SEM, *P < 0.05 (Paired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Smad2/3 nuclear translocation is Ca2+dependent. (A) When HLMFs were incubated with Ca2+-free medium for 1 hour there was a significant reduction in the amount of Smad2/3 located in the nucleus, NFC n = 3 and IPF n = 3 (a minimum of 10 random cells were measured in one field for each donor, data pooled for NFC and IPF). (B) Representative immunofluorescent staining demonstrating the attenuated nuclear expression of Smad2/3 in Ca2+-free medium. Results are represented as mean ± SEM, *P < 0.05 (Paired t-test).
Mentions: KCa3.1 channel blockade depolarises the plasma membrane and thereby reduces receptor-dependent rises in intracellular Ca2+ concentrations in many cell types including HLMFs [22,33-36]. If Ca2+ signalling is mechanistically important for the effects of KCa3.1 blockers on constitutive Smad2/3 nuclear translocation, lowering the extracellular Ca2+ concentration should also inhibit nuclear translocation. Incubating HLMFs for 1 hour in Ca2+-free media significantly reduced the amount of Smad2/3 in the nuclei compared to cells incubated in media containing normal external Ca2+ (1.8 mM), P = 0.0114 (Figure 5A and B). This suggests that the enhancement of Ca2+-influx by KCa3.1 channels is an essential requirement for the efficient nuclear translocation of Smad2/3 and subsequent transcription and expression of αSMA.Figure 5

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