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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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KCa3.1 protein expression in HLMFs. (A) KCa3.1 protein expression in HLMFs was measured using the grey scale intensity and was significantly higher in IPF (n = 4) in comparison to NFC-derived cells (n = 4), mean ± SEM, *P < 0.05 (Un-paired t-test). (B) Examples of KCa3.1 immunofluorescent staining in both NFC and IPF donors. (C) The correlation between KCa3.1 and αSMA expression in HLMFs, r = 0.798, P = 0.0176. (D) The correlation between KCa3.1 and whole cell nuclear Smad expression in HLMFs, r = 0.925, p = 0.0029.
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Fig2: KCa3.1 protein expression in HLMFs. (A) KCa3.1 protein expression in HLMFs was measured using the grey scale intensity and was significantly higher in IPF (n = 4) in comparison to NFC-derived cells (n = 4), mean ± SEM, *P < 0.05 (Un-paired t-test). (B) Examples of KCa3.1 immunofluorescent staining in both NFC and IPF donors. (C) The correlation between KCa3.1 and αSMA expression in HLMFs, r = 0.798, P = 0.0176. (D) The correlation between KCa3.1 and whole cell nuclear Smad expression in HLMFs, r = 0.925, p = 0.0029.

Mentions: Functional KCa3.1 channels are increased in IPF-derived HLMFs [22]. We therefore examined the expression of KCa3.1 by immunofluorescence and its relationship to αSMA expression. We found that IPF-derived HLMFs had a higher intensity of immunostaining than NFC cells, (Figure 2A and B), in keeping with previous patch clamp electrophysiology data [22]. Furthermore, KCa3.1 expression correlated significantly with both basal αSMA expression, r = 0.79, P = 0.0176, and basal Smad2/3 localisation, r = 0.9245, P = 0.0029 in both NFC and IPF donors (Figure 2C and D).Figure 2


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)

KCa3.1 protein expression in HLMFs. (A) KCa3.1 protein expression in HLMFs was measured using the grey scale intensity and was significantly higher in IPF (n = 4) in comparison to NFC-derived cells (n = 4), mean ± SEM, *P < 0.05 (Un-paired t-test). (B) Examples of KCa3.1 immunofluorescent staining in both NFC and IPF donors. (C) The correlation between KCa3.1 and αSMA expression in HLMFs, r = 0.798, P = 0.0176. (D) The correlation between KCa3.1 and whole cell nuclear Smad expression in HLMFs, r = 0.925, p = 0.0029.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: KCa3.1 protein expression in HLMFs. (A) KCa3.1 protein expression in HLMFs was measured using the grey scale intensity and was significantly higher in IPF (n = 4) in comparison to NFC-derived cells (n = 4), mean ± SEM, *P < 0.05 (Un-paired t-test). (B) Examples of KCa3.1 immunofluorescent staining in both NFC and IPF donors. (C) The correlation between KCa3.1 and αSMA expression in HLMFs, r = 0.798, P = 0.0176. (D) The correlation between KCa3.1 and whole cell nuclear Smad expression in HLMFs, r = 0.925, p = 0.0029.
Mentions: Functional KCa3.1 channels are increased in IPF-derived HLMFs [22]. We therefore examined the expression of KCa3.1 by immunofluorescence and its relationship to αSMA expression. We found that IPF-derived HLMFs had a higher intensity of immunostaining than NFC cells, (Figure 2A and B), in keeping with previous patch clamp electrophysiology data [22]. Furthermore, KCa3.1 expression correlated significantly with both basal αSMA expression, r = 0.79, P = 0.0176, and basal Smad2/3 localisation, r = 0.9245, P = 0.0029 in both NFC and IPF donors (Figure 2C and D).Figure 2

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