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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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Total and nuclear Smad2 and Smad3 expression is significantly higher in IPF-derived HLMFs. (A) Smad2 mRNA expression was examined by qRT-PCR in NFC (n = 4) and IPF (n = 4) myofibroblasts. IPF-derived HLMFs had significantly higher Smad2 mRNA at baseline in comparison to NFC-derived cells. (B) Similarly, Smad3 mRNA expression was significantly higher in IPF-derived HLMFs (n = 4) in comparison to NFC-derived cells (n = 4). (C) Smad3 mRNA expression was increased compared to Smad2 mRNA expression in both NFC and IPF-derived HLMFs. (D and E) Quantification of Western blot analysis showed that total Smad2/3 protein was increased in IPF-derived HLMFs in comparison to NFC-derived cells. Results were normalised to β-actin. (F) The proportion of total Smad2/3 nuclear staining to whole cell staining was measured in at least 10 cells in HLMFs from both NFC (n = 7) and IPF (n = 9) donors. IPF-derived HLMFs had a significantly higher proportion of Smad2/3 located in the nucleus compared to NFC-derived cells. (G) A representative illustration of total Smad2/3 expression assessed by immunofluorescence. (H) Representative Western blot analysis showing total Smad2/3 protein within the nucleus and cytoplasm of IPF and NFC-derived cells. TBP is localized to nuclear enriched fractions relative to cytoplasmic enriched fractions, similarly β-actin is localized to cytoplasmic enriched fractions relative to nuclear enriched fractions, N = nucleus, C = cytoplasm. Detected bands were at the correct molecular weight of 52 and 60 kDa for total Smad2 and Smad3 respectively, 38 kDa for TBP and 43 kDa for β-actin (I) Quantification of Western blot analysis confirmed that total Smad2/3 in the nuclear enriched fraction is significantly increased in IPF cells (n = 3) in comparison to NFC cells (n = 3). Results were normalized to TBP. (J) Similarly, total Smad2/3 in the cytoplasmic enriched fraction was increased in IPF cells (n = 3) in comparison to NFC-derived cells (n = 3). Results were normalized to β-actin. Results are represented as mean ± IQR #P < 0.05 (Mann Whitney) or mean ± SEM *P = 0.05 (Un-paired t-test).
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Fig3: Total and nuclear Smad2 and Smad3 expression is significantly higher in IPF-derived HLMFs. (A) Smad2 mRNA expression was examined by qRT-PCR in NFC (n = 4) and IPF (n = 4) myofibroblasts. IPF-derived HLMFs had significantly higher Smad2 mRNA at baseline in comparison to NFC-derived cells. (B) Similarly, Smad3 mRNA expression was significantly higher in IPF-derived HLMFs (n = 4) in comparison to NFC-derived cells (n = 4). (C) Smad3 mRNA expression was increased compared to Smad2 mRNA expression in both NFC and IPF-derived HLMFs. (D and E) Quantification of Western blot analysis showed that total Smad2/3 protein was increased in IPF-derived HLMFs in comparison to NFC-derived cells. Results were normalised to β-actin. (F) The proportion of total Smad2/3 nuclear staining to whole cell staining was measured in at least 10 cells in HLMFs from both NFC (n = 7) and IPF (n = 9) donors. IPF-derived HLMFs had a significantly higher proportion of Smad2/3 located in the nucleus compared to NFC-derived cells. (G) A representative illustration of total Smad2/3 expression assessed by immunofluorescence. (H) Representative Western blot analysis showing total Smad2/3 protein within the nucleus and cytoplasm of IPF and NFC-derived cells. TBP is localized to nuclear enriched fractions relative to cytoplasmic enriched fractions, similarly β-actin is localized to cytoplasmic enriched fractions relative to nuclear enriched fractions, N = nucleus, C = cytoplasm. Detected bands were at the correct molecular weight of 52 and 60 kDa for total Smad2 and Smad3 respectively, 38 kDa for TBP and 43 kDa for β-actin (I) Quantification of Western blot analysis confirmed that total Smad2/3 in the nuclear enriched fraction is significantly increased in IPF cells (n = 3) in comparison to NFC cells (n = 3). Results were normalized to TBP. (J) Similarly, total Smad2/3 in the cytoplasmic enriched fraction was increased in IPF cells (n = 3) in comparison to NFC-derived cells (n = 3). Results were normalized to β-actin. Results are represented as mean ± IQR #P < 0.05 (Mann Whitney) or mean ± SEM *P = 0.05 (Un-paired t-test).

Mentions: To elucidate the molecular mechanisms underlying the observed phenoytypic differences between NFC- and IPF-derived HLMFs we investigated the basal Smad2/3 content as Smad2/3 signalling is key for myofibroblast differentiation and 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). Smad3 mRNA was more highly expressed than Smad2 mRNA (Figure 3C).Figure 3


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)

Total and nuclear Smad2 and Smad3 expression is significantly higher in IPF-derived HLMFs. (A) Smad2 mRNA expression was examined by qRT-PCR in NFC (n = 4) and IPF (n = 4) myofibroblasts. IPF-derived HLMFs had significantly higher Smad2 mRNA at baseline in comparison to NFC-derived cells. (B) Similarly, Smad3 mRNA expression was significantly higher in IPF-derived HLMFs (n = 4) in comparison to NFC-derived cells (n = 4). (C) Smad3 mRNA expression was increased compared to Smad2 mRNA expression in both NFC and IPF-derived HLMFs. (D and E) Quantification of Western blot analysis showed that total Smad2/3 protein was increased in IPF-derived HLMFs in comparison to NFC-derived cells. Results were normalised to β-actin. (F) The proportion of total Smad2/3 nuclear staining to whole cell staining was measured in at least 10 cells in HLMFs from both NFC (n = 7) and IPF (n = 9) donors. IPF-derived HLMFs had a significantly higher proportion of Smad2/3 located in the nucleus compared to NFC-derived cells. (G) A representative illustration of total Smad2/3 expression assessed by immunofluorescence. (H) Representative Western blot analysis showing total Smad2/3 protein within the nucleus and cytoplasm of IPF and NFC-derived cells. TBP is localized to nuclear enriched fractions relative to cytoplasmic enriched fractions, similarly β-actin is localized to cytoplasmic enriched fractions relative to nuclear enriched fractions, N = nucleus, C = cytoplasm. Detected bands were at the correct molecular weight of 52 and 60 kDa for total Smad2 and Smad3 respectively, 38 kDa for TBP and 43 kDa for β-actin (I) Quantification of Western blot analysis confirmed that total Smad2/3 in the nuclear enriched fraction is significantly increased in IPF cells (n = 3) in comparison to NFC cells (n = 3). Results were normalized to TBP. (J) Similarly, total Smad2/3 in the cytoplasmic enriched fraction was increased in IPF cells (n = 3) in comparison to NFC-derived cells (n = 3). Results were normalized to β-actin. Results are represented as mean ± IQR #P < 0.05 (Mann Whitney) or mean ± SEM *P = 0.05 (Un-paired t-test).
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Fig3: Total and nuclear Smad2 and Smad3 expression is significantly higher in IPF-derived HLMFs. (A) Smad2 mRNA expression was examined by qRT-PCR in NFC (n = 4) and IPF (n = 4) myofibroblasts. IPF-derived HLMFs had significantly higher Smad2 mRNA at baseline in comparison to NFC-derived cells. (B) Similarly, Smad3 mRNA expression was significantly higher in IPF-derived HLMFs (n = 4) in comparison to NFC-derived cells (n = 4). (C) Smad3 mRNA expression was increased compared to Smad2 mRNA expression in both NFC and IPF-derived HLMFs. (D and E) Quantification of Western blot analysis showed that total Smad2/3 protein was increased in IPF-derived HLMFs in comparison to NFC-derived cells. Results were normalised to β-actin. (F) The proportion of total Smad2/3 nuclear staining to whole cell staining was measured in at least 10 cells in HLMFs from both NFC (n = 7) and IPF (n = 9) donors. IPF-derived HLMFs had a significantly higher proportion of Smad2/3 located in the nucleus compared to NFC-derived cells. (G) A representative illustration of total Smad2/3 expression assessed by immunofluorescence. (H) Representative Western blot analysis showing total Smad2/3 protein within the nucleus and cytoplasm of IPF and NFC-derived cells. TBP is localized to nuclear enriched fractions relative to cytoplasmic enriched fractions, similarly β-actin is localized to cytoplasmic enriched fractions relative to nuclear enriched fractions, N = nucleus, C = cytoplasm. Detected bands were at the correct molecular weight of 52 and 60 kDa for total Smad2 and Smad3 respectively, 38 kDa for TBP and 43 kDa for β-actin (I) Quantification of Western blot analysis confirmed that total Smad2/3 in the nuclear enriched fraction is significantly increased in IPF cells (n = 3) in comparison to NFC cells (n = 3). Results were normalized to TBP. (J) Similarly, total Smad2/3 in the cytoplasmic enriched fraction was increased in IPF cells (n = 3) in comparison to NFC-derived cells (n = 3). Results were normalized to β-actin. Results are represented as mean ± IQR #P < 0.05 (Mann Whitney) or mean ± SEM *P = 0.05 (Un-paired t-test).
Mentions: To elucidate the molecular mechanisms underlying the observed phenoytypic differences between NFC- and IPF-derived HLMFs we investigated the basal Smad2/3 content as Smad2/3 signalling is key for myofibroblast differentiation and 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). Smad3 mRNA was more highly expressed than Smad2 mRNA (Figure 3C).Figure 3

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