Human lung myofibroblast TGFβ1-dependent Smad2/3 signalling is Ca(2+)-dependent and regulated by KCa3.1 K(+) channels

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a common and invariably lethal interstitial lung disease with poorly effective therapy. Blockade of the K(+) channel KCa3.1 reduces constitutive α-SMA and Smad2/3 nuclear translocation in IPF-derived human lung myofibroblasts (HLMFs), and inhibits several transforming growth factor beta 1 (TGFβ1)-dependent cell processes. We hypothesized that KCa3.1-dependent cell processes also regulate the TGFβ1-dependent Smad2/3 signalling pathway in HLMFs. HLMFs obtained from non-fibrotic controls (NFC) and IPF lungs were grown in vitro and examined for αSMA expression by immunofluorescence, RT-PCR, and flow cytometry. Two specific and distinct KCa3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on TGFβ1-dependent signalling. Expression of phosphorylated and total Smad2/3 following TGFβ1 stimulation was determined by Western blot and Smad2/3 nuclear translocation by immunofluorescence.

Results: KCa3.1 block attenuated TGFβ1-dependent Smad2/3 phosphorylation and nuclear translocation, and this was mimicked by lowering the extracellular Ca(2+) concentration. KCa3.1 block also inhibited Smad2/3-dependent gene transcription (αSMA, collagen type I), inhibited KCa3.1 mRNA expression, and attenuated TGFβ1-dependent αSMA protein expression.

Conclusions: KCa3.1 activity regulates TGFβ1-dependent effects in NFC- and IPF-derived primary HLMFs through the regulation of the TGFβ1/Smad signalling pathway, with promotion of downstream gene transcription and protein expression. KCa3.1 blockers may offer a novel approach to treating IPF.

Keywords: Human lung myofibroblast; Idiopathic pulmonary fibrosis; Potassium channel KCa3.1.

Figure 1

Phosphorylation of Smad2/3 is K _Ca 3.1 dependent. (A) Phosphorylation of Smad2/3 in HLMFs was most abundant after 60 min of stimulation with TGFβ1 (10 ng/ml) (n = 3). (B) Representative Western blot analysis showing the increased phosphorylation of Smad2/3 after 60 min of stimulation with TGFβ1 (10 ng/ml) and its inhibition by TRAM-34 and ICA-17043. Phosphorylation of Smad2/3 was examined by fold change over total Smad2/3 and normalized to β-actin. TGFβ1-dependent increases in phosphorylated Smad2/3 were inhibited by TRAM-34 200 nM (NFC n = 3 and IPF n = 4) (C) and by ICA-17043 100 nM (NFC n = 3 and IPF n = 3) (D). Results are represented as mean ± SEM *P < 0.05, **P < 0.01 (repeated measures ANOVA corrected by Sidaks multiple comparison test).

Figure 2

Smad2/3 nuclear translocation is inhibited by K _Ca 3.1 channel blockade. (A) The ratio of nuclear to whole cell staining of Smad2/3 demonstrates a significant increase in nuclear translocation following TGFβ1 stimulation compared to control. TRAM-34 (200 nM) inhibited TGFβ1-induced Smad2/3 nuclear translocation (NFC n = 4, IPF n = 4) whereas the structurally related molecule TRAM-85 without K_Ca3.1 blocking properties did not inhibit nuclear translocation. NFC and IPF data were pooled for statistical analysis. (B) Representative fluorescent microscopy images illustrate the increased expression of Smad2/3 following TGFβ1 stimulation and its movement into the nucleus, which was significantly attenuated by TRAM-34 (200 nM). (C) ICA-17043 (100 nM) also significantly attenuated Smad2/3 nuclear translocation, which can be seen visually in (D). (E) Quantification of Western blot analysis confirms that the total Smad2/3 in the nuclear enriched fraction is significantly increased following TGFβ1 stimulation and attenuated by ICA-17043 (100 nM). Results are normalized to TATA Binding Protein (TBP) and representative Western blot analysis images are shown. (F) Similarly, the total Smad2/3 was examined in the cytoplasmic-enriched fraction; however, no changes were found following TGβ1 stimulation or treatment with ICA-17043 (NFC n = 2 and IPF n = 3, data pooled). Results are represented as mean ± SEM ***P < 0.001, **P < 0.01 (two-way ANOVA corrected by Sidaks multiple comparison test), ^## P < 0.0001, one sample t test, ^# P < 0.05, paired t test.

Figure 3

TGF 1-dependent Smad2/3 phosphorylation is Ca ²⁺ dependent. (A) A representative Western blot demonstrating Smad 2/3 phosphorylation when cells are incubated in media either containing Ca²⁺ or without. (B). Quantification of Western blots showing that the increased phosphorylation of Smad2/3 after 60 min of TGFβ1 exposure (10 ng/ml) is largely dependent on the presence of extracellular Ca²⁺. Phosphorylation of Smad2/3 was examined by fold change over total Smad2/3 and normalized to β-actin (NFC n = 2, IPF n = 4, data pooled for statistical analysis). HLMFs stimulated for 1 h in Ca²⁺-free media phosphorylated significantly less Smad2/3 than those incubated in media containing Ca²⁺. *P < 0.05, ***P < 0.001 results are represented as median (IQR) (one-way ANOVA, corrected by Sidaks multiple comparison test).

Figure 4

TGFβ1-dependent Smad2/3 nuclear translocation is Ca ²⁺ dependent. (A) The ratio of nuclear to whole cell TGFβ1-induced Smad 2/3 nuclear translocation is significantly attenuated when cells are incubated in media without Ca²⁺ (NFC n = 3, IPF n = 3, data pooled). (B) Fluorescent microscopy images illustrating the increased nuclear translocation of total Smad2/3 following TGFβ1 stimulation and its movement into the nucleus, which was significantly attenuated in the absence of Ca²⁺. Results are represented as mean ± SEM *P < 0.001 (repeated measures ANOVA corrected by Sidaks multiple comparison test).

Figure 5

TGFβ1-dependent transcription of αSMA, collagen type I and K _Ca 3.1 is K _Ca 3.1 dependent. (A) TGFβ1 stimulation significantly increased αSMA and collagen I mRNA expression in HLMFs per 10³ copies of β-Actin, which was significantly inhibited by TRAM-34 200 nM (NFC n = 4, IPF n = 4, data pooled for statistical analysis). (B) Similarly, ICA-17043 (100 nM) also significantly decreased TGFβ1-dependent increases in αSMA and collagen I mRNA expression in HLMFs (NFC n = 3, IPF n = 3, data pooled). Results are represented as mean ± SEM or median (IQR) ***P < 0.001 (one sample t test), ^# P < 0.05 and ^## P < 0.01 (paired t test or Wilcoxon signed rank test). (C, D) The fold change of TGFβ1-dependent K_Ca3.1 mRNA expression was significantly higher in IPF HLMFs compared to that in NFC HLMFs, ^# P = 0.0313 (NFC n = 4, IPF n = 4, data pooled for statistical analysis). This TGFβ1 dependent increase in K_Ca3.1 mRNA in IPF donors was significantly attenuated by K_Ca3.1 channel blockers, TRAM-34 and ICA-17043 (*P = 0.0385 and P = 0.0313, respectively, paired t test).

Figure 6

TGFβ1-dependent αSMA protein expression is attenuated by K _Ca 3.1 channel block. (A) Flow cytometry measurements of αSMA expression in HLMFs show that the fold change in gMFI was significantly increased by TGFβ1 (10 ng/ml) (NFC n = 3, IPF n = 4, data pooled for statistical analysis). TRAM-34 (20 and 200nM) dose-dependently decreased TGFβ1-induced αSMA expression. (B) Similarly, ICA-17043 (10 and 100nM) significantly reduced TGFβ1-induced αSMA expression. (C) Representative fluorescent histogram showing αSMA expression in HLMFs under the above conditions. Results are represented as mean ± SEM. ^# P < 0.05 (paired t test); *P < 0.05, **P < 0.01 (corrected by Dunn’s multiple comparisons test).

Figure 7

The involvement of K _Ca 3.1 in the TGF/Smad 2/3 signalling pathway. A diagrammatic representation of how Smad2/3 phosphorylation and subsequent nuclear translocation is reliant on an influx of extracellular Ca²⁺ and K_Ca3.1 ion channels. TGFβ1 stimulation triggers an influx of extracellular Ca²⁺, which in turns opens Ca²⁺-activated K_Ca3.1 K⁺ channels. K_Ca3.1 opening maintains a negative membrane which in turn promotes Ca²⁺ entry. Phosphorylation of Smad2/3 and therefore its downstream effects such as translocation and gene transcription are heavily reliant on Ca²⁺. K_Ca3.1 channel inhibition reduces Ca²⁺ entry, which in turn reduces Smad2/3 phosphorylation and nuclear translocation, and thus reduces TGFβ1-dependent gene transcription.