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 Ca2+ 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.
| Original language | English (US) |
|---|---|
| Article number | 5 |
| Journal | Fibrogenesis and Tissue Repair |
| Volume | 8 |
| Issue number | 1 |
| DOIs | |
| State | Published - Mar 26 2015 |
Fingerprint
Keywords
- Human lung myofibroblast
- Idiopathic pulmonary fibrosis
- Potassium channel KCa3.1
ASJC Scopus subject areas
- Medicine (miscellaneous)
- Dermatology
- Gastroenterology
- Hepatology
- Rheumatology
Cite this
Human lung myofibroblast TGFβ1-dependent Smad2/3 signalling is Ca2+-dependent and regulated by KCa3.1 K+ channels. / Roach, Katy M.; Feghali-Bostwick, Carol; Wulff, Heike; Amrani, Yassine; Bradding, Peter.
In: Fibrogenesis and Tissue Repair, Vol. 8, No. 1, 5, 26.03.2015.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Human lung myofibroblast TGFβ1-dependent Smad2/3 signalling is Ca2+-dependent and regulated by KCa3.1 K+ channels
AU - Roach, Katy M.
AU - Feghali-Bostwick, Carol
AU - Wulff, Heike
AU - Amrani, Yassine
AU - Bradding, Peter
PY - 2015/3/26
Y1 - 2015/3/26
N2 - 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 Ca2+ 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.
AB - 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 Ca2+ 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.
KW - Human lung myofibroblast
KW - Idiopathic pulmonary fibrosis
KW - Potassium channel KCa3.1
UR - http://www.scopus.com/inward/record.url?scp=84925841740&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84925841740&partnerID=8YFLogxK
U2 - 10.1186/s13069-015-0022-0
DO - 10.1186/s13069-015-0022-0
M3 - Article
AN - SCOPUS:84925841740
VL - 8
JO - Fibrogenesis and Tissue Repair
JF - Fibrogenesis and Tissue Repair
SN - 1755-1536
IS - 1
M1 - 5
ER -