Introduction Idiopathic pulmonary fibrosis (IPF) is an incurable and progressive fibrotic condition of the lung with a poor prognosis. Activation of the profibrotic cytokine transforming growth factor-β (TGFβ) is central to the pathogenesis of IPF. Mechanical signals are emerging as key mediators of TGFβ activation, and cyclical stretch is a ubiquitous stimulus in the lungs. Stretch of fibrotic lung induces TGFβ activation, and alveolar epithelial G_αq/11 signalling is important in stretch-mediated TGFβ activation. However, the role of G_αq/11 signalling in stretch-mediated TGFβ activation by lung fibroblasts is unclear.

Methods Murine embryonic fibroblasts (MEFs) and human lung fibroblasts (HLFs) were subject to cyclical stretch using the Flexcell system, and stretch regimens designed to mimic tidal breathing in the relevant organism (15% elongation unless otherwise stated, frequency: 1 Hz in MEFs, 0.3 Hz in HLFs). Phosphorylated Smad2 (pSmad2), total Smad2, and alpha smooth muscle actin (αSMA) protein expression were assessed by western blot. TGFβ activation was determined using the ratio of pSmad2 to total Smad2 on densitometry. Comparisons of stretch-mediated TGFβ activation were made between wild-type (WT), G_α12/13-, and G_αq/11-deficient MEFs, and between HLFs from patients with and without IPF.

Results Cyclical stretch induced a significant increase in pSmad2 expression after 48 hours in both MEFs and HLFs (p<0.05). However, stretch did not affect total αSMA protein expression in either cell type. There was no ‘dose-response’ relationship in stretch-induced pSmad2 expression in WT MEFs (5%, 10%, 15% and 20% elongation). MEFs deficient in G_αq/11 signalling, but not G_α12/13 signalling, activated significantly less TGFβ than WT MEFs in response to cyclical stretch (p<0.05). HLFs from both IPF and non-IPF donors activated TGFβ after 48 hours of cyclical stretch, but IPF fibroblasts activated significantly more TGFβ than fibroblasts from patients without IPF (6 IPF and 6 non-IPF cell lines; p<0.05).

Conclusion Cyclical stretch is a physiologically relevant stimulus that drives G_αq/11-mediated TGFβ activation in lung fibroblasts. IPF fibroblasts have enhanced stretch-mediated TGFβ activation, indicating that dysregulation of breathing-related stretch signalling is a potential mechanism of IPF progression. A greater understanding of these pathways may identify targets for new therapies that could halt the progression of IPF.