S77 The G proteins Gαq/11 and Gα12/13 drive unique myofibroblast functions to promote pulmonary fibrosis

Abstract

<h3>Introduction</h3> Idiopathic pulmonary fibrosis (IPF) is relentlessly progressive with a poor prognosis. In IPF, excessive scar tissue replaces normal lung parenchyma following alveolar injury. There is currently no treatment that can reverse fibrosis. Myofibroblasts drive fibrosis through contraction and extracellular matrix (ECM) generation. Increasing ECM stiffness promotes fibroblast-to-myofibroblast differentiation, ECM production, and activation of the profibrotic cytokine transforming growth factor-β (TGFβ). However, the precise mechanisms that drive this feedback loop are uncertain. Signalling by G protein coupled receptors (GPCRs) has been implicated in IPF. Messages from hundreds of GPCRs converges on four G_α subunit families, however the role of these molecules in myofibroblast activity in IPF is unknown. <h3>Aim</h3> Understand the role of G_αq/11 and G_α12/13 in profibrotic myofibroblast functions. <h3>Methods</h3> Wild-type (WT), Gnaq^-/-;Gna11^-/- (G_αq/11^-/-), and Gna12^-/-;Gna13^-/- (G_α12/13^-/-) murine embryonic fibroblasts (MEFs) and human lung fibroblasts (HLFs) were stimulated with lysophosphatidic acid (LPA, 50µM). TGFβ signalling was measured using Smad2 phosphorylation. MEFs were cultured on gels of fibrotic (100kPa, 36kPa) and physiological (5kPa) stiffness. Myofibroblast differentiation was assessed using α smooth muscle actin (αSMA) expression. MEFs and HLFs cultured on thin gels were stimulated with GPCR agonists (LPA 30µM, SFLLRN 20µM and TFLLRN 20µM), and time lapse images taken. Gel wrinkling was used to quantify contraction. <h3>Results</h3> G_αq/11 and G_α12/13 knockdown both reduced LPA-induced TGFβ signalling in MEFs and HLFs compared with controls (p&lt;0.05, n=4). Rho-associated kinase (ROCK) inhibition reduced LPA-induced TGFβ signalling, suggesting that cellular contraction mediates LPA-induced TGFβ activation. HLFs from IPF donors were more contractile than non-diseased HLFs (91 vs 42 wrinkles/image, p=0.02, n=5 per group). MEFs and HLFs lacking G_α12/13 had reduced baseline and GPCR agonist-induced contraction (p&lt;0.05), and G_α12/13^-/- MEFs had abnormal cytoskeletal appearances on immunofluorescence. G_αq/11 knockdown did not affect contractility or cytoskeletal appearance. G_αq/11^-/- MEFs had reduced αSMA expression when transferred to soft tissue culture conditions (p&lt;0.05, n=4), whereas WT and G_α12/13^-/- MEFs did not. <h3>Conclusions</h3> Myofibroblast activity is enhanced in IPF. G_αq/11 and G_α12/13 both mediate TGFβ signalling, but via different mechanisms, and they drive distinct myofibroblast profibrotic functions. A greater understanding of these processes could identify new treatments for IPF.