S106 Fibroblast Gαq/11 controls lung repair via regulation of lung extracellular matrix properties

Abstract

<h3>Introduction</h3> Lung repair requires controlled transforming growth factor-β (TGFβ) signalling and extracellular matrix (ECM) production. We previously demonstrated that constitutive and adulthood-induced mesenchymal G_αq/11 knockout alters lung ECM properties, including lung elastin and TGFβ2 deposition, causing abnormal lung development and emphysema, respectively.¹ However, the role of the G_αq/11-deficient fibroblast-generated ECM in these phenotypes is unclear. <h3>Aim</h3> Understand how wild-type and G_αq/11^-/- fibroblast-deposited ECM influence lung repair. <h3>Methods</h3> Wild-type (WT) and Gnaq^-/-;Gna11^-/- (G_αq/11^-/-) murine embryonic fibroblasts (MEFs) were cultured on ECM generated by WT or G_αq/11^-/- MEFs. Wound healing and TGFβ signalling were assessed using scratch wound and conditioned media-stimulated transformed mink lung cell (TMLC) assays. WT and G_αq/11-knockout MEFs were stimulated with 2 ng/ml TGFβ2, and elastin, TGFβ2, and platelet-derived growth factor (PDGF) signalling component expression assessed. <h3>Results</h3> WT MEFs exhibited lower TGFβ signalling and wound healing when cultured on G_αq/11^-/- ECM than on WT ECM (0.53 relative TMLC luciferase activity (RLA); 31.7% vs 46.4% 8 hour healing). Conversely, G_αq/11^-/- MEFs activated more TGFβ on WT ECM than on G_αq/11^-/- ECM (1.8 RLA). When cultured on ECM alone, TMLC TGFβ signalling was reduced on G_αq/11^-/- ECM (0.44 RLA), suggesting that G_αq/11^-/- fibroblast-deposited ECM itself alters repair. G_αq/11^-/- MEFs had lower ECM component (<i>Col3a1</i>, <i>Col1a1</i>, <i>Eln</i>) and growth factor (<i>Pdgfa</i>, <i>Pdgfb</i>, <i>Pdgfd</i>) mRNA expression than WT MEFs. G_αq/11^-/- MEFs stimulated with TGFβ2 had a time-dependent increase in <i>Eln</i> and <i>Pdgfa</i> mRNA expression, (15.8- and 3.6-fold increases, respectively), and elastin protein approached near WT levels by 48 hours. Wound healing was slower in G_αq/11^-/- than WT MEFs (33.2% vs 58.1% 8 hour healing), and was enhanced by exogenous TGFβ2 (to 38.6% and 73.3% healing, respectively). However, TGFβ2 did not restore G_αq/11^-/- MEF healing to WT levels. <h3>Conclusion</h3> Fibroblast G_αq/11 regulates ECM production, and ECM generated by G_αq/11^-/- cells is less supportive of repair than WT ECM. Reduced TGFβ2 content of G_αq/11^-/- ECM may further alter ECM generation, but restoration of TGFβ2 signalling does not fully re-establish repair. A greater understanding of these mechanisms may identify methods of manipulating lung repair to therapeutic potential. Please refer to page A211 for declarations of interest related to this abstract. <h3>Reference</h3> Goodwin AT <i>et al</i>. BioRxiv doi: https://doi.org/10.1101/2020.09.06.284778).