Dissecting The Non-redundant Role Of Gab Docking Proteins In Macrophage M2-driven Pulmonary Fibrosis In Vivo

Idiopathic pulmonary fibrosis(IPF)is a specific form of chronic progressive,irreversible,and lethal interstitial disease of unknown etiology,and is a serious hazard to human health.IPF is characterized by myofibroblast accumulation,destruction of lung architecture,and excess collagen deposition that ultimately leads to progressive dyspnea and death from respiratory failure.In recent years,the morbidity and mortality of IPF has been on the rise,and IPF remains the most challenging treatment in clinic due to the unknown pathogenesis and the lack of definitive therapies.As a result,a more detailed understanding of the pathogenesis and molecular mechanisms of pulmonary fibrosis could help pave the way for the prevention and treatment of IPF.Macrophages,the predominant innate immune cells,are characterized by plasticity and diversity.Macrophages respond to various signals with the acquisition of distinct phenotypes.In response to environmental cues,two concepts about polarization are referred to as classically activated macrophage(M1)and alternatively activated macrophage(M2).M2 macrophages serve as master regulators of fibrosis by resolution of inflammation and repair.Reversible phosphorylation,an important posttranslational modification of proteins,is a central part of the signal transductions and enzymatic reactions by protein kinases/phosphatases contribute to signal transduction,ultimately involving in physiological processes.Despite lacking of catalytic domain,docking proteins play vital roles in the fine regulation of signal transduction by interacting with kinases and phosphatases.The Grb2-associated binder(Gab)proteins,the dominant scaffolding adaptor proteins,integrate and amplify signals from extracellular stimuli into signalling pathways with distinct biological functions by providing docking sites for other adaptors,kinases,and phosphatases,contributing to regulate cell proliferation,migration,apoptosis,polarization and other physiological processes.All Gab docking molecules are characterized by a highly conserved pleckstrin homology domain and a proline-rich domain.Gab1 and Gab2(Gab1/2)are widely expressed in mammals.Gab3 has limited expressed in hematopoietic cells,and Gab3-deficient mice exhibit no obvious defects in normal development and hematopoiesis.Thus,this work focused on dissecting the functions of Gab1 and Gab2 in M2 polarization.In this work,we first observed an elevated level of Gab1/2 in alveolar macrophages isolated from fibrotic lungs and these alveolar macrophages were characterized by enriched M2-polarized macrophages,suggesting the potential involvement of Gab1/2 in M2-driven fibrosis.Herein,we employed Gab1 conditional deletion mice in monocytes/macrophages(Gab1MyKO)and Gab2 deficient mice(Gab2/-)to explore the roles of Gabl/2 in pulmonary fibrosis.We found that there is a putative compensation between Gabl and Gab2 in macrophages,implying the synergistic role of Gab1/2 in macrophages.In vitro Gab1/2 deficiency in macrophages abrogated M2 polarization.Furthermore,in vivo conditional removal of Gab1(Gab1MyKO)and geramline knockout of Gab2(Gab2-/-)in macrophages prevented a bias towards the M2 phenotype and attenuated bleomycin-induced fibrotic remodeling.Previous studies have demonstrated that JAK1/STAT6 and PI3K/AKT signaling are involved in regulating M2 polarization.Mechanistically,we found a distinctive role of Gab1/2 in IL-4-mediated downstream elements,although Gab1/2 deficiency in macrophages led to an apparently similar M2 polarization.We noticed a preferential interaction of Gabl with p85 contributing to activate PI3K/AKT signaling,while Gab2 facilitates the interaction of IL-4Ra with JAK1,attributable to the activation of JAK1/STAT6.Taken together,these observations define a non-redundant role of Gab docking proteins in M2 polarization,adding fresh insights into the pathogenesis of idiopathic pulmonary fibrosis and providing new therapeutic targets for IPF.