Discovery And Identification Of DDR2 Interacting Protein That Regulate Nonclassical TGF-β Pathways

【Objective】The treatment of pulmonary fibrosis lacks efficient and specific means.Pirfenidone and nintedanib approved in 2014 can only improve lung function,but cannot reduce mortality.Receptor tyrosine kinase inhibitors such as dasatinib are often non-specific,and lung transplantation opportunities are rare.Previous research by our research group found that discoidin domain receptors 2(DDR2)activates lung fibroblasts in two ways in pulmonary fibrosis,and promotes the process of pulmonary fibrosis.DDR2 may be a therapeutic target for pulmonary fibrosis.The purpose of this study is to find the interacting molecules of DDR2 to promote the process of fibrosis,so as to target and regulate DDR2,and then indirectly control the process of lung fibrosis.【Method】Use immunoprecipitation to find out the proteins that bind to DDR2 under TGF-β stimulation conditions,and analyze the types of these proteins by mass spectrometry.Through the above methods,our research focuses on one of the proteins that interact with DDR2,Prohibitin 2(PHB2).Use the method of immunoprecipitation to explore the interaction between PHB2 and DDR2.Then use the constructed DDR2 truncated body TID,TED,JM1+ to explore the interaction domain.And,innovatively use complementary luciferase method to verify the interaction area.Then,by controlling the stimulation duration of TGF-β or type I collagen,we can explore the changes of the interaction over time.Then we used 293 T cells expressing DDR2 and transiently transfected PHB2 to explore the effect of PHB2 on DDR2 phosphorylation and its dose dependence.On this basis,using healthy human lung primary fibroblasts to explore the effect of PHB2 on the phosphorylation of DDR2,and the effect of overexpression of PHB2 on the fibrosis of lung fibroblasts.Finally,immunoblotting was used to explore the effect of PHB2 on the activation of TGF-β pathway.The experimental results were performed using Graph Pad Prism8.02 software.Multiple comparisons between the two groups were performed using one-way analysis of variance.Differences with P values<0.05 were considered statistically significant.【Results】Co-immunoprecipitation experiments revealed that PHB2 and DDR2 interacted,and the interaction occurred in the JM1 region of the TID of the intrace llular domain of DDR2.The complementary luciferase method was used to verify that there is an interaction between TID and PHB2(P value <0.0001).Exploring with these two experimental methods also found that TGF-β stimulation affects the strength of the interaction,the interaction is enhanced within 0.5 hours of stimulation(P=0.0007),until the interaction is weakened within 1.5 hours(P=0.0015),and it is adjusted back in 4 hours.After collagen stimulation,the interaction is weakened in 4 hours,enhanced in 8 hours,and weakened in 24 hours.In terms of function,we found that PHB2 overexpression inhibited the phosphorylation of DDR2 in 293 T cells stably overexpressing DDR2,and it was dose-dependent.In fibroblasts,we also found that PHB2 inhibited DDR2 phosphorylation.Western blotting found that overexpression of PHB2 inhibited the up-regulation of fibrotic factors after TGF-β stimulation,with fibronectin and α-smooth muscle actin(α-SMA)being the most significant.Finally,Western blotting found that PHB2 inhibited the phosphorylation of ERK,P38 and AKT in the non-classical pathways induced by TGF-β,and inhibited the phosphorylation of smad3 in the classical pathway,but had no effect on the phosphorylation of smad2.【Conclusion】Our experiment found that JM1 interacts with PHB2 in the proximal membrane area of the intracellular segment of DDR2.Overexpression of PHB2 can inhibit the phosphorylation of DDR2.And PHB2 can inhibit the up-regulation of fibrotic factors Fibronectin and α-SMA after TGF-β stimulation,and inhibit the phosphorylation of ERK,P38,AKT in non-classical pathways induced by TGF-β,and phosphorylation of smad3 in the classical Smad pathway.These findings play an important role in pulmonary fibrosis and other diseases that depend on DDR2 phosphorylation,and even PHB2 may be used as a new target for the treatment of pulmonary fibrosis and these diseases.