The Role And Mechanisms Of DEPTOR/FYN In Endochondral Bone Formation And Osteoarthritis Development

Background:Osteoarthritis(OA)is a chronic degenerative disorder characterized by cartilage loss.During OA development,several pathological changes are observed,including an imbalance between anabolic and catabolic activity of chondrocytes,increased production of cartilage-degrading enzymes such as matrix metalloproteinase(MMP)and a disintegrinand metalloproteinase with thrombospondin motifs(ADAMTS)and increased apoptosis.Current pharmacological intervention that addresses chronic loss of cartilages in OA is insufficient,and no proven structure-modifying therapy is available.Therefore,further exploration of OA pathogenesis is essential for the development of appropriate preventionand treatment in clinical practice.DEPTOR is an important protein that belongs to the mTORCl and mTORC2 complexes,able to interact with mTOR and to inhibit its kinase activity.FYN is a ubiquitously expressed non-receptor tyrosine kinase of the Src family.FYN has diverse molecular functions,including regulation of cell growth,survival,adhesion,cytoskeletal remodeling,motility axon guidance,synaptic function,myelination in the central nervous system,platelet activation and T cell receptor signaling.The role of mTOR in OA had been studied well,but how did the endogenous inhibitor of mTOR and FYN kinase worked in OA haven’t been reported yet.This study had build cartilage specific knockout of DEPTOR mice,and we found DEPTOR play an important role in the development of OA.By using proteomics analysis,we had screened that DEPTOR potentially engaged in OA process through interacting with FYN.Objects:This research is mainly to conduct the following researchs:Analyze the role of DEPTOR in the endochondral bone formation;Explore the clear role and the specific mechanism of DEPTOR in OA;Analyze the specific mechanisms of FYN in the OA process,and evaluate.the effect of FYN inhibitors on OA;Explore the interaction between DEPTOR and FYN.Methods:1)Constructed transgene mice with DEPTOR specific knockout in chondrocytes with cre-loxp system.Analyzed the skeletal development with Alcian blue and alizarin red staining.Slice the limb bone to analyze the distribution of different period chondrocytes in growthplate.Detected the proliferation of chondrocytes with BrdU staining and western blot analysis;2)Producted experimental OA model in mice with DMM surgery.To analyze the loss of cartilage in OA with Safranin O-fast green staining.Eveluate the expressions of MMP13,AD AMTS 5 with IHC and Co1X with IF.Compare the OA degrees in control mice and DEPTOR-KO mice with OARSI scoring system;3)Treated the DMM-induced OA mice with mTOR inhibitors,and then analyze the effect of mTOR inhibitors on DEPTOR knockout induced OA with the upper methods;4)Screened the DEPTOR interactin proteins in the cartilages with Proteomicanalysis.Certified the interaction between FYN and DEPTOR by immunoprecipitation and double-labelling immunofluorescence;5)Mice with DMM-induced OA were treated with PP1 and AZD0530,the effect of FYN inhibitors on OA were analyzed as above;6)Explored the role of FYN in the OA process within immunoprecipitation,immunofluorescence,western blot analysis;7)DEPTOR-KO mice with DMM-induced OA were treated with PP1 and AZD0530,and the effect of FYN inhibitors on the DEPTOR-KO resulted OA were evaluated.Results:1)DEPTOR has limited effect on the endochondral bone formation process.expression of DEPTOR decreased in OA process.Deletion of DEPTOR in chondrocytes will exacerbate the OA severity and the inhibitors of mTOR cannot ameliorate the aggravating OA;2)DEPTOR interacts with FYN in chondrocytes,knockout of DEPTOR will increase FYN protein level and overexpression of DEPTOR will decrease the expression of FYN in chondrocytes;3)FYN expression was increased in the cartilage chondrocytes of experimental OA and aged OA.The inhibitors of FYN,PP1 and AZD0530 can relieve the severity of OA in mice;4)FYN can phosphorylate P-catenin at Tyr142 site in chondrocytes,and then promoting the nuclear translocation of β-catenin.FYN inhibitors can revese this process;5)The FYN inhibitors PP1 and AZD0530 will ameliorate the OA in DEPTOR-KO mice.Conclusion:1)DEPTOR is not essential for the endochondral bone formation process.2)The expression of DEPTOR decreased in OA process.Knockout of DEPTOR in chondrocytes will accelerate the progression of OA,and the way DEPTOR engaged in OA is not dependent on mTOR activation pathways;3)DEPTOR can interact with FYN in cells.Knockout of DEPTOR in chondrocytes will enhance the expression of FYN in joint cartilage and then aggravates OA.FYN expression increased in OA process.FYN can phosphorylate and activate β-catenin,and then promoting the nuclear translocation of β-catenin.The nuclear expression β-catenin will mediate the transcription of OA-releated transcription factor,moreover to aggravate the OA process.FYN inhibitors can revese this process.4)FYN inhibitors will alleviate OA process in DEPTOR-KO mice.The inhibitors of FYN may be potential drugs in preventing OA development.