Study Of The Function And Molecular Mechanisms Of SMOC2 Gene In Osteoarthritis Development

Osteoarthritis(OA),as one of the slowly progressive and degenerative disorders,is characterized by cartilage functional impairment,chondrocyte hypertrophy and apoptosis,subchondral bone changes and cystic lesions.OA affects people's quality of life through impairing mobility and lowering extremity function of joints.At present,its etiology is still unknown and there is no effective treatment.Human SPARC-related modular calcium binding 2(SMOC2)gene located at chromosome 6q27,encodes one matricellular protein which belongs to the secreted protein acidic and rich in cysteine(SPARC)family.Like other members of the SPARC family,SMOC2 has been reported to be involved in bone development process.SMOC2 can inhibit osteogenic differentiation and extracellular matrix mineralization.SMOC2 variation causes major dental and extremities development defects in human.SMOC2 also affects facial length variation in the dogs.Furthermore,suppression of SMOC2 reduces pulmonary,kidney and lipid fibrosis by inhibition of TGF-? pathway and improves inflammatory response via inactivating NF-?B pathway.These findings suggest that effective SMOC2 inhibitors could potentially be employed in treatment for these diseases.At the same time,a missense mutation in SMOC2 has been found by our research team in a Chinese family with multiple epiphyseal dysplasia(MED),which is characterized by early-onset osteoarthritis(EO-OA).These results suggested that SMOC2 played a role in osteoarthritis.To investigate the function of SMOC2 in osteoarthritis,immortalized human C28/I2 chondrocytes were treated with recombinant human(rh)SMOC2 protein.And then we used Realtime-PCR,ELISA and Western blot to analyze the expression of inflammatory factors(IL-1?,IL-6,IL8 and TNF-?),chemokines(CCL-2,CCL-3,CCL-4 and CXCL-13),matrix metalloproteinases(MMP-3,MMP-13,ADAMTS-4 and ADAMTS-5),COL2A1 and ACAN.Recombinant SMOC2 enhanced the mRNA and protein expression of above cytokines.To determine whether over-expression of SMOC2 also affects osteoarthritis related cytokines synthesis in C28/I2 cells,C28/I2 cells with SMOC2 over-expression were constructed.Results indicated that mRNA expression of above cytokines in pLVX-SMOC2-Myc C28/I2 cells were significantly higher than that in control group.Overall,SMOC2 promote the development of osteoarthritis.To investigate underlying mechanisms,we detect the expression of p-SMAD2 and p-SMAD3 in C28/I2 cells after incubated with rhSMOC2.We found rhSMOC2 could enhance the expression of p-SMAD2 and p-SMAD3 in a dose-dependent manner,which was confirmed in pLVX-SMOC2-Myc C28/I2 cells as well.These results demonstrated that SMOC2 activated the TGF-?/SMAD2/3 signal pathway.Furthermore,to investigated the mechanisms of SMOC2 activated the TGF-?/SMAD2/3 signal pathway,we found that SMOC2 not only upregulated the expression of ACTA and Nodal,but also bound with TGF-? type ?receptors(ACVR2B and TGFBR2)as a ligand,and then activating ALK5 type ?receptor subunits that propagate the signal by phosphorylating SMAD2/3 proteins subsequently.