The Role And Molecular Mechanism Of ADAMTS18 In Chondrocyte Differentiation

Bone is a highly sophisticated connective tissue composed of the collagen fibrils and the inorganic calcium phosphate.It supports the body structurally,protect our vital organs,and allow us to move.Endochondral osteogenesis is an important process of bone formation mainly accomplished through the proliferation and differentiation of chondrocytes in various growth plates.The process of cartilage progenitor cells forming bone tissue mainly includes four stages: resting stage,proliferation stage,prehypertrophy stage and hypertrophy stage.ADAMTS18 is a member of a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS)family of proteases.Dysregulation and mutation of ADAMTS18 has been implicated in various important physiological and pathological processes,such as development,tumor,blood coagulation and atherosclerosis.In our previous study,we found that the ADAMTS18 KO mice has small size,and ADAMTS18 deletion affected chondrogenesis.To investigate the role of ADAMTS18 in chondrocyte differentiation and its molecular mechanisms,ATDC5 cells were used to establish an in vitro model of endochondral ossification.In this paper,to study the role of ADAMTS18 in chondrocyte differentiation,pc DNA3.1-ADAMTS18 and sh RNA-ADAMTS18 plasmids were constructed and transfected into ATDC5 cells to construct stable cell lines.The successful construction of a stable tranfected ATDC5 cell lines were verified by RT-PCR and Western blot.Whether ADAMTS18 affected the proliferation of ATDC5 cells was dected by MTT assay.The results demonstrated that ADAMTS18 significantly promoted ATDC5 cells proliferation.Flow cytometry was used to detect the effect of ADAMTS18 on ATDC5 cell cycle.The result suggested that the ATDC5 cells were arrested in G1 phase.ATDC5 cell differentiation was stimulated and induced by INS and cultured for 0,5,7,10 and 14 days.Results of microscopic observation,alcian blue and saffron O staining showed that the number of cartilage nodules was more in ADAMTS18 overexpressing cells,compared with the control cells after 10 days of induction.The m RNA expression level of chondrogenic differentiation marker gene was detected by Q-PCR.The results showed that after the overexpression of ADAMTS18,the expression of Col II,Col X,MMP13 and Cbfa1 were increased.The protein levels of Col X and MMP13 were detected by Western blot.It was also found the expressions of Col X and MMP13 were up-regulated.Meanwhile,we found that the m RNA expression levels of Col II,Col X,MMP13 and Cbfa1 were decreased in ADAMTS18-silenced cells;the number of cartilage nodules were reduced after 10 days of induction;and the expression levels of Col X and MMP13 proteins were significantly down-regulated,compared with the control cells.ADAMTS18 may affect chondrocyte differentiation by regulating the IHH/PTHr P signaling pathway.The expression changes of IHH and PTH1 R were detected by Western blot.The results showed that ADAMTS18 could enhance the expression of IHH and PTH1 R.In addition,we also found that ADAMTS18 could partially reverse the effects of cyclopamine on the expression of IHH,PTH1 R and hypertrophic markers like Col X and MMP13.In summary,ADAMTS18 promotes chondrogenic differentiation and affects IHH/PTHr P signaling pathway.It provides a theoretical basis for the study of the effects of ADAMTS family in bone and new ideas for the diagnosis and treatment of chondrodevelopmental diseases by investigated the role and molecular mechanism of ADAMTS18 in chondrocyte differentiation.