Studies Of The Effect And Mechanism Of Mef2a On Col10a1 Gene Expression During Endochondral Bone Formation

Background and Significance:Hypertrophic chondrocytes have been described as a major "regulator" of bone growth,and the type X collagen gene(COL10A1)is a specific marker of hypertrophic chondrocytes.Improper expression of COL10A1 results in abnormal chondrocyte hypertrophy,which was seen in a range of skeletal diseases including osteoarthritis and osteosarcoma.Therefore,elucidating the regulatory mechanisms of Col10a1 is essential for further our understanding of cartilage development and associated human disease and injuries.Previous studies have identified multiple transcription factors(TFs),including myocyte enhancer factor 2a(Mef2a),that have previous been identified by in silico analysis as a potential murine Col10 al gene regulator.Objective:The purpose of this research is to investigate the correlation between the expression of the Mef2 a and Col10a1 as well as the potential effects of Mef2 a on endochondral bone formation in vitro.Methods:(1)By using bioinformatics program to predict transcription factors and searching the literature,we chose Mef2 a as the subject of our study.(2)The basal expression levels of Mef2 a in two chondrogenic models(ATDC5,MCT)and mouse chondrocytes in situ were detected by quantitative real-time PCR(q RT-PCR),Western Blot(WB)and immunohistochemistry(IHC).(3)The effects of Mef2 a knockdown or overexpression on Col10a1 expression were investigated using q RT-PCR and Western Blot assays after small interfering fragments of Mef2 a or overexpression plasmids of Mef2 a were transfected into two in vitro chondrocytic models.(4)We predicted the binding site of Mef2 a within the 150-bp Col10a1cis-enhancer by TRAP,PROMO and h TFtarget bioinformatics programs and verified the site by a dual luciferase reporter assay.(5)We constructed ATDC5 cell lines with stable knockdown of Mef2 a using the lentiviral vector,and then performed alcian blue staining,alkaline phosphatase(ALP)staining and alizarin red staining on these cells to analyze the role of Mef2 a in chondrocyte differentiation and maturation in vitro.(6)Preliminary analysis of the effect of Mef2 a on several chondrocyte differentiation-related markers in ATDC5 stable cell lines by q RT-PCR.Results:(1)The expression of Mef2 a in hypertrophic chondrocytes was significantly higher than that in proliferative chondrocytes in two chondrocytic models,ATDC5 and MCT cells,as well as in mouse chondrocytes in situ.(2)Interference with Mef2 a caused decreasesd Col10a1 expression,while overexpression of Mef2 a upregulated Col10a1.(3)The result of the dual luciferase reporter assay showed that Mef2 a enhanced Col10a1 gene enhancer activity by binding to the corresponding site within 150-bp Col10a1 cis-enhancer.(4)Although no significant differences were seen in ALP staining,significantly weaker alcian blue staining intensity was noticed in Mef2 a knockdown stable cell lines compared to the control cells at day 21,while slightly weaker alizarin red staining was seen in the stable cell lines at days 14 and 21.(5)We detected decreased Runx2,increased Sox9 in Mef2 a knockdown ATDC5 stable cell lines compared with the controls.Conclusion:Our results support that Mef2 a upregulates Col10a1 expression by binding to its cis-enhancer and plays an active role in chondrocyte proliferation and matrix mineralization in vitro.