The Roles Of The Acetyltransferase Mof During Osteoblast Differentiation

Bone marrow mesenchymal stem cells(BMSCs)are multipotent stem cells derived from bone marrow with the ability of self-renewal and multidirectional differentiation potential.It can be differentiation into osteoblast,chondrocyte,adipocyte and muscle cells in vivo or induced in vitro.Bone marrow mesenchymal stem cells can be applied to bone defect,cartilage deterioration,liver and heart or other tissue repair,metabolic diseases.So it has high clinical value in regenerative medicine and tissue engineering.TGF-?/BMPs pathway and classical Wnt signal pathway are the major regulation approach during bone marrow mesenchymal stem cells differentiate into osteoblast.Bmp2 which is an important subtype of BMPs family and ?-catenin from the Wnt signal pathway can increase the expression of osteogenic differentiation related genes runx2 and osterix to stimulate osteoblast differentiation.Mof also called KAT8 belongs to the MYST family which was one of the histone acetyltransferase families with highly conserved MYST domain.It is different from the majority of HATs families acetylize histone lysine at the tail randomly,Mof has strict substrate specificity for lysine at position 16 of histone H4.Mof plays an important role in the activities on the basis of chromatin,including embryonic development,cell differentiation,DNA damage repair and tumorigenesis.Histone acetylation has important significance in bone marrow mesenchymal stem cells differentiate to the osteoblast.The increased expression of osteogenic differentiation related protein Runx2 is connected with H3K9ac and H3K14ac.HDAC8 inhibits the osteogenetic differentiation by suppressing H3K9 acetylation and Runx2 activity.Histone deacetylase inhibitors can promote osteogenic differentiation.So,there has important research significance and clinical significance for the regulating mechanism in the exploration of Mof during osteoblast differentiation.Objective:We induced osteogenic differentiation in ST2 cells and MC3T3-E1 cells by osteogenesis induction medium,checking by alkaline phosphatase staining and alizarin red staining,to explore the expression of Mof.Investigating the osteoblast differentiation after interfereing Mof expression by siRNA.We researched the protein and RNA expression of Bmp,p-catenin,Runx2 and Osterix after interfereing Mofexpression and overexpressing Mof by Western Blotting and Q-PCR experiments.We also explored the interactive protein by Co-IP experiment method.Research Methods:1.We cultured mouse bone marrow mesenchymal stem cells ST2 cells,preosteoblast MC3T3-E1 cells and induced osteogenetic differentiation by osteogenesis inductionmedium,the result was testified by alkaline phosphatase staining.We observed the expression of Mof,H4K16ac and ?-catenin,Runx2 and Osterix,at 0day,3day,7day,14day after osteogenetic differentiation.2.We interfered with the expression of Mof by using siRNA in ST2 cells,MC3T3-E1 cells to test whether the osteogenetic differentiation is restrained.Observing the changes in the expression of Runx2 and Osterix after decreasing Mof expression.3.Detecting Bmp2,?-catenin,Runx2 and Osterix expression when we transiently transfected Flag-Mof plasmid in ST2 cells,MC3T3-E1 cells to overexpress Mof.4.To verified the interactive relationship between Mof and osteogenetic differentiation related proteins Runx2,Osterix and P-catenin,we used Co-IP experiment in wild type and osteogenetic differentiation ST2 cells,MC3T3-E1 cells.5.We observed the changes of bone structure between control group and whole body knockout Mof mice by HE stainning.Results:1.The expression level of Mof,H4K16ac were enhanced with osteogenetic differentiation days increasing in ST2 cells and MC3T3-E1 cells.The expression level of Bmp2,?-catenin,Runx2,Osterix were also increased.2.Osteogenetic differentiation is restrained after interferencing the expression of Mof through siRNA.Bmp2 and ?-catenin have no change,but Runx2 and Osterix decreasingly expressed.3.The protein expression level of Runx2 and Osterix was enhenced with Mof overexpressing.4.Co-IP experiment has demonstrated that there were interactive relationship between Mof and Runx2,Osterix but not ?-catenin.5.The study of individual level of mice found that the morphological structure of bone did not change significantly after knockout Mof.Phenotype change was not obvious because of the number of days was too short.Conclusions:Mof expresses increasingly in the process of osteoblast differentiation.Osteoblast differentiation is inhibited after interfering expression of Mof.Mof has interaction with Runx2 and Osterix in the protein level.Our research indicate Mof plays a significance role during osteoblast differentiation.