GATA4 Regulates Osteoblastic Differentiation And Bone Remodeling Via P38-mediated Signaling

Objective:To study the role of GATA4 in osteoblast differentiation and bone rebuilding by GATA4 knockdown,both in vitro and in vivo.Methods:Flow cytometric analysis was conducted to identify osteoblast precursor cells.Then cells were transfected with shGATA4 and shCTRL respectively,and the expression of GATA4 as well as osteogenic markers((osterix(OSX),osteopontin(OPN),and osteocalcin(OCN))was detected by western blotting.The osteogentic differentiation capacity of osteoblast precursor cells transfected was observed by ALP staining,alizarin red staining,western blotting and Real-time PCR.Mouse tooth movement model was built,and lentivirus was injected locally to knockdown GATA4.Immunohistochemistry was carried out to detect the expression of GATA4 as well as osteogenic markers(OSX,OPN and OCN).Micro-CT and histological analyses such as total collagen and Goldner's trichrome staining was conducted to observe new bone formation.To explore the pathway involved in the process,Western Blotting was conducted to dectect the expression of MAPK pathway related proteins(p38,p-p38,ERK and p-ERK).Results:Identification of osteoblast precursor cells by flow cytometric analysis demonstrated that the cells have similar characteristics as BMSCs.Western blotting relvealed that the expression of GATA4 and osteoblast-specific marker proteins transiently increased during osteoblast differentiation.In vitro experiment showed that the expression level of GATA4 protein was much lower in shGATA4 group than that in the control group(shCTRL).In shGATA4 group,the expression of osteogenic markers(OSX,OPN and OCN)were significantly downregulated at day 14(P<0.05).In vivo experiment showed the expression of GATA4 and osteoblast-specific marker proteins was decreased in the shGATA4 group,the new bone formation of alveolar bone between the mesial and distal roots of the first maxillary molar of the shGATA4 group was also significantly decreased compared to the control group at day 14(P<0.05).Western blotting revealed the espression of p-p38 was significantly reduced in the shGATA4 group at day 7 after mineralization induction,while levels of phosphorylated ERK were unaffected.Conclusion:The expression level of GATA4 was successfully downregulated both in vitro and in vivo,and the downregulation of GATA4 inhibit the differentiation of osteoblasts and bone remodeling via p38-mediated signaling.