Runx2 Modulates Osteogenic Differentiation Of Stem Cells In High Glucose Environment

Objective:Diabetes mellitus is a chronic population health hazard systemic metabolic disease characterized by high blood glucose levels,which result from defects in secretion(type 1 diabetes),insulin action(type 2 diabetes),or both.Although diabetic osteopathy involves complex of pathogenesis,including genetic and environmental or acquired factors,the increasing evidence suggested hyperglycemia was one of the most important pathogenesis of diabetic osteopathy.Research indicated that diabetes mellitus modulated osteogenic function in diabetes osteopathy,such as extracellular matrix formation and mineralization.It is reported that Runx2 is one of the most major regulator in the process of osteogenesis and plays a key crosstalk in the multiple signaling pathways.PI3K/AKT,which is a main target molecule of insulin signaling,have been shown to regulate glycogen metabolism;Wnt/β-catenin sigaling pathway has been reported to be a classical osteogenesis pathway.Our research studies indicated that high glucose impaired osteogenesis of BMSCs.However,the exact mechanism still remain unknown.Therefore,our study is to transiently transfected with Runx2 plasmid and detect the effect of Runx2 on osteogenesis in high glucose condition,and then explore the exact mechanism from the aspects of signaling pathways.Method:Part I: We isolated bone marrow stromall cells from SD male mice,BMSCs were primarily cultured and indentified in vitro to undergo osteogenic/adipogenic/chondrogenic differentiation.In vitro the different glucose concentrations in our study were chosen to simulate high glucose and high glucose-associated hyperosmolarity condition,we tested cell activity and proliferation index by CCK-8 and Flow cytometer,as well as ALP activity,ALP staining and Alizarin red S staining were detected to the ability of osteogenesis.Part II: Based on the result,we only chose 25mmol/L as high glucose condition.First cells were transiently transfected with Runx2 plasmid,qRT-PCR and western blotting were used to detect the success rate.In this study,the effect of Runx2 on osteoblast differentiation in high glucose condition were evaluated by the expression of osteogenesis-related maker including Runx2,ALP,OC and OPN,as well as ALP staining and Alizarin red S staining.Western blot analysis were performed to detect the protein expression levels of p-AKT,AKT,p-GSK3β,GSK3β and β-catenin.Immunofluorescence staining analysis were performed to detect subcellular localization of β-catenin.Results:Part I: The BMSCs were be induced to osteogenic.adipogenic.Chondrogenic differentiation in vitro.In high glucose or high glucose-associated hyperosmolarity condition,with a range of glucose concentration(5.5mM-25mM),the concentration of glucose continuing increase,BMSCs proliferation was accelerated,high osmosis displayed a slight decrease of cell proliferation.Cells were cultured in the mineralizing medium,with the concentration of glucose continuing increase,the ability of mineralization differentiation decreased,however,high osmosis groups showed little significantly difference in osteogenesis.Part II: Cells were transfected with Runx2 plasmid,and then qRT-PCR and Western blot analysis suggested that Runx2 was successfully transfected into cells and high-efficiency expression.In the study,pretreatment of Runx2 plasmid significantly increased the expression of Runx2,ALP,OC and OPN mRNA and the matrix biomineralization.Pretreatment of cells with Runx2 plasmid transfected in high glucose increased the protein expression of p-AKT,p-GSK3β and β-catenin.To confirm the role of Runx2 on PI3K/AKT/GSK3β/β-catenin pathway exposed to high glucose,pretreatment of cells with Runx2 plasmid transfected were treated with 10μM PI3K/AKT inhibitor LY294002.We found that the increase protein expression of p-AKT,p-GSK3β and β-catenin by Runx2 were significantly decreased by addition of LY294002.Immunofluorescence staining analysis indicated β-catenin was located in not only cytoplasm but also nucleus in normal glucose condition,however,β-catenin was only detected in the cytoplasm in high glucose condition.When pretreatment of cells with Runx2 plasmid transfected were exposed to high glucose,almost nuclear localization of β-catenin was found.Conclusion:1.Cell proliferation were increased but osteogenic differentiation were inhibited in high glucose condition2.Cells cultured with Runx2 plasmid transfected in high glucose condition alleviated high glucose-suppressed osteogenic differentiation.3.PI3K/AKT signaling pathway,which is a critial molecule of insulin signaling,have been shown to coorperate with classics Wnt//β-catenin signaling through GSK3β to form mutual effect.High glucose significantly reduced the protein expression of p-GSK3β via PI3K/AKT signaling,thereby inhibited the protein expression of β-catenin and impaired BMSCs osteogeneic differentiation.Cells cultured with Runx2 plasmid transfected reversed the effect to some extent.