Effects Of PDK1 On Proliferation,differentiation And Metabolic Function Of MC3T3-E1 Cells Under Hypoxia

Objective:This study is conducted to observe the effects of hypoxia on osteoblast proliferation,differentiation and metabolic functions by mimicking hypoxic environment in vitro,and to explore the role of PDK1 signaling pathway on osteogenic differentiation in hypoxic environment,revealing the molecular basis of the biological activities of osteoblasts in the local hypoxic microenvironment of bone diseases.Methods:In the study,the MC3T3-E1 cells were subjected to a hypoxic environment（2%O₂）constructed by triple gas incubator.Part 1:（1）The effect of hypoxia on the vital activity of MC3T3-E1 cells was evaluated by measuring live cell counting,CFSE staining,cell cycle and apoptosis after 3 days of continuous hypoxia,and the specific stages of the effect of hypoxia on proliferation was investigated by live cell counting,CFSE staining and cell cycle analysis of MC3T3-E1 cells cultured under normoxia and hypoxia for 1 and 3 days;（2）Detecting the expression levels of c-fos,Runx2 m RNA and the protein were to assess the effect of hypoxia on the proliferation and early differentiation of MC3T3-E1 cells;（3）The capacity of glucose intake,lactate content,mitochondrial mass,reactive oxygen species concentration and PDK1 signaling-related proteins and their m RNA expression levels were tested to reveal the effects of hypoxia on metabolic function and PDK1 signaling in MC3T3-E1 cells.Part 2:The protein expression levels of p-PDK1,p-AKT,HIF-1α,Glut1 and Runx2 in MC3T3-E1 cells were measured after 3 days of intervention with PDK1 activator PS48,these were to investigate the actions of PDK1 on the early differentiation of MC3T3-E1cells under hypoxic environment.Results:（1）The number of live MC3T3-E1 cells cultured in hypoxia for 3 days was significantly lower than that of cells in the normoxia group（P<0.0001）,while the mean fluorescence intensity of CFSE（P<0.01）,the number of S-phase cells（P<0.0001）and the apoptosis rate（P<0.01）were remarkably higher than that of the normoxia group;however,MC3T3-E1 cells cultured in hypoxia for 1 day cells（P<0.05）and the number of S-phase cells（P<0.0001）were significantly lower than those in the normoxic group,while the mean fluorescence intensity of CFSE（P<0.0001）was significantly higher than that in the normoxic group.（2）Compared with the normoxic group,hypoxia inhibited Runx2 m RNA expression in MC3T3-E1 cells（P<0.05）but increased its protein level（P<0.01）;meanwhile,hypoxia promoted p-PDK1（P<0.01）,p-AKT（P<0.01）,HIF-1α（P<0.01）and Glut1（P<0.05）protein expression.（3）Hypoxia increased glucose uptake（P<0.01）and stimulated lactate secretion（P<0.05）in MC3T3-E1 cells;as well as increased mitochondrial mass（P<0.05）and reactive oxygen concentration（P<0.01）.（4）MC3T3-E1 cells with combined intervention of PDK1 activator and hypoxia showed increased expression of p-PDK1（P<0.01）,p-AKT（P<0.01）,HIF-1α（P<0.01）and Runx2（P<0.05）proteins compared to cells with PDK1 activator alone,and there was no interaction between hypoxia and PDK1 activator intervention on MC3T3-E1 cell differentiation（P=0.4996）.Conclusions:（1）Hypoxia inhibits the proliferation of MC3T3-E1 cells,acts mainly in the division phase,promotes the differentiation of osteogenesis and enhances the ability of glycolysis.（2）Hypoxia promotes early osteogenic differentiation of MC3T3-E1 cells by increasing their glucose uptake capacity through the PDK1/AKT signaling pathway.