The Role Of PDK1/AKT In The Process Of Osteoclastogenesis

Objective:To investigate the effect of PDK1/AKT on osteoclast differentiation and provide a theoretical basis for the study of specific mechanisms of osteoclatogenesis.Method:Using two different ways to culture RAW264.7 macrophages cells:normal culture medium or addition of 50ng/ml Macrophage Colony-Stimulating Factor(M-CSF)and100ng/ml Nuclear Factor K?Receptor Activating Factor Ligand.(RANKL)as differentiation culture medium.(1)On the fourth day,mature osteoclasts were identified by TRAP staining and immunofluorescence staining,and the expression of p-AKT protein in cells of normal cultured medium or differentiation cultured medium was detected.(2)Add AKT inhibitor or PDK1 inhibitor to intervene RAW264.7 differentiation.The relative expression of DC-STAMP and NFATc1 mRNA were detected on the 2nd and 4th day after the intervention.On the 4th day after intervention,the TRAP staining positive cells with the area of?2000?m~2 were counted.The expression of DC-STAMP protein in cells was detected.Result:(1)50ng/ml Macrophage Colony-Stimulating Factor(M-CSF)and 100ng/ml Nuclear Factor K?Receptor Activating Factor Ligand(RANKL)can induce differentiation to osteoclasts,during which p-AKT protein expression is decrease.(2)The addition of AKT inhibitors or PDK1 inhibitors can differentiating many large area mature osteoclasts.(3)The expression of DC-STAMP protein and DC-STAMP mRNA increased significantly with intervention by inhibitors.(4)NFATc1 mRNA expression was affected by inhibitors intervention.Conclusion:(1)PDK1/AKT negatively regulates osteoclast.(2)PDK1/AKT effects osteoclastogenesis through regulating DC-STAMP expression.(3)NFATc1 participated in PDK1/AKT regulating osteoclastogenesis.