RAC1 Regulate Tumor Necrosis Factor-a-mediated Impaired Osteogenic Differentiation Of Dental Pulp Stem Cells

Aim To understand the effect of high concentration TNF-α on the osteogenic differentiation of dental pulp stem cells(DPSCs),and the mechanism.Methods We isolated and cultured DPSCs.To induce osteogenic differentiation,DPSCs were cultured in osteogenic medium and treated with TNF-α in different concentrations during differentiation.Osteogenic differentiation of DPSCs was demonstrated by ALP and Alizarin red S staining.DPSCs were cultured in osteogenic medium and stimulated with 100 ng/mL TNF-α for 3,5,7 and 14 days.The expression level of RAC1 and osteogenic marker protein RUNX2,BMP2 were detected by Western blot.The expression level of Wnt/β-catenin pathway relative protein β-catenin and GSK3β were detected by immunofluorescent staining and Western blot.We used siRNA to genetic silence of RAC1 expression.The expression level of RAC1 were detected by Western blot and RT-PCR.Osteogenic differentiation of DPSCs was demonstrated by ALP staining.The expression level of osteogenic marker protein RUNX2,BMP2 and Wnt/β-catenin pathway relative protein β-catenin and GSK3β were detected by Western blot.Results ALP activity and Alizarin red S staining for mineralized matrix results show that influence of TNF-α on osteogenic differentiation of DPSCs was different in different concentrations.TNF-α promotes the formation of mineralized nodules and the activity of ALP at 10 ng/mL concentration.But the osteogenic potential of DPSCs was significantly decreased with the increasing concentration of 50 and 100 ng/mL.The RAC1 protein level did not reveal any statistically significant changes with the treatment of TNF-α at the concentration of 10 ng/mL.But the protein level was relatively higher with the treatment of TNF-α at the concentration of 50 and 100 ng/mL.After TNF-α stimulation the Wnt/β-catenin signaling pathway was activated.Genetic silencing of RAC1 expression using siRNA could restore osteogenic differentiation of DPSCs.Conclusion In summary,our findings conclude that TNF-α suppresses osteogenic differentiation of human DPSCs by increasing the expression of RAC1,and then activating the Wnt/β-catenin signaling pathway.Block RAC1 can restore osteogenic differentiation of DPSCs,which might have clinical implications for inflammatory bone disease.