Abstract: Objective To study the effect of TLR4 activation with LPS on BMP9-induced osteogenic differentiation of immortalized mouse embryonic fibroblasts(iMEFs). Methods The activation of TLR4/NF-κB signaling pathway was detected by ICC.iMEFs were treated with LPS,BAY11-7082,Adnovirus GFP and BMP9. The early osteogenic differentiation capability ofiMEFs was detected by ALP staining and quantitative assay. The later osteogenic differentiation capability was detected by alizarin red S staining. The expression of later osteogenic differentiation marker gene OCNand OPNwere detected by PCR and Western blot. The change of p-Smad1/5/8 was detected by Western blot. The expression of Runx2and Dlx5 were detected by PCR and Western blot. Results LPS can stimulate TLR4/NF-κB signaling pathway effectively. TLR4 activation inhibited BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation related gene and Smad1/5/8 signaling activation were inhibited by TLR4 activation. The inhibition effect could be partly reversed by BAY11-7082(P<0.05).Conclusions TLR4 activation with LPS can inhibit BMP9-induced osteogenic differentiation of iMEFs cellsvia NF-κB signaling pathway.

Key words: TLR4, BMP9, iMEFs , osteogenic differentiation