Pam3CSK4, a TLR2 Agonist, Induces Osteoclastogenesis in RAW 264.7 Cells

Abstract

To clarify whether Pam3CSK4, a TLR2 agonist, induces the differentiation of osteoclasts, we investigated the osteoclastogenesis and gene expression induced by Pam3CSK4 in RAW264.7 monocyte/macrophage cells. We found that 1 μg/ml Pam3CSK4 induced osteoclastogenesis without adding RANKL exogenously, whereas 1 μg/ml LPS (Re mutant), a ligand for TLR4, failed to produce osteoclasts in RAW 264.7 cells. The number of TRAP-positive multinuclear cells in the Pam3CSK4 group (156.2 +/− 26.5 cells/well) was significantly (p<0.01) less than that of 100 ng/ml RANKL (196.5 +/− 32.0 cells/well), which was a positive control. Quantitative real-time RT-PCR analysis showed that i) the gene expression levels of TRAP, cathepsin K and matrix metalloproteinase 9, which are osteoclast differentiation markers, were upregulated (p<0.01) by both RANKL and Pam3CSK4, whereas LPS did not increase gene expression of TRAP or cathepsin K, ii) the expression level of RANK was decreased significantly (p<0.01) by both Pam3CSK4 and LPS, but increased by RANKL, iii) the expression levels of TNFα and IL-6, inflammatory cytokines, were upregulated significantly (p<0.01) by both Pam3CSK4 and LPS and iv) the expression level of RANKL was similar to that of other experimental groups in RAW 264.7 cells (p>0.05). Collectively, these results indicate that Pam3CSK4, but not LPS, induces osteoclastogenesis in RAW 264.7 cells in the absence of exogenous RANKL.