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

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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.

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 <i>μ</i>g/ml Pam3CSK4 induced osteoclastogenesis without adding RANKL exogenously, whereas 1 <i>μ</i>g/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.

Journal

  • Dental Medicine Research

    Dental Medicine Research 32(3), 181-188, 2012-11-30

    Showa University Dental Society

References:  25

Codes

  • NII Article ID (NAID)
    10031141316
  • NII NACSIS-CAT ID (NCID)
    AA12322983
  • Text Lang
    ENG
  • Article Type
    ART
  • ISSN
    18820719
  • NDL Article ID
    024268155
  • NDL Call No.
    Z19-1251
  • Data Source
    CJP  NDL  IR  J-STAGE 
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