Oxidized Low-Density Lipoprotein Enhances Toll-like Receptor-Mediated Osteoclastogenic Responses in the Absence of Exogenous RANKL: Implications for Inflammatory Osteoclastogenesis in Periodontitis.

Background and

Objectives

Oxidized LDL (oxLDL) and Toll-like receptor (TLR) signaling are implicated in inflammatory bone loss and periodontitis, but their cooperative effects on osteoclastogenesis remain unclear. We investigated whether TLR2/4 stimulation regulates the transcription of LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a receptor for oxLDL, and whether oxLDL enhances TLR-mediated osteoclastogenic responses in the absence of exogenous RANKL. Materials and

Methods

Mouse bone marrow cells (BMCs) were differentiated into bone marrow macrophages (BMMs) with M-CSF and stimulated with TLR ligands (Pam3CSK4 for TLR2 and Lipid A for TLR4) and/or oxLDL, with or without exogenous RANKL. Raw264.7 cells were stimulated with TLR ligands or Porphyromonas gingivalis lipopolysaccharide (LPS). LOX-1 transcriptional activity and osteoclastogenic responses were evaluated using luciferase assays, quantitative RT-PCR, and tartrate-resistant acid phosphatase (TRAP) staining.

Results

In Raw264.7 cells, stimulation with Pam3CSK4, Lipid A, or LPS significantly increased LOX-1 promoter activity. In BMCs, Pam3CSK4 significantly increased LOX-1 and MyD88 mRNA expression on day 1, whereas expression decreased by day 3; these changes were suppressed by a TLR2 inhibitor. Lipid A significantly increased LOX-1 and MyD88 mRNA expression on day 3, and the increase in LOX-1 expression was inhibited by a TLR4 inhibitor. Functionally, Pam3CSK4 or Lipid A alone increased mononuclear TRAP-positive cells but did not induce multinucleated TRAP-positive cells. In contrast, co-stimulation of oxLDL with either Pam3CSK4 or Lipid A promoted multinucleated TRAP-positive cell formation even in the absence of exogenous RANKL.

Conclusions

TLR2/4 stimulation transiently enhanced LOX-1 transcription and expression in association with increased MyD88 expression. TLR ligands cooperated with oxLDL to promote multinucleated TRAP-positive cell formation in the absence of exogenous RANKL, suggesting that inflammatory and metabolic signals may act synergistically during osteoclastogenesis. These findings improve our understanding of how dyslipidemia-associated factors may contribute to inflammatory bone-resorptive diseases, including periodontitis.