FIGURE 3.

The lipopeptide Pam2 and three additional TLR2 agonists stimulate bone resorption, osteoclast formation, and expression of osteoclastic and osteoclastogenic genes in organ cultures of neonatal mouse parietal bones by an effect dependent on RANKL but independent on cytokine and prostaglandin formation. A, LPS P. gingivalis and Pam2 did not affect the mRNA expression of Tlr1, Tlr2, Tlr4, and Tlr6. B–E, Pam2 increased the release of ⁴⁵Ca (B) and CTX (C), up-regulated Ctsk mRNA (D), and enhanced the number of cathepsin K positive (CTSK⁺) osteoclasts (E). F, Pam2 increased the mRNA expression of Tnfrsf11a, Tnfsf11, Csf1, Csf1r, and Oscar without affecting Tnfrsf11b. G–I, Pam2 time-dependently increased Tnfsf11 mRNA (G) resulting in increased RANKL protein after 48 h (H) without affecting Tnfrsf11b mRNA (G) or OPG protein (I). J–L, Pam3 (10 ng/ml), HKLM (10⁷ colony-forming units (CFU)) and FSL-1 (0.1 μg/ml)-stimulated ⁴⁵Ca release (J) and the mRNA expression of Tnfsf11 (K) without affecting Tnfrsf11b (L). M and N, the stimulatory effect by LPS P. gingivalis and Pam2 on ⁴⁵Ca release was unaffected by adding a mixture of antibodies neutralizing IL-1β, IL-6, IL-11, LIF, OSM, and TNF-α or by adding indomethacin (1 μmol/liter). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with unstimulated controls (C, E, H, J, K, M, and N). LPS P. gingivalis (P.g.) was used at a concentration of 10 μg/ml in A, M, and N. Pam2 was used at a concentration of 10 ng/ml in A–I, M, and N. Data are the means of four-five observations, and S.E. is given as vertical bars when larger than the radius of the symbol.