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. 2007 Aug 28;104(36):14436–14441. doi: 10.1073/pnas.0702811104

Fig. 4.

Fig. 4.

The CD200-CD200R axis is required for osteoclast fusion/multinucleation. (A) Bone-marrow-derived macrophages from 6-week-old wild-type mice were cultured in the presence of M-CSF (30 ng/ml) and RANKL (50 ng/ml) with or without the recombinant extracellular domain of CD200 (sCD200; 0.5 μg/ml) or recombinant prostate-specific antigen as a control. sCD200 allowed the differentiation of osteoclasts in macrophages that lacked CD200 (SD; n = 3). (B) Bone marrow macrophages isolated from CD200-deficient and wild-type mice were cultured in the presence of M-CSF (5 ng/ml) for 12–18 h. Nonadherent cells were cultured for a further 2 days in the presence of M-CSF (30 ng/ml), starved for 2 h, and then treated with RANKL (50 ng/ml) with or without sCD200 (0.5 μg/ml) for 30 min. The cells were then subjected to Western blot analysis with the indicated antibodies against IkBα and JNK and their phosphorylated forms. Numbers represent relative expression of P-IκB and P-JNK over JNK. The addition of sCD200 restored the activation of JNK but not of IκBα. (C) Bone-marrow-derived macrophages from 6-week-old wild-type mice were cultured in the presence of M-CSF (30 ng/ml) and RANKL (50 ng/ml) with or without the recombinant extracellular domain of the CD200 receptor (sCD200R). sCD200R blocked the fusion of macrophages (SD; n = 5), whereas recombinant prostate-specific antigen had no effect (osteoclast surface, 0.38 ± 0.1; osteoclast number, 170.0 ± 23.3). (D) Bone-marrow-derived macrophages from 6-week-old wild-type mice were cultured in the presence of M-CSF (30 ng/ml) for 2 days before being transduced with the retroviral vector MigR1, which encoded, or not, shRNAs designed after the CD200R1 cDNA. A construct encoding random (rdm) oligonucleotides was used as a control. Each of the three targeting retroviral constructs, namely shRNAi1, shRNAi2, and shRNAi3, abolished the expression of CD200R1 (see Western blot, Bottom Left) and prevented the formation of multinucleate osteoclasts (Top and Right). These experiments were reproduced several times with similar results.

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