Figure 1.

mCRP but not native C-reactive protein (nCRP) induces osteoclast differentiation of Raw 264.7 cell line. Raw cells were treated with the indicated reagents for 2 days and the expression of osteoclast marker genes was determined with q-PCR. (A) 100 µg/ml mCRP upregulated the expression of TRAP and Cathepsin K, while nCRP at the same concentration was ineffective. (B) mCRP mutant lacking cholesterol-binding sequence motif (Δ35–47) or boiled wild-type mCRP showed impaired capacity to upregulate the expression of TRAP. The effects of LPS at 100 ng/ml, which is 50-fold higher than the residual level of endotoxin in our mCRP preparation, could be abrogated by the copresence of 2.5 µg/ml polymyxin B (PMB) that was included in all differentiation experiments. (C) FITC-labeled nCRP and mCRP were incubated with Raw cells at 4°C and visualized by confocal microscopy. Cell membranes and nuclei were counterstained with FM-4-64 and DAPI, respectively. mCRP showed intense binding to Raw cells, but nCRP did not. (D) Dose-dependent induction of TRAP expression by mCRP. Following treatment with 100 µg/ml nCRP or mCRP for 6 days, Raw cells were stained for TRAP (E) and osteoclast number was counted as TRAP-positive multinucleated cells (F). Raw cells were plated on bone slices and treated with nCRP or mCRP for 6 days. The slices were then stained by toluidine blue (G) to measure eroded surface (H). mCRP but not nCRP induced the differentiation of Raw cells to mature osteoclasts with bone resorption activities.