Fig. 4: Heparan sulfate suppresses osteoclastogenesis through binding to M-CSF.

a Cell proliferation. Osteoclast precursors (left panel) and RAW264.7 cells (right panel) were treated with heparan sulfate or chondroitin sulfate in the presence and absence of M-CSF for 3 days, respectively. Cell growth was measured by MTT assay. b Osteoclast differentiation. Osteoclast precursors were treated with either heparan sulfate or chondroitin sulfate and differentiated into osteoclasts for 4 days. Cells were stained for TRAP (left panel) and the number of TRAP(+) MNCs was counted (right panel). Scale bar, 200 μm. c Osteoclastogenic marker gene expression. Osteoclast precursors were treated with heparan sulfate or chondroitin sulfate and differentiated into osteoclasts for 3 days. The expression levels of the osteoclast markers (NFATc1, c-Fos, and ATP6V0D2) were assessed by immunoblot analysis. β-Actin was used as a loading control. d M-CSF-dependent signaling. Osteoclast precursors were incubated with heparan sulfate or chondroitin sulfate (100 μg/mL) for 4 h and then stimulated with M-CSF (5 ng/mL). The activity of M-CSF-dependent signaling was examined by immunoblot analysis. e Competitive binding assay. A pre-mixture of M-CSF and heparan sulfate or chondroitin sulfate at various concentrations was incubated with His-tagged syndecan ectodomains from HEK293E cells bound on the Ni-coated plate. After washing, the level of M-CSF on Ni-coated plate was analyzed as described in Fig. 2d to assess the binding affinity of M-CSF to syndecan ectodomains. Results represent the means ± SD (n = 4 as in a; n = 3 as in b and e). The p value indicates the comparison between the treatment group and control. *p < 0.05; **p < 0.01