CXCL2 increased osteoclast differentiation and enhanced osteoclastic bone resorption
In experiments conducted to evaluate the effects of CXCL2 on osteoclastogenesis, cultures from all groups formed osteoclasts on plastic and the average numbers of osteoclasts were similar in the control cultures and cultures exposed to a low concentration of CXCL2 (10 ng/mL). However, high doses of CXCL2 tended to promote the formation of osteoclasts (Fig. 4A). Formation of the F-actin ring by osteoclasts is a necessary step in bone resorption. Immunofluorescence microscopy of CD14+ monocytes cultured for 14 days treated with CXCL2 (100 ng/ml) in the presence of M-CSF and RANKL revealed that F-actin rings were significantly increased in number (Fig. 4B). CXCL2 stimulation also increased cell fusion, resulting in multinucleated cells (Fig. 4C), which suggested that CXCL2 promoted osteoclast formation.
Functional evidence of osteoclast formation was obtained via the lacunar resorption assay system using cell cultures on dentine slices. The result showed that the resorption area was increased in bone slices exposed to CXCL2 (Fig. 4D). Expression of the osteoclast markers RANK, cathepsin K, and TRAP was significantly increased after 3 days of stimulation with the CXCL2 (100 ng/mL) in the presence of M-CSF (50 ng/ml) and RANKL (100 ng/ml) (p < 0.05; Fig. 4E).
Consistent with the results obtained in CD14+ monocytes, the effects of exogenous CXCL2 on osteoclast formation were also observed in RAW264.7 cells. Compared with the control group, much larger multinucleated cells were evident in the CXCL2 groups (Fig. 5A). Expression of osteoclast markers such as cathepsin K and TRAP was also significantly increased in the CXCL2 group after 3 days of stimulation in the presence of M-CSF and RANKL (p < 0.05; Fig. 5B). These results suggest that CXCL2 enhances osteoclast formation and bone resorption. In addition, CXCL2 significantly increased the expression of NFATc1 and c-Fos by RAW264.7 cells after 5 days of stimulation with M-CSF (10 ng/ml) and RANKL (50 ng/ml) (p < 0.05; Fig. 5C).