The role of the small Rho GTPase Rac2 in mature osteoclasts has not been extensively studied. Rac2−/− mice are of normal size and have normal tooth eruption. However, femoral cortical thickness was significantly greater in Rac2−/− compared to wild-type mice, while percent cortical porosity was lower. As assessed by histomorphometry, trabecular bone mass was significantly higher in male Rac2−/− than wild-type animals, although trabecular bone mass was similar when data from male and female animals were combined. There were no significant differences in the number of osteoblasts per bone surface; however, the number of osteoclasts per total bone area tended to be higher in Rac2−/− mice and was significantly higher in male Rac2−/− mice. In the aggregate, these data suggested a defect in osteoclast function and, consistent with that, rates of bone resorption were significantly reduced in Rac2−/− osteoclasts. In addition, Rac2−/− osteoclasts had a significantly delayed spreading response to treatment with CSF1 for 15 min. Phalloidin staining showed areas of abnormal actin accumulation and impaired actin ring formation in Rac2−/− osteoclasts. Finally, Rac2−/− osteoclasts showed a marked defect in chemotaxis toward a point source of CSF1, with a dramatic reduction in migratory rate. Together, these findings indicate an important role for Rac2 in mature osteoclasts.
The cytoskeleton determines cell shape and is involved in cell motility. It also plays a role in differentiation and in modulating specialized cellular functions. LIM kinase 1 (LIMK1) participates in cytoskeletal remodeling by phosphorylating and inactivating the actin-severing protein, cofilin. Severing F-actin to release G-actin monomers is required for actin cytoskeletal remodeling. Although less well established, LIMK1 may also influence the cell cycle and modulate metalloproteinase activity. Since the role of LIMK1 in bone cell biology has not been reported, the skeletal phenotype of LIMK1−/− mice was examined. LIMK1−/− mice had significantly reduced trabecular bone mass when analyzed by micoCT (p <0.01). Histomorphometric analyses demonstrated a 31% reduction in the number osteoblasts (p=0.0003) and a 23% reduction in osteoid surface (p=0.0005). The number of osteoclasts was no different in control and knock out animals. Consistent with the in vivo findings in osteoblasts, the number of osteoblast colony forming units in LIMK1−/− bone marrow was reduced by nearly 50%. Further, osteoblasts isolated from LIMK1−/− mice showed significantly reduced rates of mineralization in vitro. Osteoclasts from LIMK1−/− mice evidenced more rapid cytoskeletal remodeling in response to treatment with CSF1. In keeping with this latter finding, basal levels of phospho-cofilin were reduced in LIMK1−/− osteoclasts. LIMK1−/− osteoclasts also resorbed dentine slices to a greater extent in vitro and were more active in a pit assay. These data support the hypothesis that LIMK1 is required for normal osteoblast differentiation. In addition, its absence leads to increased cytoskeletal remodeling and bone resorption in osteoclasts.
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