RESUMO
Osteoclasts (OCs) differentiate from monocyte/macrophagelineage hematopoietic precursor cells, which are known as OC precursors (OCPs). Several studies have investigated cell chemotaxis in the bone microenvironment; however, OCP migration ability in the bone microenvironment during OC differentiation is yet to be elucidated. As an initial investigation of this characteristic, the present study aimed to determine the effects of transforming growth factor (TGF)ß1 on OCP migration in vitro. Preosteoclastic RAW264.7 cells were cultured with and without TGFß1 (2, 5 or 20 ng/ml), receptor activator of NFκB ligand (RANKL; 50 ng/ml), and/or SB431542 (10 µM), a potent and specific inhibitor of TGFß1 receptor kinase activity. Cell proliferation was significantly inhibited in the presence of TGFß1 for 3 days, and the effect was reversed by SB431542. Tartrateresistant acid phosphatase (TRAP) activity in RAW264.7 cells was significantly increased by RANKL treatment, compared with TRAP activity in control cells on day 3. The highest TRAP activity in RAW264.7 cells was induced by the combined treatment with TGFß1 (2 ng/ml) and RANKL. When TGFß1 signaling was inhibited by addition of SB431542 to the medium during culture, OC differentiation was notably suppressed. These findings suggest that TGFß1 accelerates RANKLinduced OC differentiation, but does not act in a dosedependent manner. The migration of RAW264.7 cells was promoted at 24 h, but was suppressed at 72 h, during RANKLinduced osteoclast differentiation in the presence of TGFß1. These results were accompanied with the increased expression of small Gproteins, RhoA and Rac, at 24 h, but their expression decreased at 72 h. RAW264.7 cells treated with TGFß1 for 24 h underwent morphological changes, from round to polygonal morphology. Furthermore, protrusions were completely lost and the cell morphology reverted from polygonal to round after TGFß1 treatment for 72 h. Therefore, our findings indicated that OCP migration may be modified by differentiation in vitro.
