Adipocytes Promote The Osteoclast Differentiation And Function Of Through CXCL12/CXCR4 Signaling Pathway

Objective: The purpose of this study was to investigate the direct effect of adipocytes on the osteoclast differentiation and function and further attempted to elucidate the possible signaling pathway.Methods: ST2 cells,a cloned stromal cell line from mouse bone marrow and possessing the ability to differentiate into adipocytes,were treated with adipogenic differentiation medium for 21 days.Differentiated ST2-adipocyte cells were cultured in serum-free α-MEM for 12 – 16 h and then the medium was collected to be used as the adipocyte-conditioned medium(ADIPO CM).RAW264.7 cells were cultured in adipocyte-conditioned medium in the presence of 10ng/ml RANKL and bone marrow-derived macrophages(BMMs)were cultured in adipocyte-conditioned medium in the presence of 10ng/ml RANKL and 30ng/ml M-CSF to induce osteoclast differentiation.TRAP staining was used to visualize the induced multinucleated osteoclast cells.Resorption pit assay was performed to evaluate the mineral resorptive ability of osteoclast.m RNA expression of osteoclast adhesion molecules was determined by q RT-PCR.Western blot analysis was used to detect the protein level of osteoclast adhesion molecules and osteoclast functional factors.Results: ELISA analysis revealed that CXCL12 was abundantly detected in adipocyte-conditioned medium.CCK-8 assays showed that there is no proliferation effect of exogenous CXCL12(20 ng/ml,50 ng/ml or 100 ng/ml)on RAW264.7 cells.Adipocyte-conditioned media enhanced osteoclast formation and resorption ability both by RAW264.7 cells and BMMs.Besides,exogenous CXCL12 efficiently potentiated the formation of TRAP-positive osteoclast and resorptive ability by RAW264.7 cells.Western blot and q RT-PCR suggested that adipocyte-conditioned medium or combined treatment with exogenous CXCL12 caused a significant increase in the expression of NFAT2,src and osteoclast adhesion-related molecules including β3 Integrin,CD44 and OPN.However,these promotive effects were largely abrogated upon treatment of AMD3100,an antagonist against CXCR4.Conclusion: Adipocytes promote osteoclast differentiation and function through CXCL12/CXCR4 signaling pathway.