Direct stimulation of osteoclastogenesis by MIP-1α: Evidence obtained from studies using RAW264 cell clone highly responsive to RANKL

Macrophage inflammatory protein-1α (MIP-1α) is a member of the CC chemokines. We have previously reported the use of a whole bone marrow culture system to show that MIP-1α stimulates the formation of osteoclast-like multinucleated cells. Here we use rat bone marrow cells deprived of stromal cells, and clones obtained from murine macrophage-like cell line RAW264 to show that MIP-1α acts directly on cells in osteoclast lineage. We obtained several types of RAW264 cell clones, one of these clones, designated as RAW264 cell D clone (D clone), showed an extremely high response to receptor activator of NFκB ligand (RANKL) and tumor necrosis factor-α. (TNF-α), while the other clone, RAW264 cell N clone (N clone), demonstrated no response to RANKL or TNF-α. Although both clones expressed receptor activator NFκB (RANK) before being stimulated for differentiation, only the D clone expressed cathepsin K when cells were stimulated to differentiate to osteoclasts. MIP-1α stimulated the formation of mononuclear preosteoclast-like cells from rat bone marrow cells deprived of stromal cells. MIP-1α also stimulated formation of osteoclast-like multinucleated cells from the D clone, when these cells were stimulated with RANKL and TNF-α. These findings provide strong evidence to show that MIP-1α acts directly on cells in the osteoclast lineage to stimulate osteoclastogenesis. Furthermore, pretreatment of RAW264 cell D clone with MIP-1α significantly induced adhesion properties of these cells to primary osteoblasts, suggesting a crucial role for MIP-1α in the regulation of the interaction between osteoclast precursors and osteoblasts in osteoclastogenesis.