ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells

Multinucleated giant cells derive from fusion of precursor cells of the macrophage lineage. It has been proposed that the purinoreceptor P2X₇ is involved in this fusion process. Prolonged exposure of macrophages to ATP, the ligand for P2X₇, induces the formation of plasma membrane pores and eventual cell death. We took advantage of this cytolytic property to select RAW 264.7 (RAW) cells that lacked P2X₇ function by maintaining them in ATP (RAW ATP-R cells). RAW ATP-R cells failed to fuse to form multinucleated osteoclasts in response to receptor activator nuclear factor-B ligand, although they did become positive for the osteoclast marker enzyme tartrate-resistant acid phosphatase, and upregulated expression of other osteoclast marker genes. RAW ATP-R cells and wild-type RAW cells expressed similar amounts of P2X₇ protein, but little P2X₇ was present on the surface of RAW ATP-R cells. After ATP was removed from the medium of RAW ATP-R cells, the cells reexpressed P2X₇ on the cell surface, regained sensitivity to ATP, and formed multinucleated osteoclasts. These results suggest that P2X₇ or another protein that is downregulated in concert with P2X₇ is involved either in the mechanics of cell fusion to form osteoclasts or in a signaling pathway proximal to this event. These results also suggest that P2X₇ may be regulated by ligand-mediated internalization and that extracellular ATP may regulate the formation of osteoclasts and other multinucleated giant cells. macrophage fusion; P2X receptor; purinergic receptor; receptor activator nuclear factor-B