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 P2X(7) is involved in this fusion process. Prolonged exposure of macrophages to ATP, the ligand for P2X(7), 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 P2X(7) 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-kappaB 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 P2X(7) protein, but little P2X(7) 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 P2X(7) on the cell surface, regained sensitivity to ATP, and formed multinucleated osteoclasts. These results suggest that P2X(7) or another protein that is downregulated in concert with P2X(7) 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 P2X(7) may be regulated by ligand-mediated internalization and that extracellular ATP may regulate the formation of osteoclasts and other multinucleated giant cells.
Hiken
Jeffrey F
JF
Dept. of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA. hiken@id.wustl.edu
Steinberg
Thomas H
TH
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D005110:Extracellular Space / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015726:Giant Cells / Q000166:cytology / Q000378:metabolism; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D015854:Up-Regulation
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C546469:P2rx7 protein, mouse; D010975:Platelet Aggregation Inhibitors; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate