Adenosine is generated in increased concentrations at sites of injury/hypoxia and mediates a variety of physiological and pharmacological effects via G proteincoupled receptors (A 1, A 2A, A 2B, and A 3). Because all adenosine receptors are expressed on osteoclasts, we determined the role of A 2A receptor in the regulation of osteoclast differentiation. Differentiation and bone resorption were studied as the macrophage colony-stimulating factor-1receptor activator of NF-κB ligand formation of multinucleated tartrate-resistant acid phosphatase (TRAP)positive cells from primary murine bone marrowderived precursors. A 2A receptor and osteoclast marker expression levels were studied by RT-PCR. Cytokine secretion was assayed by enzyme-linked immunosorbent assay. In vivo examination of A 2A knockout (KO)/control bones was determined by TRAP staining, microcomputed tomography, and electron microscopy. The A 2A receptor agonist, CGS21680, inhibited osteoclast differentiation and function (half maximal inhibitory concentration, 50 nmol/L), increased the percentage of immature osteoclast precursors, and decreased IL-1β and tumor necrosis factor-α secretion, an effect that was reversed by the A 2A antagonist, ZM241385. Cathepsin K and osteopontin mRNA expression increased in control and ZM241385-pretreated osteoclasts, and this was blocked by CGS21680. Microcomputed tomography of A 2AKO mouse femurs showed a significantly decreased bone volume/trabecular bone volume ratio, decreased trabecular number, and increased trabecular space. A 2AKO femurs showed an increased TRAP-positive osteoclast. Electron microscopy in A 2AKO femurs showed marked osteoclast membrane folding and increased bone resorption. Thus, adenosine, acting via the A 2A receptor, inhibits macrophage colony-stimulating factor-1receptor activator of NF-κB ligandstimulated osteoclast differentiation and may regulate bone turnover under conditions in which adenosine levels are elevated.
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