Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Adenosine A2A Receptor Ligation Inhibits Osteoclast Formation.
Mediero1, Aranzazu, Kara1, Firas M., Hawly2, Maya, Sellam2, Elie, Cronstein3, Bruce N.
Adenosine is a nucleoside that is generated at sites of injury and hypoxia and which mediates its physiologic and pharmacologic effects via activation of one or more G protein coupled receptors (A1, A2A, A2B and A3). We have previously reported that adenosine A1 receptor ligation is required for osteoclast formation and that blockade or deletion of adenosine A1 receptors leads to increased bone density. Because all four adenosine receptors are expressed on osteoclasts and osteoclast precursors we determined whether adenosine A2A receptors also regulate osteoclast formation in vivo and in vitro.
Osteoclast differentiation was studied in vitro as the GM-CSF/RANKL stimulated formation of multinucleated (>=3 nuclei), TRAP-positive cells from primary murine (C57Bl/6) bone marrow-derived precursors. Signaling events were studied by Western Blot for activated (phosphorylated) signaling molecules and changes in message were determined by RT-PCR. Histological examination of bones from A2A knockout and wild type mice was carried out with TRAP and alcian blue stains. Murine bone was further analyzed by micro CT and electron microscopy.
The highly selective A2A receptor agonist CGS21680 inhibited osteoclast differentiation as much as 38±1% with an IC50 of approximately 50nM (p<0.05), reversed by the selective high affinity antagonist ZM241385. CGS21680 (1uM) stimulated phosphorylation of ERK1/2 (122±3 and 134±3% of control at day 3 and 7 of differentiation, p<0.001 for both) and JAK1 (107±1% of control phosphorylation at day 7 of differentiation, p<0.01), activation that was reversed by ZM241385 (not shown). Moreover, CGS21680 inhibited NFkB translocation to the nucleus (nuclear NFkB decreased by 44±5% at day 3, p<0.001). There was also a marked increase in cellular IkB levels (maximal IkB increased by 19±1% at day 7, p<0.001) whereas pretreatment with ZM241385 increased phosphorylation of IkB (maximal pIkB increased by 13±0.4% at day 6, p <0.001) and enhanced nuclear translocation of NFkB (by 14±2% at day 3, p<0.01). MicroCT analysis of femurs from A2AKO mice showed significantly decreased bone volume/total volume ratio (17.49±0.95 for A2AKO vs. 22.61±2.15 for wild type, p<0.05), trabecular number (4.4±0.2 for A2AKO vs 5.8±0.07 for wild type, p<0.001) and increased trabecular space (0.19±0.006 for A2AKO vs. 0.13±0.0004 for wild type, p<0.001). There were more osteoclasts in the TRAP-stained femurs of A2AKO (50±13/lpf) than wild type mice (35±1/lpf, 6 fields each from femurs of 2 different mice each). Electron microscopy of osteoclasts in femurs from A2AKO mice showed marked osteoclast membrane folding and increased osteoclast bone resorption.
These results indicate that adenosine A2A receptors inhibit GM-CSF/RANKL-stimulated osteoclast differentiation and thereby regulate bone turnover. Because adenosine mediates the anti-inflammatory effects of methotrexate we further speculate that the capacity of methotrexate to inhibit bone erosion in patients with Rheumatoid Arthritis may be mediated by methotrexate-stimulated increases in adenosine concentration.
To cite this abstract, please use the following information:
Mediero, Aranzazu, Kara, Firas M., Hawly, Maya, Sellam, Elie, Cronstein, Bruce N.; Adenosine A2A Receptor Ligation Inhibits Osteoclast Formation. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1471