Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Inhibition of Osteoclast Formation by Adenosine A 2AReceptor Is Due to Inhibition of NFkB Nuclear Translocation by a PKA-Mediated Mechanism.
Mediero1, Aranzazu, Cronstein2, Bruce N.
Adenosine, a nucleoside released at sites of injury and hypoxia and which mediates the anti-inflammatory effects of methotrexate, mediates its effects via activation of G-protein-coupled receptors (A1, A2A, A2B, A3). Previously we reported that the A2AR agonist CGS21680 inhibited osteoclast differentiation in a dosedependent manner. We therefore determined which intracellular pathways are involved in A2AR-mediated regulation of osteoclast differentiation
Osteoclast differentiation was studied as the M-CSF/RANKL-stimulated formation of multinucleated TRAP-positive cells from primary murine (C57Bl/6) bone marrow-derived precursors in the presence or absence of CGS21680, ZM241385 (A2A receptor antagonist), activators and inhibitors of PKA/EPAC pathways. Signaling events (PKA/EPAC, MAPK and NFATc1 activation) were studied by Western Blot. A2A Receptor, Cathepsin K and NFATc1 expression were studied by real time RT-PCR. Cytokine expression was assayed by ELISA.
During M-CSF/RANKL-stimulated osteoclast formation the expression of A2AR changed over time, increasing in control and ZM241385 pre-treated cells, an effect which was abrogated in CGS21680-treated cells. Cathepsin K is upregulated in control and ZM241385-treated cells (up to 80 fold, p<0.001) but CGS21680 reduces the increase. NFATC1 was upregulated during osteoclast differentiation under all conditions studied. CGS21680 decreased NFkB nuclear translocation in a time-dependent manner, an effect that was abrogated by ZM241385 (88±2% vs 176±12% of control, CGS21680 alone or with ZM241385, respectively, p<0.001, n=4), a finding confirmed by direct assay of NFkB transcriptional activity. Western Blot showed PKA activation increased over time in the presence of CGS21680 and this correlated with decreased EPAC activation over the same period of time. CGS21680 increased pERK after 5 and 15min, 1h and 12h (to as much as 119±0.6%, p<0.001, n=4), and pre-treatment with ZM241385 reversed this activation. CGS21680 also increased p38MAPK phosphorylation to as much as 110±1% of control, p<0.5, n=4), an effect which was abrogated by ZM241385. Finally, CGS21680 activated pJNK over time to as much as 181±2% of control, p<0.001, n=4) and ZM241385 partially reversed this effect. A PKA inhibitor (PKI) and/or EPAC activator (8-CPT-cAMP) reversed the effect of CGS21680 on osteoclast differentiation.
Adenosine, acting at A2AR, inhibits osteoclast differentiation and regulates bone turnover mainly by the activation of PKA and MAPK, the inhibition of NFkB translocation to the nucleus and decreasing the expression of pro-inflammatory cytokines. Because adenosine mediates the anti-inflammatory effects of methotrexate we speculate that the capacity of methotrexate to inhibit bone erosion in Rheumatoid Arthritis may be mediated by increases in adenosine which inhibit osteoclast formation and function.
To cite this abstract, please use the following information:
Mediero, Aranzazu, Cronstein, Bruce N.; Inhibition of Osteoclast Formation by Adenosine A 2AReceptor Is Due to Inhibition of NFkB Nuclear Translocation by a PKA-Mediated Mechanism. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :2436