Activation of adenosine A 2A receptor reduces osteoclast formation via PKA ‐ and ERK1 /2‐mediated suppression of NF κ B nuclear translocation

Background and Purpose We previously reported that adenosine, acting at adenosine A 2A receptors ( A 2A R ), inhibits osteoclast ( OC ) differentiation in vitro ( A 2A R activation OC formation reduces by half) and in vivo . For a better understanding how adenosine A 2A R stimulation regulates OC differentiation, we dissected the signalling pathways involved in A 2A R signalling. Experimental Approach OC differentiation was studied as TRAP + multinucleated cells following M ‐ CSF / RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264 .7, in presence/absence of the A 2A R agonist CGS21680 , the A 2A R antagonist ZM241385 , PKA activators (8‐ Cl ‐ cAMP 100 nM, 6‐ Bnz ‐ cAMP ) and the PKA inhibitor ( PKI ). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA ) RAW264 .7 cells (scrambled shRNA as control). OC marker expression was studied by RT‐PCR . Key Results A 2A R stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385 . The direct PKA stimuli 8‐ Cl ‐ cAMP and 6‐ Bnz ‐ cAMP inhibited OC maturation whereas PKI increased OC differentiation. A 2A R stimulation inhibited p50/p105 NF κ B nuclear translocation in control but not in PKA KO cells. A 2A R stimulation activated ERK1 /2 by a PKA ‐dependent mechanism, an effect reversed by ZM241385 , but not p38 and JNK activation. A 2A R stimulation inhibited OC expression of differentiation markers by a PKA ‐mechanism. Conclusions and Implications A 2A R activation inhibits OC differentiation and regulates bone turnover via PKA ‐dependent inhibition of NF κ B nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like R heumatoid A rthritis.