Activation of adenosine A_2A receptor reduces osteoclast formation via PKA‐ and ERK1/2‐mediated suppression of NFκB nuclear translocation

Abstract

BACKGROUND AND PURPOSEWe previously reported that adenosine, acting at adenosine A2A receptors (A2AR), inhibits osteoclast (OC) differentiation in vitro (A2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2AR signalling. EXPERIMENTAL APPROACHOC 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 A2AR agonist CGS21680, the A2AR 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 RESULTSA2AR 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. A2AR stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A2AR stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A2AR stimulation inhibited OC expression of differentiation markers by a PKA-mechanism. CONCLUSIONS AND IMPLICATIONSA2AR activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis.

Direct or indirect stimulation of adenosine A 2A receptors enhances bone regeneration as well as bone morphogenetic protein‐2

“…On one hand, A2AR activation inhibits osteoclast differentiation and function both in vitro and in vivo. A2AR stimulation suppresses osteoclast differentiation and function in vitro and in vivo (30,38), confirmed here by altered expression of osteoclast markers in vitro and a marked reduction of osteoclasts in lesional bone in vivo. On the other hand, A2AR activation promotes osteoblast expression of key markers for bone formation, such as osteoclacin and osteonectin (Supplemental Figs.…”

Signaling pathways involving adenosine A2A and A2B receptors in wound healing and fibrosis

“…A 2A and A 2B receptors, the subtypes most relevant to fibrosis, both signal via a G s protein and activate adenylyl cyclase leading to an increase in intracellular cAMP [9,10]. The rise of cAMP, in turn, activates PKA and other downstream targets that influence gene expression [11]. Termination of adenosine signaling involves equilibrative nucleoside transporters which transports adenosine from the extracellular to the intracellular space down its concentration gradient [12].…”

Adenosine A2B receptors play an important role in bone homeostasis

“…Thus, our laboratory has previously reported that blockade or deletion of A1R leads to increased bone density in mice since endogenous activity of A1R is important in osteoclast formation by bone marrow cells [2][3][4]. In contrast, A2AR ligation enhances bone regeneration by inhibiting differentiation of osteoclasts and by promoting osteoblast functionality and, when deleted in vivo, leads to a marked reduction in bone density in mice [5][6][7][8].…”