Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.

The axonal guidance proteins semaphorin (Sema)4D and Sema3A play important roles in communication between osteoclasts and osteoblasts. As stimulation of adenosine A2A receptors (A2AR) regulates both osteoclast and osteoblast function, we asked whether A2AR regulates both osteoclast and osteoblast expression of Semas. In vivo bone formation and Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1 protein expression were studied in a murine model of wear particle-induced osteolysis. Osteoclast/osteoblast differentiation were studied in vitro as the number of tartrate-resistant acid phosphatase+/Alizarin Red+ cells after challenge with CGS21680 (A2AR agonist, 1 µM) or ZM241385 (A2AR antagonist, 1 µM), with or without Sema4D or Sema3A (10 ng/ml). Sema3A/PlexinA1/Neuropilin-1, Sema4D/PlexinB1, and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) expression was studied by RT-PCR and Western blot. β-Catenin activation and cytoskeleton changes were studied by fluorescence microscopy and Western blot. In mice with wear particles implanted over the calvaria, CGS21680 treatment increased bone formation in vivo, reduced Sema4D, and increased Sema3A expression compared with mice with wear particle-induced osteolysis treated with vehicle alone. During osteoclast differentiation, CGS21680 abrogated RANKL-induced Sema4D mRNA expression (1.3 ± 0.3- vs. 2.5 ± 0.1-fold change, P < 0.001, n = 4). PlexinA1, but not Neuropilin-1, mRNA was enhanced by CGS21680 treatment. CGS21680 enhanced Sema3A mRNA expression during osteoblast differentiation (8.7 ± 0.2-fold increase, P < 0.001, n = 4); PlexinB1 mRNA was increased 2-fold during osteoblast differentiation and was not altered by CGS21680. Similar changes were observed at the protein level. CGS21680 decreased RANKL, increased OPG, and increased total/nuclear β-catenin expression in osteoblasts. Sema4D increased Ras homolog gene family, member A phosphorylation and focal adhesion kinase activation in osteoclast precursors, and CGS21680 abrogated these effects. In summary, A2AR activation diminishes secretion of Sema4D by osteoclasts, inhibits Sema4D-mediated osteoclast activation, and enhances secretion of Sema3A by osteoblasts, increasing osteoblast differentiation and diminishing inflammatory osteolysis.-Mediero, A., Wilder, T., Shah, L., Cronstein, B. N. Adenosine A2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis.