Rolofylline, an adenosine A1 receptor antagonist, inhibits osteoclast differentiation as an inverse agonist

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5'-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors (A1 R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1 R agonists neither affect basal osteoclastogenesis nor do they reverse A1 R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1 R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1 R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation. Experimental approach: Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa. Key

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5'-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A1 receptors (A1 R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A1 R agonists neither affect basal osteoclastogenesis nor do they reverse A1 R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A1 R antagonist-mediated inhibition, and, when we saw no effect, determined whether A1 R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation. Experimental approach: Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand-macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa. Key

Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC50 = 20-70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold. Conclusions and implications: Based on these findings, we hypothesize that the A1 R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A1 R antagonists act as inverse agonists to release A1 R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A1 R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.