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Rolofylline, an adenosine A 1 receptor antagonist, inhibits osteoclast differentiation as an inverse agonist
Author(s) -
He Wenjie,
Wilder Tuere,
Cronstein Bruce N
Publication year - 2013
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12342
Subject(s) - osteoclast , adenosine , medicine , endocrinology , adenosine receptor , chemistry , receptor , activator (genetics) , agonist , macrophage colony stimulating factor , extracellular , biology , microbiology and biotechnology , biochemistry , macrophage , in vitro
Background and Purpose 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 A 1 receptors ( A 1 R ) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A 1 R agonists neither affect basal osteoclastogenesis nor do they reverse A 1 R antagonist‐mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase‐mediated adenosine production was required for A 1 R antagonist‐mediated inhibition, and, when we saw no effect, determined whether A 1 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 ( C tsk , A cp5 , MMP ‐9 and NFATc1 ). Intracellular cAMP concentration was determined by elisa . Key Results Rolofylline inhibited osteoclast formation in a dose‐dependent manner ( IC 50 = 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 A 1 R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A 1 R antagonists act as inverse agonists to release A 1 R ‐mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A 1 R promotes osteoclast formation and down‐regulation of this activity blocks osteoclast formation.