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Abstract Number: 1964

Rolofylline, an Adenosine A1R Antagonist, Acts As an Inverse Agonist to Inhibit Osteoclast Differentiation

Wenjie He1 and Bruce N. Cronstein2, 1Dept of Med, Div of Rheum, NYU, New York, NY, 2Internal Medicine, NYU School of Medicine, Division of Rheumatology, New York, NY

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: Adenosine receptors and osteoclastogenesis

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Session Information

Title: Osteoporosis and Metabolic Bone Disease

Session Type: Abstract Submissions (ACR)

Background/Purpose:

Previous work from our laboratory has uncovered a critical role of adenosine A1 receptor (A1R) in osteoclast formation both in vivo and in vitro. Adenosine may be generated by hydrolysis of extracellular adenosine nucleotides including ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5′-nucleotidase (CD73) and nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1). Interestingly selective A1R agonists neither affect basal osteoclast formation nor do they reverse A1R-mediated inhibition of osteoclast formation.  In this study, we determined whether ectonucleotidase-mediated adenosine production was required for osteoclast formation and, when we saw no effect, determined whether the A1R was constitutively activated and the antagonist was acting as an inverse agonist to mediate its effects on osteoclast formation. 

Methods: Osteoclasts were generated from bone marrow mononuclear cells (BMMs) extracted from wildtype, CD39KO, CD73KO and NPP-1KO mice using differentiation factors macrophage colony-stimulating factor (M-CSF) and RANKL.  The A1R specific antagonist, Rolofylline, was added to the culture media. TRAP+ staining was performed and Acp5 and Ctsk mRNA expression were examined to study osteoclast differentiation. Intracellular cAMP concentration was determined by ELISA.

Results:

A1R blockade inhibits osteoclast differentiation of BMMs derived from wildtype mice in a dose-dependent manner (IC50=1μM p<0.05, n=3). A1R blockade similarly inhibits osteoclast formation by marrow precursors from CD73KO, CD39KO and NPP-1KO mice in a dose-dependent manner (IC50=1μM, and 1μM, and 0.1μM, respectively, p<0.05 for all, n=3) for all three knockouts, although baseline osteoclast formation was significantly less (310 in CD73 KO vs 91 in wildtype, p<0.05, n=3) in precursors from CD73KO mice. Moreover, in the absence of agonist, A1R antagonist, rolofylline (1μM) caused an increase of cAMP content of BMMs by 9.9 fold (p<0.05, compared with control: M-CSF + RANKL, n=3). Similarly, rolofylline (1μM) leads to increased cAMP production in human healthy BMMs by 3.6 fold (n=1, compared with control: M-CSF+ RANKL), which is consistent with our findings that A1R blockade by rolofylline inhibits human BMMs-derived osteoclast formation (p<0.001, n=3; IC50= 1nM). 

Conclusion: Based on these findings we hypothesize that the A1R is constitutively activated in osteoclast precursors, thereby diminishing basal adenylate cyclase activity, and that the A1R antagonist acts as an inverse agonist to release the A1R-mediated inhibition of basal adenylate cyclase activity. The constitutive activity of A1R promotes osteoclast formation and downregulation of this activity blocks osteoclast formation.


Disclosure:

W. He,
None;

B. N. Cronstein,

Canfite BioPharma,

1,

NIH, URL Pharma, OSI,

2,

Bristol-Myers Squibb, Novartis, URL, Regeneron, Gismo Therapeutics,

5,

Arthritis Foundation, SLE Foundation,

6,

Patents on use of adenosine receptor antagonists to treat or prevent fibrosis. Multiple other patents.,

.

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