Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement

The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.


Adenosine Inhibits RANKL-Induced Osteoclastogenesis in Vitro

Soltner1,  Elise, Le Goff1,  Benoit, Duplomb1,  Laurence, Berthelot2,  Jean-Marie, Heymann1,  Dominique

INSERM U957, Nantes, France
University Hospital, Nantes, France

Purpose:

Adenosine is an endogenous purine nucleoside that modulates many physiological processes. Cellular signaling occurs through four known adenosine receptor subtypes (A1, A2a, A2b, and A3). Adenosine is believed to be an anti-inflammatory agent, implicated in many pathologies such as asthma or inflammatory joint disease. However, there is very few information in the literature on the role and expression of adenosine receptors in bone cells. We investigated therefore whether osteoclasts express adenosine receptors and how adenosine could regulate osteoclastogenesis.

Method:

Human peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over Ficoll gradient. CD14+ cells were magnetically labelled with CD14+ microbeads and positively selected by MACS technology. CD14+ cells were cultured with human M-CSF (25 ng/ml) and, after 3 days of culture, with or without hRANK-L (100 ng/ml). The formation of TRAP positive cells occurred around the 12th day of culture and was observed by TRAP staining. Expression of adenosine receptors was determined by RT-PCR and micro-array analysis of CD14+ cells and osteoclasts. Activity of the PI3K pathway, a key signaling protein downstream of adenosine receptors, was analysed by western blotting. TRAP staining was used to evaluate the effects of adenosine and 4 specific agonists of the adenosine receptors [CCPA (A1), CGS21680 (A2a), NECA (A2b), IB-MECA (A3)] on osteoclast differentiation.

Results:

The mRNA for all four adenosine receptors (A1, A2a, A2b, and A3), was detectable by RT-PCR in both CD14+ cells and osteoclasts. Microarray analysis showed a 2-fold increase in expression of the A3 receptor subtype in osteoclasts compared to CD14+ cells, whereas expression of the 3 other subtypes remained stable during differentiation. Western blot analysis showed a decreased phosphorylation of PI3K in CD14+ cells and osteoclasts stimulated with adenosine. TRAP staining demonstrated that adenosine inhibited RANKL-induced osteoclastogenesis. All four specific agonists of individual receptor subtypes inhibited osteoclastogenesis with comparable efficacies.

Conclusion:

Our work shows that osteoclasts and CD14+ cells express all four subtypes of adenosine receptor. Furthermore, phosphorylation of PI3K was decreased following stimulation with adenosine, confirming that these receptors are functional. We also demonstrated that adenosine has a potent inhibitory effect on RANKL-induced osteoclastogenesis in vitro, through its 4 receptor subtypes. We hypothesise that adenosine may be a regulator of osteoclastogenesis and contribute to the balance between bone formation and resorption in inflammatory conditions.

To cite this abstract, please use the following information:
Soltner, Elise, Le Goff, Benoit, Duplomb, Laurence, Berthelot, Jean-Marie, Heymann, Dominique; Adenosine Inhibits RANKL-Induced Osteoclastogenesis in Vitro [abstract]. Arthritis Rheum 2009;60 Suppl 10 :1296
DOI: 10.1002/art.26370

Abstract Supplement

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