Involvement of deoxyadenosine and adenosine deaminase in the methotrexate-induced suppression of inflammatory bone destruction

Methotrexate (MTX) is an anti-tumor medicine classified into the anti-folate drugs. MTX suppresses DNA synthesis through inhibiting dihydrofolate reductase (DHF) of de novo nucleic acid pathway, which suppresses proliferation of malignant tumors. MTX is also utilized as for anti-rheumatic medicine as the lower doses of MTX are able to suppress effectively the inflammation of the synovial tissues in RA patients. In our previous study, plasma adenosine level was markedly increased during induction of inflammation in rats with adjuvant-induced arthritis (arthritic rats). MTX-induced suppression of osteoclastogenesis was canceled by the addition of adenosine in vitro as well as in vivo. Deoxyadenosine (dAdo) is known to regulate proliferation of immune cells and partly share metabolic pathways with adenosine. Adenosine deaminase (ADA) is a key enzyme which metabolize both of adenosine and dAdo to produce inosine and deoxyinosine perpetually. Here we examined the regulatory role of dAdo in MTX-induced suppression of inflammatory bone destruction and investigated a possible involvement of ADA as a target molecule of MTX. In rats with adjuvant-induced arthritis, in whole bone marrow cultures for evaluating osteoclastogenesis, MTX markedly suppressed osteoclastogenesis and dAdo completely canceled its suppression. This cancellation was partially blocked by caffeine, antagonist for the adenosine receptors, A₁AR, A_2aAR and A₃AR. Semiquantitative RT-PCR showed that MTX suppressed expression of RANKL without affecting osteoprotegerin (OPG) expression. Addition of dAdo clearly recovered the expression of RANKL and slightly suppressed OPG expression, which result in augment in osteoclastogenesis. In arthritic rats, MTX-induced suppression of inflammatory bone destruction was completely cancelled by the injection of dAdo. A marked induction of ADA was apparent in bone marrow cells observed in the bone destruction sites around ankle joints in arthritic rats. MTX strikingly suppressed expression of ADA in the corresponding area. Immunoblot analysis also showed a clear induction of ADA protein in arthritic rats and a marked suppression of ADA protein when arthritic rats were treated with MTX. In bone marrow cultures for evaluating osteoclastogenesis, expression of ADA mRNA was markedly suppressed by MTX and dAdo partially cancelled its inhibition. These observations suggest that ADA is involved in the MTX-mediated inhibition of inflammatory bone destruction regulated by adenosine and dAdo. Suppression of ADA by MTX may contribute to the stability of dAdo. A list of abbreviation: AA, adjuvant-induced arthritis; ADA, adenosine deaminase; AR, adenosine receptor; CFA, complete Freund adjuvant; dAdo, deoxyadenosine; MNC, multinucleated cells; MTX, methotrexate; RA, rheumatoid arthritis; RANK, receptor activator NF-kB ligand, TRAP, tartrate acid-resistant phosphatase.