Department of Molecular Cell Biology and Oral Anatomy, Kyushu University, Fukuoka, Japan.
Lab Invest. 2011 May;91(5):719-31. doi: 10.1038/labinvest.2011.9. Epub 2011 Feb 21.
Methotrexate (MTX) is widely utilized for the treatment of patients with rheumatoid arthritis (RA); however, recent observation of the MTX-resistant patients proposed some difficulty in MTX-dependent therapeutic approach for RA. To access cellular events related to MTX resistance in RA in respect to inflammatory bone destruction, we investigated on an involvement of the potent inflammatory mediator adenosine in the regulation of osteoclastogenesis and inflammatory bone destruction. In rats with adjuvant-induced arthritis (AA rats), MTX efficiently suppressed bone destruction when it was administrated within 3 days after adjuvant injection, while it could not suppress inflammatory bone destruction if MTX was injected at the time of onset of inflammation (at day 10 after adjuvant injection). Time-course change in the level of plasma adenosine of AA rats was estimated by use of high-performance liquid chromatography and elucidated that adenosine level was markedly elevated till 10 days after adjuvant injection. In vitro bone marrow culture system for evaluating osteoclastogenesis, MTX markedly suppressed osteoclastogenesis in a stromal cell-dependent manner. This MTX-induced suppression of osteoclastogenesis was abrogated by the addition of adenosine. MTX suppressed the expression of mRNA for the receptor activator NF-κB ligand (RANKL), but it did not suppress the expression of osteoprotegerin (OPG). The addition of MTX and adenosine together markedly suppressed the level of OPG expression. Abolishment of MTX action by adenosine was significantly blocked by MRS1754, a highly selective antagonist for the A(2b) adenosine receptor (A(2b)AR), but not by caffeine, an antagonist for A₁, A(2a), A₃ AR (A₁AR, A(2a)AR, and A₃AR), which suggests that adenosine acts through A(2b)AR. Immunohistochemical studies showed abundant expression of A(2b)AR in cells localized in the bone-bone marrow boundary of the distal tibia in AA rats but not in control rats. When adenosine was injected in the ankle joints of MTX-treated AA rats, the suppressive effects of MTX on bone destruction was abolished. The current data therefore suggest that upregulation of adenosine production abolished the suppressive effect of MTX on osteoclastic bone destruction. Involvement of the adenosine-A(2b)AR system may explain MTX resistance in RA.
甲氨蝶呤（MTX）广泛用于治疗类风湿关节炎（RA）患者；然而，最近观察到 MTX 耐药患者在 MTX 依赖性治疗 RA 方面存在一些困难。为了研究与 RA 中 MTX 耐药相关的细胞事件及其与炎症性骨破坏的关系，我们研究了强效炎症介质腺苷在破骨细胞生成和炎症性骨破坏中的调节作用。在佐剂诱导关节炎（AA 大鼠）大鼠中，MTX 在佐剂注射后 3 天内给药时可有效抑制骨破坏，而在炎症发作时（佐剂注射后 10 天）给药时则不能抑制炎症性骨破坏。通过高效液相色谱法估计 AA 大鼠血浆腺苷水平的时程变化，并阐明腺苷水平在佐剂注射后 10 天内显著升高。用于评估破骨细胞生成的体外骨髓培养系统中，MTX 以基质细胞依赖性方式显著抑制破骨细胞生成。这种 MTX 诱导的破骨细胞生成抑制作用可被腺苷消除。MTX 抑制核因子-κB 配体（RANKL）受体的 mRNA 表达，但不抑制骨保护素（OPG）的表达。MTX 和腺苷的联合添加显著抑制 OPG 表达水平。通过 A（2b）腺苷受体（A（2b）AR）的高选择性拮抗剂 MRS1754 显著阻断腺苷对 MTX 作用的消除，但通过咖啡因（A₁、A（2a）、A₃ AR（A₁AR、A（2a）AR 和 A₃ AR）的拮抗剂）则不能阻断，这表明腺苷通过 A（2b）AR 起作用。免疫组织化学研究显示，AA 大鼠胫骨远端骨-骨髓交界处的细胞中大量表达 A（2b）AR，但在对照大鼠中则没有表达。当腺苷被注射到 MTX 治疗的 AA 大鼠的踝关节中时，MTX 对骨破坏的抑制作用被消除。因此，目前的数据表明，腺苷产生的上调消除了 MTX 对破骨细胞性骨破坏的抑制作用。腺苷-A（2b）AR 系统的参与可能解释了 RA 中的 MTX 耐药性。
