Increased Sensitivity to Apoptosis Induced by Methotrexate Is Mediated by JNK

Objective. Low-dose methotrexate (MTX) is an effective therapy for rheumatoid arthritis (RA), yet its mechanism of action is incompletely understood. The aim of this study was to explore the induction of apoptosis by MTX. Methods. Flow cytometry was performed to assess changes in the levels of intracellular proteins, reactive oxygen species (ROS), and apoptosis. Quantitative polymerase chain reaction was performed to assess changes in the transcript levels of select target genes in response to MTX. Results. MTX did not directly induce apoptosis but rather primed cells for markedly increased sensitivity to apoptosis via either mitochondrial or death receptor pathways, by a JNK-dependent mechanism. Increased sensitivity to apoptosis was mediated, at least in part, by MTX-dependent production of ROS, JNK activation, and JNK-dependent induction of genes whose protein products promote apoptosis. Supplementation with tetrahydrobiopterin blocked these MTX-induced effects. Patients with RA who were receiving low-dose MTX therapy expressed elevated levels of the JNK target gene, jun. Conclusion. Our results support a model whereby MTX inhibits reduction of dihydrobiopterin to tetrahydrobiopterin, resulting in increased production of ROS, increased JNK activity, and increased sensitivity to apoptosis. The finding of increased jun levels in patients with RA receiving low-dose MTX supports the notion that this pathway is activated by MTX in vivo and may contribute to the efficacy of MTX in inflammatory disease.