Methotrexate suppresses NF-kappaB activation through inhibition of IkappaBalpha phosphorylation and degradation.
Methotrexate (MTX), a folate antagonist, is a commonly used anti-inflammatory, antiproliferative, and immunosuppressive drug whose mode of action is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that MTX must mediate its effects through suppression of NF-kappaB activation. We investigated the effects of MTX on NF-kappaB activation induced by TNF in Jurkat cells. The treatment of these cells with MTX suppressed TNF-induced NF-kappaB activation with optimum effects occurring at 10 microM MTX for 60 min. These effects were not restricted to Jurkat cells because other cell types were also inhibited. Besides TNF, MTX also suppressed the NF-kappaB activation induced by various other inflammatory stimuli. The suppression of TNF-induced NF-kappaB activation by MTX correlated with inhibition of IkappaBalpha degradation, suppression of IkappaBalpha phosphorylation, abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Because ecto 5' nucleotidase inhibitor (alpha,beta-methylene adenosine-5'-diphosphate) blocked the effect of MTX, adenosine mimicked the effect of MTX, and adenosine A2b receptor antagonist (3,7-dimethyl-1-propargylxanthine) reversed the inhibitory effect of MTX, we suggest that MTX suppresses NF-kappaB activation by releasing adenosine. A partial reversal of MTX-induced NF-kappaB suppression by thymidine and folinic acid indicates the role of the thymidylate synthase pathway also. Overall, our results clearly demonstrate that MTX suppresses NF-kappaB activation through the release of adenosine, which may contribute to the role of MTX in anti-inflammatory, immunomodulatory, and antiproliferative effects.
Majumdar
S
S
Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Aggarwal
B B
BB
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors; D000241:Adenosine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D019169:Jurkat Cells; D002955:Leucovorin / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D058915:Purinergic P1 Receptor Antagonists; D043704:Receptor, Adenosine A2B; D013805:Theobromine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013936:Thymidine / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020298:U937 Cells
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009838:Oligodeoxyribonucleotides; D058915:Purinergic P1 Receptor Antagonists; D043704:Receptor, Adenosine A2B; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C057837:3,7-dimethyl-1-propargylxanthine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D015720:5'-Nucleotidase; D000241:Adenosine; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D013805:Theobromine; D002955:Leucovorin; D013936:Thymidine; D008727:Methotrexate