Methotrexate-mediated inhibition of nuclear factor κB activation by distinct pathways in T cells and fibroblast-like synoviocytes

Rheumatology (Oxford). 2015 Jan;54(1):178-87. doi: 10.1093/rheumatology/keu279. Epub 2014 Aug 12.

Abstract

Objectives: Nuclear factor κB (NF-κB) is a critical activator of inflammatory processes and MTX is one of the most commonly prescribed DMARDs for treatment of RA. We sought to determine whether MTX inhibited NF-κB activity in RA and in lymphocytes and fibroblast-like synoviocytes (FLSs) and to define underlying mechanisms of action.

Methods: An NF-κB luciferase reporter plasmid was used to measure NF-κB activation across experimental stimuli. Flow cytometry was used to quantify changes in intracellular protein levels, measure levels of reactive oxygen species and determine apoptosis. Quantitative RT-PCR was used to identify changes in MTX target genes.

Results: In T cell lines, MTX (0.1 μM) inhibited activation of NF-κB via depletion of tetrahydrobiopterin (BH4) and increased Jun-N-terminal kinase (JNK)-dependent p53 activity. Inhibitors of BH4 activity or synthesis also inhibited NF-κB activation and, similar to MTX, increased JNK, p53, p21 and JUN activity. Patients with RA expressed increased levels of phosphorylated or active RelA (p65) compared with controls. Levels of phosphorylated RelA were reduced in patients receiving low-dose MTX therapy. In contrast, inhibition of NF-κB activation by MTX was not mediated via BH4 depletion and JNK activation in FLSs, but rather was completely prevented by adenosine receptor antagonists.

Conclusion: Our findings support a model whereby distinct pathways are activated by MTX in T cells and FLSs to inhibit NF-κB activation.

Keywords: Jun-N-terminal kinase; T cell; fibroblast-like synoviocytes; methotrexate; nuclear factor-kappaB; p53; rheumatoid arthritis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adult
  • Aged
  • Apoptosis / drug effects
  • Arthritis, Rheumatoid / drug therapy
  • Arthritis, Rheumatoid / metabolism
  • Arthritis, Rheumatoid / pathology
  • Biopterin / analogs & derivatives
  • Biopterin / metabolism
  • Case-Control Studies
  • Cell Line
  • Female
  • Fibroblasts / drug effects*
  • Fibroblasts / metabolism
  • Fibroblasts / pathology
  • Humans
  • Jurkat Cells
  • MAP Kinase Kinase 4 / metabolism
  • Male
  • Methotrexate / pharmacology*
  • Methotrexate / therapeutic use
  • Middle Aged
  • NF-kappa B / antagonists & inhibitors*
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / drug effects*
  • Signal Transduction / physiology
  • Synovial Membrane / drug effects*
  • Synovial Membrane / metabolism
  • Synovial Membrane / pathology

Substances

  • NF-kappa B
  • Reactive Oxygen Species
  • Biopterin
  • MAP Kinase Kinase 4
  • sapropterin
  • Methotrexate