Dataset Information

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

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.

SUBMITTER: Spurlock CF

PROVIDER: S-EPMC4269792 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Spurlock Charles F CF Gass Henry M HM Bryant Carson J CJ Wells Benjamin C BC Olsen Nancy J NJ Aune Thomas M TM

Rheumatology (Oxford, England) 20140812 1

<h4>Objectives</h4>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.<h4>Methods</h4>An NF-κB luciferase reporter plasmid was used to measure NF-κB activation across experimental stimuli. Flow cytometry was used to quantify chan ...[more]

PMID: 25118313

Similar Datasets

Project description:BackgroundActivated fibroblast-like synoviocytes (FLS) are drivers of synovitis and structural joint damage in rheumatoid arthritis (RA). Despite the use of disease-modifying drugs, only about 50% of RA patients reach remission in real-world settings. We used an unbiased approach to investigate the effects of standard-of-care methotrexate (MTX) and a Janus kinase inhibitor, tofacitinib (TOFA), on gene expression in RA-FLS, in order to identify untargeted disease mediators.MethodsPrimary RA-FLS were activated by stimulation with interleukin-1β (IL-1β) or platelet-derived growth factor + IL-1β in the presence or absence of MTX or TOFA, with or without additional inhibitors. Co-cultures of synovial cells were performed in direct and indirect systems. Cells were collected for RNA sequencing or qPCR, and supernatants were analyzed for protein concentrations.ResultsSix thousand three hundred fifty genes were differentially expressed, the majority being upregulated, in MTX-treated activated RA-FLS and 970 genes, the majority being downregulated, in TOFA-treated samples. Pathway analysis showed that MTX had largest effects on 'Molecular mechanisms of cancer' and TOFA on 'Interferon signaling'. Targeted analysis of disease-associated genes revealed that MTX increased the expression of cell cycle-regulating genes but also of pro-inflammatory mediators like IL-1α (IL1A) and granulocyte-macrophage colony-stimulating factor, GM-CSF (CSF2). The MTX-promoted expression of CSF2 in activated RA-FLS peaked at 48 h, could be mediated via either NF-κB or AP-1 transcription factors, and was abrogated by IL-1 inhibitors (IRAK4 inhibitor and anakinra). In a co-culture setting, MTX-treatment of activated RA-FLS induced IL1B expression in macrophages.ConclusionsMTX treatment induces secretion of IL-1 from activated RA-FLS which by autocrine signaling augments their release of GM-CSF. This unexpected effect of MTX might contribute to the persistence of synovitis.

Dataset Information

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

Objectives

Methods

Results

Conclusion

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets