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Methotrexate inhibits NF-κB activity via long intergenic (noncoding) RNA-p21 induction.

Spurlock CF, Tossberg JT, Matlock BK, Olsen NJ, Aune TM
Arthritis Rheumatol. 2014 66 (11): 2947-57

PMID: 25077978 · PMCID: PMC4211976 · DOI:10.1002/art.38805

OBJECTIVE - To determine interrelationships between the expression of long intergenic (noncoding) RNA-p21 (lincRNA-p21), NF-κB activity, and responses to methotrexate (MTX) in rheumatoid arthritis (RA) by analyzing patient blood samples and cell culture models.

METHODS - Expression levels of long noncoding RNA and messenger RNA (mRNA) were determined by quantitative reverse transcription-polymerase chain reaction. Western blotting and flow cytometry were used to quantify levels of intracellular proteins. Intracellular NF-κB activity was determined using an NF-κB luciferase reporter plasmid.

RESULTS - Patients with RA expressed reduced basal levels of lincRNA-p21 and increased basal levels of phosphorylated p65 (RelA), a marker of NF-κB activation. Patients with RA who were not treated with MTX expressed lower levels of lincRNA-p21 and higher levels of phosphorylated p65 compared with RA patients treated with low-dose MTX. In cell culture using primary cells and transformed cell lines, MTX induced lincRNA-p21 through a DNA-dependent protein kinase catalytic subunit (DNA PKcs)-dependent mechanism. Deficiencies in the levels of PRKDC mRNA in patients with RA were also corrected by MTX in vivo. Furthermore, MTX reduced NF-κB activity in tumor necrosis factor α-treated cells through a DNA PKcs-dependent mechanism via induction of lincRNA-p21. Finally, we observed that depressed levels of TP53 and lincRNA-p21 increased NF-κB activity in cell lines. Decreased levels of lincRNA-p21 did not alter NFKB1 or RELA transcripts; rather, lincRNA-p21 physically bound to RELA mRNA.

CONCLUSION - Our findings support a model whereby depressed levels of lincRNA-p21 in RA contribute to increased NF-κB activity. MTX decreases basal levels of NF-κB activity by increasing lincRNA-p21 levels through a DNA PKcs-dependent mechanism.

Copyright © 2014 by the American College of Rheumatology.

MeSH Terms (20)

Adult Antirheumatic Agents Arthritis, Rheumatoid Cell Line Cells, Cultured Cyclin-Dependent Kinase Inhibitor p21 DNA-Activated Protein Kinase Dose-Response Relationship, Drug eIF-2 Kinase Female Humans Male Methotrexate Middle Aged Monocytes NF-kappa B Nuclear Proteins RNA, Long Noncoding T-Lymphocytes Tumor Suppressor Protein p53

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