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

Abstract

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.

Figure 1

Reduced expression of lincRNA-p21 in RA. A, TP53, lincRNA-p21, and PANDA transcript levels were measured by qRT-PCR and normalized to GAPDH. Whole blood samples from CTRL (n = 45), RA (n = 18), SLE (n = 24), and SS (n = 12) subjects were collected into PaxGene tubes. B, Comparison of RA differentially expressed genes (DEGs) and genes significantly over-expressed (OE) or under-expressed (UE) following lincRNA-p21 or TP53 RNA interference. Chi-squared test was used to calculate P values. C, Activated T cells were transfected with the indicated siRNAs and transcript levels measured by qRT-PCR. D, Correlation between lincRNA-p21 and TP53 transcripts in CTRL (n=20; left panel) and RA subjects (n = 22; right panel).

Figure 2

Methotrexate increases expression of lincRNA-p21. A, LincRNA-p21 expression levels in CTRL (n = 45), RA-MTX (n = 18), and RA+MTX (n = 18) subjects were determined by quantitative RT-PCR. Results are expressed as mean lincRNA-p21 relative to GAPDH transcript levels. B, LincRNA-p21 transcript levels in activated T cells (left panel) or Jurkat cells (right panel) cultured with the indicated concentrations of MTX. Results are expressed as in A. C, Jurkat cells or activated T cells were cultured with 0.1μM MTX and intracellular protein measurements determined by flow cytometry. A representative flow diagram for JNK and p53 shows background fluorescence (left, shaded gray) and results obtained with untreated (middle) or MTX-treated (right) cells. Fold increase in mean fluorescence intensity (MFI) of four independent experiments is shown in the right graph. D, LincRNA-p21 expression in Jurkat cells cultured with MTX and the JNK inhibitor, BI-78D3, (left panel) or adenosine receptor antagonists, caffeine (CAFF) and theophylline (THEO) (right panel). Results are expressed as in A. * = p < 0.05, ** = p < 0.005. Values are the mean ± S.D. NS = not significant

Figure 3

Methotrexate induces lincRNA-p21 via DNA-PKcs activation. A, Activated T cells and Jurkat cells were treated with the indicated concentrations of MTX and levels of the indicated phosphorylated proteins were measured by flow cytometry. Representative flow diagrams for phosphorylated-ATM and phosphorylated-DNA-PKcs show background fluorescence (left, shaded gray), untreated cells (middle) and MTX-treated (right) cultures. * = p < 0.05 versus untreated cells. B-C, PRKDC transcript levels relative to GAPDH were measured in activated T cells and Jurkat cells treated with MTX (B) or in CTRL (n = 45), RA-MTX (n = 18), and RA+MTX (n = 18) subjects (C). D, MTX-treated Jurkat cells were co-cultured for 48 hours with varying concentrations of KU-55933 (KU) or NU-7441 (NU) and transcript levels of TP53 (left panel) and lincRNA-p21 (right panel) determined by qRT-PCR. * = p < 0.05 versus MTX-treated cells. E, Fold induction of lincRNA-p21 expression relative to GAPDH in activated T cells cultured with MTX ± NU-7441. * = p < 0.05 versus untreated cultures. ** = p <0.05 versus MTX-treated T cells. See Figure 2 for other definitions.

Figure 4

Methotrexate reduces NF-κB activity via DNA-PKcs activation and lincRNA-p21 induction. A, Jurkat cells were transfected with an NF-κB luciferase reporter construct in the presence of TP53 (gray) and lincRNA-p21 (black) siRNAs or a scrambled siRNA control (Neg. Ctrl, white). Cells were treated with methotrexate (MTX) for 48 hours. TNF-α (5ng) was added to cell cultures after 24 hours. Results are expressed in light units. B-C, As in A, Jurkat cells (B) or activated T cells (C) were transfected with an NF-κB luciferase reporter construct and treated with MTX in the presence or absence of KU-55933 (KU) or NU-7441 (NU). Results are expressed as in A. D, Western blotting for phosphorylated p65 (P-p65). Whole cell lysates were prepared in CTRL (n = 9), RA-MTX (n = 4) and RA+MTX subjects (n = 4). Right panel shows quantitation of band intensity relative to β-actin. E, Intracellular flow cytometry measurements of P-p65 in CTRL (n=20), RA-MTX (n=6), and RA+MTX (n=6) subjects gating on the indicated cell surface markers. A representative flow diagram gating on CD3+ T cells is shown (left panel) along with quantification of P-p65 relative fluorescence in the indicated subject cohorts gating on CD3+ or CD14+ cells. A-E, * = p < 0.05

Figure 5

Association of RELA and lincRNA-p21 transcripts. A, THP-1 (left) or Jurkat (right) cells were transfected with an NF-κB luciferase reporter construct in the presence of specific siRNAs targeting lincRNA-p21 (black), TP53 (gray), or a scrambled siRNA control (Neg. Ctrl, white). Luminescence was quantified 48 hours post-transfection. TNF-α (5ng) was administered to cultures at 24 hours post-transfection. * = p < 0.05 B, THP-1 cells were transfected with a lincRNA-p21 siRNA and qRT-PCR measurements of the indicated transcripts measured 48 hours post-transfection. * = p < 0.05 versus cells transfected with a negative control siRNA. C, Sites of high complementarity between RELA and JUNB mRNA and lincRNA-p21 and D, relative enrichment as determined by qRT-PCR of RELA and JUNB mRNAs purified with a lincRNA-p21 biotinylated probe. * = p < 0.05 versus 18S and NFKB1 transcript levels. E, Western blotting for total p65 (RelA). Whole cell lysates were prepared from PBMC isolated from RA-MTX (n=4), RA+MTX (n=4) subjects. * = p < 0.05. The graph shows quantitative comparisons derived from the western image.