Research ArticleImmunology

The intracellular pyrimidine 5′-nucleotidase NT5C3A is a negative epigenetic factor in interferon and cytokine signaling

See allHide authors and affiliations

Science Signaling  20 Feb 2018:
Vol. 11, Issue 518, eaal2434
DOI: 10.1126/scisignal.aal2434
  • Fig. 1 Type I IFN–mediated induction of NT5C3A mRNA and protein.

    (A) Schematic diagram of the human NT5C3A gene showing the intron-exon structure of the 12 exons and alternative splicing variants. The locations of putative ISREs are indicated with black vertical lines below each variant, and translated exons are shown in orange. aa, amino acid. Real-time PCR assay primers are indicated with black horizontal lines above each variant, and isoform-specific primers are indicated with blue lines below each variant (see table S1). (B) Induction of NT5C3A expression by IFN-α-2b in HeLa cells. Top: Real-time quantitative PCR (RT-qPCR) analysis of NT5C3A expression in response to the indicated concentrations of IFN-α-2b for 6 hours. Middle: Representative blot from three independent experiments showing NT5C3A protein abundance in response to overnight treatment with the indicated concentrations of IFN-α-2b. Bottom: Relative amounts of NT5C3A protein in response to the indicated IFN concentrations. (C) Time course of IFN-induced NT5C3A expression in HeLa cells. Left: RT-qPCR analysis of NT5C3A expression in response to IFN-α (100 U/ml) for the indicated times. Right: Relative amounts of NT5C3A protein in cells left untreated (0) or stimulated with IFN-α (100 U/ml) for the indicated times. Bottom: Representative Western blot assessing protein abundance. (D) Induction of NT5C3A expression by IFN in multiple cell types. RT-qPCR analysis of NT5C3A expression in NHFs, HT-1080 cells, HMDMs, and THP-1 cells exposed to IFN-α (100 U/ml) for 4 or 16 hours. (E) Top: Induction of NT5C3A transcript variants in HeLa cells exposed to IFN-α (100 U/ml) for 6 hours. Bottom: Representative Western blot assessing NT5C3A abundance in HeLa and WISH cells exposed to IFN-α (1000 U/ml) for 24 hours. Lysates were resolved by 4 to 12% SDS–polyacrylamide gel electrophoresis. Arrow indicates NT5C3A variant 3 (NT5C3A-3). (F) Induction of NT5C3A-3 expression by IFN-α, IFN-β, and IFN-γ in HeLa cells. Top: RT-qPCR analysis of NT5C3A-3 expression in cells exposed to the indicated IFNs (100 U/ml) for 6 hours. Bottom: Representative Western blot assessing NT5C3A-3 protein abundance under the indicated conditions. (G) Schematic diagram of the NT5C3A-3 promoter. The sequence shown represents −465 bp of the 5′UTR of exon 2 and part of intron 2. In silico predicted transcription factor–binding sites are indicated. (H) Analysis of ISRE use in NT5C3A. Top: Schematic diagram of the NT5C3A-ISRE–containing reporter constructs. Bottom: Analysis of enhanced GFP (EGFP) intensity in Huh7 cells expressing NT5C3A-ISRE1–, NT5C3A-ISRE2–, or NT5C3A-ISRE3–containing EGFP constructs. Twenty-four hours after transfection, the cells were mock-treated or treated with IFN-α (100 U/ml) for 6 hours. All Western blotting densitometry data are means ± SEM of three independent experiments. All RT-qPCR data are normalized to housekeeping mRNA abundance as indicated, and statistical significance is indicated relative to unstimulated cells. P values were calculated by two-tailed Student’s t test. *P < 0.01, **P < 0.001, and ***P < 0.0001.

  • Fig. 2 STAT1, STAT2, and JAK1 are required for IFN-dependent induction of NT5C3A expression through IRF1.

    (A) Representative Western blot of three experiments showing the effect of siRNAs against STAT1 (siSTAT1), STAT2 (siSTAT2), JAK1 (siJAK1), or control scrambled siRNA (siCtrl) (all at 50 nM, overnight treatment) on the abundances of NT5C3A after overnight treatment of HeLa cells with IFN-α-2b (1000 U/ml). Samples were also analyzed by Western blotting to assess knockdown efficiency. (B) Left: RT-qPCR analysis of NT5C3A-3 expression in parental HT-1080 and STAT1-null 2fTGH (U3A) fibroblasts treated with the indicated concentrations of IFN-α-2b for 6 hours. Statistical significance is indicated relative to unstimulated HT-1080 cells. Right: Representative Western blot from three experiments assessing NT5C3A and STAT1 abundance in cells treated overnight with IFN-α-2b (1000 U/ml). Arrow indicates NT5C3A-3. (C) Top: RT-qPCR analysis of the expression of murine NT5C3A-1 (Lupin) and NT5C3A-2 in WT and STAT1-null MEFs treated with murine IFN-β (1000 U/ml) for 6 hours. Statistical significance is indicated relative to unstimulated WT cells (Mock). Bottom: Representative Western blot of three experiments assessing murine NT5C3A (Lupin) abundance in STAT1+/+ and STAT1−/− MEF cells treated overnight with murine IFN-β (1000 U/ml). (D) Binding of IRFs to NT5C3A-ISRE3. EMSA was performed with nuclear extracts from HeLa cells that were treated overnight with IFN-α. Excess unlabeled ISRE (competitor) served as the control. IRF binding was assessed with isoform-specific antibodies (Ab). The probe-protein-antibody complex (supershift, marked with an asterisk) and unbound free probe (Free) are indicated. Arrow indicates probe-protein complexes. (E) Top: RT-qPCR analysis of NT5C3A-3 expression in HeLa cells transfected with 50 nM siRNAs against IRF1 (siIRF1), IRF2 (siIRF2), IRF3 (siIRF3), IRF9 (siIRF9), or control scrambled (siCtrl) overnight and then mock-treated (Ctrl) or stimulated with IFN-α-2b (1000 U/ml) for 6 hours. Statistical significance is indicated relative to unstimulated cells (Ctrl) in each group. Bottom: Representative Western blot assessing NT5C3A abundance in HeLa cells transfected with the indicated siRNAs and then treated overnight with IFN-α-2b (1000 U/ml). (F) Huh7 cells cotransfected with an NT5C3A-ISRE3 EGFP-containing linear PCR construct (NT5C3A-ISRE3) and with plasmids expressing IRF1 or IRF9 or with control plasmid. As a control, cells were cotransfected with a non–ISRE-containing −74 cytomegalovirus linear PCR construct (−74 CMV) and with plasmids expressing IRF1 or IRF9 or with control plasmid. Twenty-four hours after transfection, EGFP fluorescence intensities were quantified. (G) ChIP experiment to assess the binding of IRF1 to NT5C3A-ISRE3 in cells. Whole-cell lysates of HeLa cells treated for 3 hours with IFN-α-2b (1000 U/ml) were incubated with antibodies against IRF1, IRF9, or STAT1. RT-qPCR analysis was then performed with primers specific for the NT5C3A-ISRE3 promoter (table S3). Data are means ± SEM of at least two independent biological experiments, each performed in triplicate. Unless otherwise stated, all quantitative data are means ± SEM of three independent experiments. Statistical analysis between individual groups was performed with a two-tailed Student’s t test. *P < 0.01, **P < 0.001, and ***P < 0.0001. Statistical comparison between pairs of groups was performed by two-way analysis of variance (ANOVA), with groups indicated with a bracket; ^^P < 0.001. EMSAs and Western blots are representatives of three independent experiments.

  • Fig. 3 Suppression of NT5C3A expression enhances cytokine-stimulated IL-8 gene expression and protein secretion.

    (A) Left: Representative Western blot from three independent experiments showing silencing efficiency of siNT5C3A in HeLa cells transfected with siCtrl or siNT5C3A and then treated overnight with IFN-α (1000 U/ml). Right: Enzyme-linked immunosorbent assay (ELISA) of IL-8 in the culture medium of HeLa cells transfected with the indicated siRNAs and then left unstimulated or stimulated for 24 hours with IFN-α (1000 U/ml). (B) Left: RT-qPCR analysis of IL8 expression in HeLa cells 24 hours after they were transfected with the indicated siRNAs and then treated with TNF-α (10 ng/ml) for the indicated times. Statistical differences at the specified time points are indicated. Right: ELISA of IL-8 abundance in the cultured medium from HeLa cells transfected with the indicated siRNAs and then left unstimulated or stimulated with TNF-α (10 ng/ml) for 24 hours. (C) Representative Western blot of three independent experiments showing the abundance of NT5C3A and IRF1 in HeLa cells treated with TNF-α (10 ng/ml) for the indicated times. (D) Left: RT-qPCR analysis of NT5C3A-3 expression in HeLa cells transfected with the indicated siRNAs and then left unstimulated or stimulated with TNF-α (10 ng/ml) for 6 hours. Right: Representative blot of three independent experiments showing NT5C3A abundance in siIRF1-transfected HeLa cells. (E) Representative Western blot of three independent experiments showing NT5C3A abundance in NT5C3A-3–HEK293 cells. (F) Left: RT-qPCR analysis of IL8 expression in control cells (Vector-HEK293) and NT5C3A-3–HEK293 cells that were left unstimulated or were stimulated with TNF-α (10 ng/ml) for 2 hours. Right: ELISA of IL-8 abundance in the culture medium from NT5C3A-3–HEK293 cells and control Vector-HEK293 cells that were left unstimulated or were stimulated with TNF-α (10 ng/ml) for 24 hours. All quantitative data are means ± SEM of three independent experiments. Statistical analysis between individual groups was performed with a two-tailed Student’s t test. *P < 0.01, **P < 0.001, and ***P < 0.0001. Statistical comparison between pairs of groups was performed by two-way ANOVA, with groups indicated with a bracket; ^P < 0.01 and ^^^P < 0.0001.

  • Fig. 4 NT5C3A-mediated suppression of the TNF-induced activation of NF-κB.

    (A) Top: Western blotting analysis of the abundance of NT5C3A in NT5C3A-3–HEK293 cells. Bottom: NF-κB luciferase reporter activity in NT5C3A-HEK293 cells. Cells were cotransfected with 100 ng of the pGL3-NF-κB 3×-Luc reporter and 20 ng of pRL-TK and treated with TNF-α (10 ng/ml) or IL-1β (10 ng/ml) for 6 hours. Luciferase assays were performed on triplicate samples, and data are presented as the relative light units (RLU) of firefly luciferase activity normalized to the RLU of Renilla luciferase activity. Data are means ± SEM of three experiments, each performed in triplicate. (B) Top: Western blotting analysis of the abundance of NT5C3A in HEK293 cells transfected with the indicated siRNAs. Bottom: NF-κB luciferase reporter activity in HEK293 cells cotransfected with siCtrl or siNT5C3A (25 nM), 100 ng of pGL3-NF-κB 3×-Luc reporter, and 20 ng of pRL-TK for 24 hours, followed by treatment with TNF-α (10 ng/ml) for 6 hours. Data are means ± SEM of three experiments. (C) Left: Luciferase reporter activity in NT5C3A-3–HEK293 cells 24 hours after they were cotransfected with 100 ng of pGL3-NF-κB 3×-Luc reporter and 20 ng of pRL-TK, together with control plasmid (100 ng) or plasmids expressing TAK1 and TAB1 (50 ng each) or IKKβ (100 ng). Data are means ± SEM of three independent experiments, each performed in triplicate. Right: Representative Western blot from three experiments assessing NT5C3A abundance in NT5C3A-HEK293 cells, as well as the abundances of overexpressed TAK1-HA (hemagglutinin), TAB1, and IKKβ-Flag. (D) Left: Representative Western blot from three independent experiments assessing the knockdown efficiency of siNT5C3A in HEK293 cells, as well as the abundances of TAK1-HA, TAB1, and IKKβ-Flag proteins. Right: Luciferase reporter activity in HEK293 cells 24 hours after they were transfected with siCtrl or siNT5C3A (25 nM), together with control plasmid (100 ng), plasmids encoding TAK1 and TAB1 (50 ng each), or plasmid encoding IKKβ plasmid (100 ng). Data are means ± SEM of three independent experiments, each performed in triplicate. (E) Top: Schematic diagram of the IL8 promoter. Bottom left: Luciferase reporter activity in NT5C3A-3–HEK293 stable polyclonal cells cotransfected with 100 ng of IL8 (−133/+44-Luc) proximal promoter or the pGL3-basic empty vector, together with 100 ng of vector or pCMV4-p65 plasmid and 20 ng of pRL-TK. Data are means ± SEM of three experiments, each performed in triplicate. Right: Representative Western blot from three experiments assessing NT5C3A abundance in NT5C3A-3–HEK293 stable cells, as well as the abundance of p65 protein in overexpressing cells. All quantitative data are means ± SEM of three independent experiments. (F) Right: EMSA was performed using HeLa nuclear extracts from cells transfected with vector and then treated with TNF-α (10 ng/ml), TNF-α (10 ng/ml) and IFN-α (100 U/ml), or IFN-α (100 U/ml) alone for 2 hours or from cells transfected with the NT5C3A-3 construct and treated with TNF-α (10 ng/ml) for 2 hours. Arrows indicate probe-protein complexes. Left: Band intensity quantification from three independent EMSA experiments. Statistical analysis between individual samples was performed with two-tailed Student’s t test. *P < 0.01, **P < 0.001, and ***P < 0.0001. Statistical comparison between pairs of groups was performed by two-way ANOVA, with the groups indicated with a bracket; ^^^P < 0.0001.

  • Fig. 5 Mechanism of the NT5C3A-mediated attenuation of TNF-induced IL8 expression.

    (A) Determination of intracellular NAD+ concentrations using an enzymatic cycling assay. HeLa cells transfected with control vector or the NT5C3A-3 construct were treated with TNF-α (10 ng/ml) for the indicated times before NAD+ concentrations were determined. (B) Left: Measurement of HAT activity in HeLa cells transfected with control vector or the NT5C3A-3 construct and then treated with TNF-α (10 ng/ml) for 2 hours. Right: Measurement of HAT activity in NT5C3A-silenced HeLa cells treated with TNF-α (10 ng/ml) for 2 hours. OD, optical density. (C) RT-qPCR analysis of SIRT1 mRNA abundance in Vector-HeLa cells and NT5C3A-3–HeLa cells that were treated with TNF-α (10 ng/ml) for the indicated times. (D) RT-qPCR analysis of SIRT6 mRNA abundance in Vector-HeLa cells and NT5C3A-3–HeLa cells treated as described for (C). (E) Western blotting analysis of SIRT1 and SIRT6 protein abundance in NT5C3A-3–HEK293 cells treated with TNF-α (10 ng/ml) for the indicated times. (F) Western blotting analysis of SIRT1 and SIRT6 protein abundance in NT5C3A-3–HeLa cells treated as described for (E). (G) Left: Western blotting analysis of the abundances of nuclear acetyl Lys310-p65 and total p65 in NT5C3A-3HeLa cells that were treated with TNF-α (10 ng/ml) for the indicated times. Proliferating cell nuclear antigen (PCNA) was used as a loading control. Right: Quantification of the relative abundance of acetyl-p65 (Ac-p65) protein from three different experiments was determined by densitometric analysis of Western blots. PCNA protein abundance was used for normalization. (H) ChIP–RT-qPCR assay using antibody against acetylated histone H3 Lys14 to determine IL8 promoter occupancy. Primers that amplify the nucleosome near the TSS of the IL8 promoter (p-IL8 −114/+68) were used. All quantitative data are means ± SEM of three independent experiments. Statistical analysis between individual samples was performed by two-tailed Student’s t test. *P < 0.01, **P < 0.001, and ***P < 0.0001. Statistical comparisons between pairs of groups were performed by two-way ANOVA, with groups indicated with a bracket; ^P < 0.01 and ^^^P < 0.0001. All Western blots are from one experiment and are representative of three independent experiments.

  • Fig. 6 SIRT1 and SIRT6 are required for the NT5C3A-3–mediated attenuation of IL8 expression.

    (A) Top: RT-qPCR analysis of IL8 expression in Vector-HeLa and NT5C3A-3–HeLa cells transfected with 70 nM siCtrl, siSIRT1, or siSIRT6 overnight and then treated with TNF-α (10 ng/ml) for 90 min. Bottom: ELISA to determine IL-8 abundance in the culture medium from Vector-HeLa or NT5C3A-3–HeLa cells transfected as indicated and then treated with TNF-α (10 ng/ml) for 24 hours. (B) Representative Western blot from three experiments assessing the abundances of SIRT1, SIRT6, and NT5C3A-3–Flag–tagged protein. β-Actin was used as a loading control. (C) Top: Schematic diagram of the organization of NT5C3A protein, showing motifs and domain structures and NT5C3A pathological mutations. NT5C3A mutations in red represent the most studied mutations. Bottom left: RT-qPCR analysis of IL8 expression in Vector-HeLa, NT5C3A-3–HeLa cells, and HeLa polyclonal cells stably overexpressing one of the indicated three mutant forms of NT5C3A (D87V, G230R, or N179S) and then treated with TNF-α (10 ng/ml) for the indicated times. Bottom right: ELISA to determine IL-8 abundance in the cultured medium from the indicated HeLa cells treated with TNF-α (10 ng/ml) for the indicated times. All quantitative data are means ± SEM of three independent experiments. Statistical comparisons between pairs of groups (Vector-HeLa and NT5C3A-3–HeLa cells) were performed by two-way ANOVA; ^^P < 0.001 and ^^^P < 0.0001. (D) Proposed model of the role of NT5C3A during the adaptation phase of acute inflammation.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/518/eaal2434/DC1

    Fig. S1. Dose-dependent induction of NT5C3A expression across different cell lines.

    Fig. S2. Knockdown efficiency for siRNAs targeting STAT1, STAT2, and JAK1 in HeLa cells.

    Fig. S3. Organization of NT5C3A.

    Fig. S4. Knockdown efficiency for siRNAs targeting IRF1, IRF2, IRF3, and IRF9 in HeLa cells.

    Fig. S5. Induction of NT5C3A expression by different inducers and the effect of NT5C3A on TNFA and CXCL1 expression.

    Fig. S6. KEGG pathway analysis for nicotinate and nicotinamide metabolism.

    Fig. S7. Efficiency of SIRT1 and SIRT6 knockdown.

    Fig. S8. Protein association network (STRING).

    Table S1. List of oligonucleotide primers.

    Table S2. List of cloning-free primers and EMSA oligonucleotides.

    Table S3. List of ChIP-qPCR primers.

    Table S4. List of site-directed mutagenesis primers.

    Table S5. List of plasmids.

    Table S6. List of siRNA sequences.

  • Supplementary Materials for:

    The intracellular pyrimidine 5′-nucleotidase NT5C3A is a negative epigenetic factor in interferon and cytokine signaling

    Latifa Al-Haj and Khalid S. A. Khabar*

    *Corresponding author. Email: khabar{at}kfshrc.edu.sa

    This PDF file includes:

    • Fig. S1. Dose-dependent induction of NT5C3A expression across different cell lines.
    • Fig. S2. Knockdown efficiency for siRNAs targeting STAT1, STAT2, and JAK1 in HeLa cells.
    • Fig. S3. Organization of NT5C3A.
    • Fig. S4. Knockdown efficiency for siRNAs targeting IRF1, IRF2, IRF3, and IRF9 in HeLa cells.
    • Fig. S5. Induction of NT5C3A expression by different inducers and the effect of NT5C3A on TNFA and CXCL1 expression.
    • Fig. S6. KEGG pathway analysis for nicotinate and nicotinamide metabolism.
    • Fig. S7. Efficiency of SIRT1 and SIRT6 knockdown.
    • Fig. S8. Protein association network (STRING).
    • Table S1. List of oligonucleotide primers.
    • Table S2. List of cloning-free primers and EMSA oligonucleotides.
    • Table S3. List of ChIP-qPCR primers.
    • Table S4. List of site-directed mutagenesis primers.
    • Table S5. List of plasmids.
    • Table S6. List of siRNA sequences.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

Navigate This Article