Research ArticleImmunology

Noncanonical STAT1 phosphorylation expands its transcriptional activity into promoting LPS-induced IL-6 and IL-12p40 production

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Science Signaling  24 Mar 2020:
Vol. 13, Issue 624, eaay0574
DOI: 10.1126/scisignal.aay0574
  • Fig. 1 LPS-induced TLR4 endocytosis promotes ARID5A, IL-6, and IL-12p40 production.

    (A to C) MDMs were stimulated with LPS (100 ng/ml) in the presence or absence of 10 μM MitMAB. (A and C) Total RNA was isolated at the indicated times. The indicated transcripts (mRNAs) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Data are representative of three independent experiments (six donors per experiment) and are presented as means ± SD. (B) Whole-cell lysates were harvested 3 hours after stimulation and were separated by SDS–polyacrylamide gel electrophoresis (PAGE). The indicated endogenous proteins were detected by Western blotting analysis. Blots are representative of three independent experiments. (D to F) MDMs were left unstimulated or were stimulated with LPS (100 ng/ml) or P3C (100 ng/ml). (D and F) Total RNA was isolated at the indicated times. The indicated transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments (six donors per experiment) and are presented as means ± SD. (E) Cell-free supernatants were harvested 24 hours after stimulation and were analyzed by enzyme-linked immunosorbent assay (ELISA) for the indicated cytokines. Data are representative of three independent experiments (six donors per experiment) and are presented as means ± SD. (G) MDMs were left unstimulated or were stimulated with LPS (100 ng/ml) or P3C (100 ng/ml) in the presence or absence of 10 μM MitMAB. Whole-cell lysates were harvested 3 hours after stimulation and separated by SDS-PAGE. The indicated endogenous proteins were detected by Western blotting analysis. Blots are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as measured by one-way analysis of variance (ANOVA) with post hoc Tukey’s test (A and C to F). n.s, not significant.

  • Fig. 2 TLR4 endocytosis–dependent IFN-β–JAK–STAT1-pTyr701 signaling is dispensable for ARID5A expression.

    (A and B) WT or MYD88−/− dTHP-1 cells were left unstimulated or were stimulated with LPS (100 ng/ml) for 3 hours. (A) Total RNA was isolated and ARID5A transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (B) Whole-cell lysates were harvested and separated by SDS-PAGE. The indicated endogenous proteins were detected by Western blotting analysis. Blots are representative of three independent experiments. (C to F) WT dTHP-1 cells were transfected with scrambled siRNA or siRNAs targeting TRIF (C and D) or STAT1 (E and F). Forty-eight hours later, the cells were then left unstimulated or were stimulated with LPS (100 ng/ml). (C and E) Whole-cell lysates from the indicated transfected cells were separated by SDS-PAGE and analyzed by Western blotting. Blots are representative of three independent experiments. (D and F) Total RNA was isolated from the indicated cells 3 hours after stimulation. ARID5A transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (G and H) WT or IFNAR2−/− dTHP-1 cells were left unstimulated or were stimulated with LPS (100 ng/ml) for 3 hours. (G) Total RNA was isolated and ARID5A transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (H) Whole-cell lysates were harvested and separated by SDS-PAGE. The indicated endogenous proteins were detected by Western blotting analysis. Blots are representative of three independent experiments. (I) IFNAR2−/− dTHP-1 cells were transfected with scrambled siRNA or siRNA targeting STAT1. Forty-eight hours later, the cells were then left unstimulated or were stimulated with LPS (100 ng/ml). Whole-cell lysates were harvested 3 hours after stimulation and separated by SDS-PAGE. The indicated endogenous proteins were then detected by Western blotting analysis. Blots are representative of three independent experiments. (J and K) MDMs were stimulated with the indicated concentrations of human IFN-β for 1 hour (J) or were stimulated with human IFN-β (25 ng/ml) for the indicated times (K). Whole-cell lysates were harvested and separated by SDS-PAGE. The indicated endogenous proteins were detected by Western blotting analysis. Blots are representative of three independent experiments. (L and M) WT dTHP-1 cells were transfected with scrambled siRNA or siRNA targeting IRF3. Forty-eight hours later, the cells were then left unstimulated or were stimulated with LPS (100 ng/ml). (L) Whole-cell lysates from the transfected cells were separated by SDS-PAGE and analyzed by Western blotting. Blots are representative of three independent experiments. (M) Total RNA was isolated, and the indicated transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (N) MDMs were transfected with scrambled siRNA or siRNA targeting IRF3. Forty-eight hours later, the cells were then left unstimulated or were stimulated with LPS (100 ng/ml) for 3 hours. Whole-cell lysates were harvested and separated by SDS-PAGE. The indicated endogenous proteins were detected by Western blotting. Blots are representative of three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 as measured by one-way ANOVA with post hoc Tukey’s test (A, D, F, G, and M).

  • Fig. 3 Noncanonical phosphorylation of STAT1 at Thr749 activates ARID5A transcription.

    (A) Whole-cell lysates from WT dTHP-1 cells transduced with lentiviruses expressing scrambled shRNA or shRNA targeting STAT1 were separated by SDS-PAGE, and the indicated endogenous proteins were analyzed by Western blotting. Blots are representative of three independent experiments. (B to F) dTHP-1 cells expressing the indicated shRNAs were left unstimulated or were stimulated with LPS (100 ng/ml) for 3 hours (B to D) or 1 hour (E and F). (B and C) Total RNA was isolated, and the indicated transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (D and F) Whole-cell lysates were separated by SDS-PAGE, and the indicated endogenous proteins were detected by Western blotting. (E) Cellular cytoplasmic and nuclear fractions were isolated and analyzed by Western blotting. Blots in (D) to (F) are representative of three independent experiments. (G to J) Measurement of the luciferase activity of U3A cells 48 hours after transfection with a luciferase reporter plasmid containing the human ARID5A promoter, together with control plasmid (EV) or an expression plasmid for WT STAT1 or one of its mutants. Results are presented relative to Renilla luciferase activity. Data are representative of three independent experiments and are presented as means ± SD. (K) Homology model of human STAT1 protein. The protein chain is colorized with a rainbow spectrum from the N-terminal (blue) to C-terminal (red) regions. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as measured by one-way ANOVA with post hoc Tukey’s test (B, C, and H to J) or by paired Student’s t test (G).

  • Fig. 4 Phosphorylation of STAT1 at Thr749 facilitates its binding to a noncanonical DNA motif in the ARID5A promoter region.

    (A and B) U3As1 cells expressing EGFP-tagged STAT1-WT, STAT1-T749A, or STAT1-T749E. (A) Representative fluorescence micrographs showing the intracellular distribution of the indicated EGFP-tagged STAT1 proteins and the localization of the corresponding Hoechst-stained nuclei. Scale bar, 10 μm. Data are representative of three independent experiments. (B) Cellular cytoplasmic and nuclear fractions were isolated and analyzed by Western blotting to detect the indicated proteins. Blots are representative of three independent experiments. (C) Scheme of the full-length human ARID5A promoter (FULL) and its deletion construct (DEL). (D) Measurement of the luciferase activity of U3A cells 48 hours after transfection with luciferase reporter plasmids containing the promoter regions described in (C) together with control plasmid (EV) or an expression plasmid for WT STAT1. Results are presented relative to Renilla luciferase activity. Data are representative of three independent experiments and are presented as means ± SD. (E) Binding assay of STAT1 to biotinylated nucleotides containing a noncanonical DNA motif (5′-TTTGAGGC-′3) of the human ARID5A promoter. Cleared lysates from U3A cells expressing STAT1-WT, STAT1-T749A, or STAT1-T749E were incubated with the indicated biotinylated nucleotides. Bound proteins were immunoprecipitated with streptavidin beads and analyzed by Western blotting to detect STAT1. Input samples were prepared before incubation with biotinylated nucleotides. Blots are representative of three independent experiments. (F and G) U3A cells or U3As1 cells expressing STAT1-WT, STAT1-DoMut, STAT1-T749A, or STAT1-T749E. (F) Whole-cell lysates were separated by SDS-PAGE, and the indicated proteins were analyzed by Western blotting. Blots are representative of three independent experiments. (G) Total RNA was isolated, and the indicated transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (H and I) U3As1 cells expressing STAT1-WT, STAT1-DoMut, STAT1-T749A, or STAT1-T749E. (H) Cells were stimulated with human IFN-γ (10 ng/ml) for 1 hour. Whole-cell lysates were separated by SDS-PAGE, and the indicated proteins were analyzed by Western blotting. Blots are representative of three independent experiments. (I) ChIP was performed with anti-STAT1 or control immunoglobulin G (IgG), which was followed by qPCR analysis with primers specific for the indicated promoter region of ARID5A. Fold enrichment was calculated using the percentage input method, and the graph shows the relative fold enrichment from the qPCR results. Data are representative of three independent experiments and are presented as means ± SD. (J and K) WT dTHP-1 cells were left unstimulated or were stimulated with LPS (100 ng/ml) for 4 hours. (J) Whole-cell lysates were harvested and cleared, and the pTyr- or pThr-containing proteins were subjected to immunoprecipitation. Immunoprecipitates from the stimulated samples were left untreated or were treated with lambda protein phosphatase (λPP) and analyzed by Western blotting. Input samples were prepared before the pTyr- or pThr-containing proteins were immunoprecipitated. Blots are representative of three independent experiments. (K) ChIP was performed using anti-STAT1 or control IgG, which was followed by qPCR analysis with primers specific for the indicated promoter regions of ARID5A or IRF1. Fold enrichment was calculated using the percentage input method, and the graph shows the relative fold enrichment from the qPCR assay results. Data are representative of three independent experiments and are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as measured by one-way ANOVA with post hoc Tukey’s test (D, G, and I) or by paired Student’s t test (K).

  • Fig. 5 A noncanonical TBK1-IKKβ heterodimer promotes the phosphorylation of STAT1 at Thr749 downstream of TLR4 endocytosis.

    (A) MDMs were stimulated with LPS (100 ng/ml) in the presence or absence of 10 μM BX795 or 10 μM BMS-345541. Total RNA was isolated 3 hours after stimulation, and ARID5A transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments (five donors per experiment) and are presented as means ± SD. (B and C) WT dTHP-1 cells were left unstimulated or were stimulated with LPS (100 ng/ml). (B) Whole-cell lysates were harvested at the indicated times. IKKβ was immunoprecipitated from cleared lysates. IKKβ immunoprecipitates were subjected to Western blotting analysis of the indicated endogenous proteins. Input samples were prepared before IKKβ immunoprecipitation was performed. (C) Whole-cell lysates were harvested 4 hours after stimulation, and STAT1 was then immunoprecipitated. STAT1 immunoprecipitates from stimulated samples were left untreated or were treated with λPP and then analyzed by Western blotting for the indicated endogenous proteins. Input samples were prepared before STAT1 immunoprecipitation was performed. Blots are representative of three independent experiments. (D) WT, MYD88−/−, or IFNAR2−/− dTHP-1 cells were stimulated with LPS (100 ng/ml) for 4 hours in the presence or absence of 10 μM MitMAB. Whole-cell lysates were harvested, and IKKβ, TBK1, and pThr-containing proteins were immunoprecipitated from the cleared lysates. Immunoprecipitated samples were analyzed by Western blotting to detect the indicated endogenous proteins. Input samples were prepared before the immunoprecipitations were performed. Blots are representative of three independent experiments. (E and F) U3A cells were transiently co-transfected with the indicated expression plasmids. Twenty-four hours later, HA-tagged proteins were immunoprecipitated from the cleared cell lysates and then analyzed by Western blotting to detect the indicated proteins. Input samples were prepared before immunoprecipitations were performed. Blots are representative of three independent experiments. (G) U3A cells were transiently co-transfected with the indicated expression plasmids. Twenty-four hours later, Flag-tagged proteins were immunoprecipitated from the cleared cell lysates and then analyzed by Western blotting to detect the indicated proteins. Input samples were prepared before immunoprecipitations were performed. Blots are representative of three independent experiments. (H and I) U3As1 cells expressing STAT1-WT or STAT1-T749A were transiently transfected with plasmid expressing IKKβ and then cultured for 24 hours. (H) Total RNA was isolated, and the indicated transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (I) Whole-cell lysates were separated by SDS-PAGE, and the indicated proteins were analyzed by Western blotting. Blots are representative of three independent experiments. (J) Scheme of the full-length STAT1 (FULL) and STAT1 (G3S) constructs. (K and L) U3A cells were transiently co-transfected with the indicated expression plasmids. Twenty-four hours later, HA-tagged (K) or Flag-tagged (L) proteins were immunoprecipitated from the cleared cell lysates and analyzed by Western blotting to detect the indicated proteins. Input samples were prepared before the immunoprecipitations were performed. Blots are representative of three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 as measured by one-way ANOVA with post hoc Tukey’s test (A and H).

  • Fig. 6 LPS-induced TLR4 endocytosis augments IL12B transcription in a manner dependent on STAT1-pThr749.

    (A) Transduced dTHP-1 cells expressing the indicated shRNAs were left unstimulated or were stimulated with LPS (100 ng/ml) for the indicated times. Total RNA was isolated, and the indicated transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (B) Transduced dTHP-1 cells expressing the indicated shRNAs were stimulated with LPS (100 ng/ml) in the presence or absence of 10 μM MitMAB. Total RNA was isolated 3 hours after stimulation, and the indicated transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (C) Scheme of the full-length human IL12B promoter (FULL) and its deletion construct (DEL). (D) Measurement of the luciferase activity of U3A cells 48 hours after transfection with luciferase reporter plasmids containing the promoter regions described in (C) together with control plasmid (EV) or expression plasmid for WT STAT1. Results are presented relative to Renilla luciferase activity. Data are representative of three independent experiments and are presented as means ± SD. (E) Measurement of the luciferase activity of U3A cells 48 hours after transfection with a luciferase reporter plasmid containing the full-length human IL12B promoter, together with control plasmid (EV) or expression plasmid for WT or T749A STAT1. Results are presented relative to Renilla luciferase activity. Data are representative of three independent experiments and are presented as means ± SD. (F) U3A or U3As1 cells expressing STAT1-WT, STAT1-DoMut, STAT1-T749A, or STAT1-T749E. Total RNA was isolated, and IL12B transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (G) U3As1 cells expressing STAT1-WT, STAT1-DoMut, STAT1-T749A, or STAT1-T749E. ChIP was performed using anti-STAT1 or control IgG, which was followed by qPCR analysis with primers specific for the indicated IL12B promoter region. Fold enrichment was calculated using the percentage input method, and the graph shows the relative fold enrichment from the qPCR assay results. Data are representative of three independent experiments and are presented as means ± SD. (H) U3As1 cells expressing STAT1-WT or STAT1-T749A were transiently transfected with expression plasmid for HA-IKKβ. Twenty-four hours later, total RNA was isolated and IL12B transcripts were quantified by qRT-PCR analysis. Data are representative of three independent experiments and are presented as means ± SD. (I) WT dTHP-1 cells were left unstimulated or were stimulated with LPS (100 ng/ml) for 4 hours. ChIP was performed with anti-STAT1 or control IgG, which was followed by qPCR analysis with primers specific for the indicated promoter regions of IL12B. Fold enrichment was calculated using the percentage input method, and the graph shows the relative fold enrichment from the qPCR assay results. Data are representative of three independent experiments and are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as measured by one-way ANOVA with post hoc Tukey’s test (A, B, and D to H) or by paired Student’s t test (I).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/624/eaay0574/DC1

    Fig. S1. LPS-induced TLR4 endocytosis promotes ARID5A, IL-6, and IL-12p40 production.

    Fig. S2. ARID5A stabilizes IL6 mRNA, but not IL12B or TNF mRNA, through binding to its 3′UTR.

    Fig. S3. TLR4 endocytosis–dependent IFN-β–JAK–STAT1-pTyr701 signaling is dispensable for ARID5A expression.

    Fig. S4. Phosphorylation of STAT1 at Thr749 facilitates its binding to a noncanonical DNA motif in the ARID5A promoter region.

    Fig. S5. A noncanonical TBK1-IKKβ heterodimer mediates the phosphorylation of STAT1 at Thr749 downstream of TLR4 endocytosis.

    Table S1. List of primer sequences for RT-qPCR analysis, plasmid construction, and ChIP-qPCR assays.

    Table S2. List of antibodies used for Western blotting and immunoprecipitations.

  • This PDF file includes:

    • Fig. S1. LPS-induced TLR4 endocytosis promotes ARID5A, IL-6, and IL-12p40 production.
    • Fig. S2. ARID5A stabilizes IL6 mRNA, but not IL12B or TNF mRNA, through binding to its 3′UTR.
    • Fig. S3. TLR4 endocytosis–dependent IFN-β–JAK–STAT1-pTyr701 signaling is dispensable for ARID5A expression.
    • Fig. S4. Phosphorylation of STAT1 at Thr749 facilitates its binding to a noncanonical DNA motif in the ARID5A promoter region.
    • Fig. S5. A noncanonical TBK1-IKKβ heterodimer mediates the phosphorylation of STAT1 at Thr749 downstream of TLR4 endocytosis.
    • Table S1. List of primer sequences for RT-qPCR analysis, plasmid construction, and ChIP-qPCR assays.
    • Table S2. List of antibodies used for Western blotting and immunoprecipitations.

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