Research ArticleCytokines

IL-17 integrates multiple self-reinforcing, feed-forward mechanisms through the RNA binding protein Arid5a

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Science Signaling  09 Oct 2018:
Vol. 11, Issue 551, eaat4617
DOI: 10.1126/scisignal.aat4617
  • Fig. 1 IL-17 increases the abundance of Arid5a, which inducibly associates with TRAF2.

    (A) Quantitative reverse transcription PCR (qRT-PCR) analysis of Arid5a mRNA expression in the tongue tissue of WT or Il17ra−/− mice at 24 hours after oral exposure to phosphate-buffered saline (sham) or C. albicans (CAF2-1). Fold change data are means ± SEM of at least eight mice per group from two independent experiments. (B) Left: qRT-PCR analysis of Arid5a mRNA expression in ST2 cells treated with IL-17 for the indicated times. Fold change data are means ± SEM from three independent experiments. Right: Western blot analysis of Arid5a on lysates from ST2 cells treated with IL-17A for 4 hours. Blots are representative of three independent experiments. Quantified band intensity values are means ± SEM from all experiments. UT, untreated. (C) Co-immunoprecipitation analysis of Arid5a interaction with TRAF2 in lysates from HEK293T cells transfected with empty vector (EV), Flag/Myc-Arid5a, or TRAF2 and immunoprecipitated for Flag. Blots are representative of two independent experiments (fig. S1B). WCL, whole cell lysate. (D) CoIP analysis of Arid5a interaction with TRAF2 in lysates from ST2 cells transfected with Flag/Myc-Arid5a, treated with IL-17 for the indicated times and immunoprecipitated with Ab against Myc. Blots are representative of three independent experiments (fig. S2E). Quantified band intensity values are means ± SEM from all experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by analysis of variance (ANOVA) with post hoc Tukey’s test (A), Dunnett’s test (B, left), or paired Student’s t test (B, right).

  • Fig. 2 Arid5a promotes cellular responses to IL-17.

    (A) qRT-PCR analysis of the indicated mRNAs in ST2 cells transfected with pooled siRNAs targeting Arid5a (siArid5a) or scrambled control (siControl) and treated with IL-17 for 3 hours. Fold change data are means ± SEM from three independent experiments. (B) qRT-PCR analysis of Il6 mRNA expression in ST2 cells transfected with pooled siRNAs targeting Arid5a, TRAF2, or scrambled control and treated with IL-17 for 3 hours. Fold change data are means ± SEM from three independent experiments. (C) qRT-PCR analysis of Il6 or Lcn2 mRNA expression in ST2 cells treated with IL-17 for the indicated times. Fold change data are means ± SEM from three independent experiments. (D) qRT-PCR analysis of Il6 or Lcn2 in ST2 cells transfected with pooled siRNAs targeting Arid5a ± MCPIP1 or scrambled control and treated with IL-17 for 3 hours. Fold change data are means ± SEM from three independent experiments. (E) qRT-PCR analysis of Il6 or Cxcl1 mRNA in ST2 cells transfected with pooled siRNAs targeting Arid5a or scrambled control, pretreated with TNFα for 3 hours, and then treated with actinomycin D (ActD) and IL-17 for the indicated times. Remaining mRNAs compared to time = 0 data are means ± SEM representative of three independent experiments. (F) Luciferase (LUC) assay of Il6 3′UTR activity in HEK293T cells at 24 hours after transfection with a luciferase reporter and EV, Flag/Myc-Arid5a, Act1-Myc, or MCPIP1 and analyzed after 24 hours. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA with post hoc Tukey’s test (A, B, D, and F) or Dunnett’s test (C); half-lives (t½) were determined using equations that defined decay kinetics as shown by colored lines in the graph, as described (42) (E). n.s., not significant.

  • Fig. 3 Arid5a mediates translation of C/EBPβ mRNA.

    (A) Luciferase assay of Lcn2 proximal promoter activity in HEK293T cells transfected with a luciferase reporter together with EV or Flag/Myc-Arid5a and analyzed after 24 hours. Fold change data are means ± SEM from three to five independent experiments. (B) qRT-PCR analysis of Cebpb mRNA expression in ST2 cells treated with IL-17 for the indicated times. Fold change data are means ± SEM from three independent experiments. (C) qRT-PCR analysis of Cebpb mRNA expression in ST2 cells transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 1 hour. Fold change data are means ± SEM from three independent experiments. (D) Western blot analysis of C/EBPβ isoforms (LAP*, LAP, and LIP) in nuclear extracts from ST2 cells transfected with siRNAs targeting Arid5a or control siRNA and treated with IL-17A for 4 hours. Blots are representative of three independent experiments. Quantified band intensity values are means ± SEM from all experiments. (E) RIP assay (left) of Cebpb mRNA amount by qRT-PCR analysis on immunoglobulin G (IgG) or eIF4G immunoprecipitates from cytoplasmic extracts of ST2 cells after transfection with siRNAs targeting Arid5a or control siRNA and treatment with IL-17 for 3 hours. Inset: Western blot analysis of eIF4G in cytoplasmic fractions immunoprecipitated with IgG or eIF4G. Data are fold change means ± SEM representative of three independent experiments. (F) CoIP analysis of Arid5a interaction with eIF4G in lysates from ST2 cells transfected with Flag/Myc-Arid5a, treated with IL-17 for the indicated times, and immunoprecipitated with Ab against eIF4G. Blots are representative of three independent experiments (fig. S2F). Quantified band intensity values are means ± SEM from all experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001 by ANOVA with post hoc Tukey’s test (A and C to F) or Dunnett’s test (B).

  • Fig. 4 Arid5a mediates translation of IκBζ.

    (A) qRT-PCR analysis of Nfkbiz mRNA expression in ST2 cells treated with IL-17 for the indicated times. Fold change data are means ± SEM from three independent experiments. (B) Western blot analysis of IκBζ in nuclear extracts from ST2 cells treated with IL-17A for indicated times. Blots are representative of three independent experiments. Quantified band intensity values are means ± SEM from all experiments. (C) Left: qRT-PCR analysis of Nfkbiz mRNA expression in ST2 cells transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 30 min. Fold change data are means ± SEM from two independent experiments. Right: Western blot analysis of IκBζ in nuclear extracts from ST2 cells transfected with siRNAs targeting Arid5a or control siRNA and treated with IL-17A for 4 hours. Blots are representative of four independent experiments. (D) Left: qRT-PCR analysis of Nfkbiz mRNA expression in primary MEFs transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 24 hours. Fold change data are means ± SEM from four independent experiments. Right: Western blot analysis of IκBζ in whole-cell lysates from primary MEFs transfected with siRNAs targeting Arid5a or control siRNA and treated with IL-17A for 24 hours. Blots are representative of three independent experiments. (E) Western blot analysis of IκBζ and Myc-tagged Arid5a in lysates from HEK293T cells transfected with EV, IκBζ, or Flag/Myc-Arid5a. Blots are representative of three independent experiments. Quantified band intensity values are means ± SEM from all experiments. (F) qRT-PCR analysis of Nfkbiz mRNA expression in ST2 cells transfected with pooled siRNAs targeting Arid5a or scrambled control that were pretreated with TNFα for 3 hours and then treated with actinomycin D and IL-17 for the indicated times. Remaining mRNAs compared to time = 0 data are means ± SEM representative of two independent experiments. (G) RIP assay of Nfkbiz mRNA amount by qRT-PCR analysis on IgG or eIF4G immunoprecipitates from cytoplasmic extracts of ST2 cells after transfection with siRNAs targeting Arid5a or control siRNA and treatment with IL-17 for 3 hours. Data are fold change means ± SEM representative of three independent experiments. Inset: Western blot analysis of eIF4G in cytoplasmic fractions immunoprecipitated with IgG or eIF4G. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA with post hoc Tukey’s test or Dunnett’s test (A and B); half-lives (t½) were assessed as described (42) (E).

  • Fig. 5 Arid5a binds directly to target mRNA transcripts.

    (A and B) qRT-PCR analysis of mRNAs from ST2 cytoplasmic extracts transfected with Flag/Myc-Arid5a, treated with IL-17 for 3 hours, and subjected to RIP with IgG2a or Myc. Data are fold change means ± SEM representative of three independent experiments. (C) In vitro RNA pulldown assay (box) of Arid5a-Myc by Western blot analysis streptavidin bead immunoprecipitates from lysates of Arid5a-Myc transfected HEK293T cells incubated with the indicated in vitro–generated, biotinylated mRNAs. Data are derived from the same blot and are representative of three independent experiments for Il6 and two independent experiments for Cxcl1 and Csf2. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA with post hoc Tukey’s test.

  • Fig. 6 Arid5a promotes responses to IL-17 in primary MEFs and human KCs.

    (A) qRT-PCR analysis of primary MEFs transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 24 hours. Fold change data are means ± SEM from three independent experiments. (B) Enzyme-linked immunosorbent assay (ELISA) analysis of conditioned supernatants from MEFs transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 24 hours. Fold change data are means ± SEM representative of two independent experiments. (C) qRT-PCR analysis of human KC (N/TERT2G) cells transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 5 hours. Fold change data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by ANOVA with post hoc Tukey’s test.

  • Fig. 7 Model of Arid5a in the IL-17R signaling pathway.

    Upon IL-17 stimulation, Arid5a expression is increased, and this RBP is recruited to TRAF2. Arid5a promotes the mRNA stability of multiple genes, including cytokines and chemokines, and the TF Nfkbiz (IκBζ). In addition, Arid5a enhances the translation of Nfkbiz and Cebpb, TFs that in turn regulate downstream genes such as Lcn2, which are not intrinsically unstable. Thus, Arid5a is a central player in the posttranscriptional IL-17 signaling cascade.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/551/eaat4617/DC1

    Fig. S1. Arid5a interactions within the IL-17 signaling pathway.

    Fig. S2. Role of TRAF2 and HuR in Arid5a-mediated IL-17 response.

    Fig. S3. Arid5a promotes IL-17–induced mRNA stability.

    Fig. S4. Arid5a promotes IL-17–induced IκBζ expression.

    Fig. S5. Arid5a promotes IL-17 responses through distinct mechanisms.

    Fig. S6. Arid5a binds directly to target mRNA transcripts.

    Fig. S7. Arid5a promotes translation of IL-17–induced mRNAs.

    Fig. S8. Arid5a promotes mRNA stability of IL-17–induced genes.

  • This PDF file includes:

    • Fig. S1. Arid5a interactions within the IL-17 signaling pathway.
    • Fig. S2. Role of TRAF2 and HuR in Arid5a-mediated IL-17 response.
    • Fig. S3. Arid5a promotes IL-17–induced mRNA stability.
    • Fig. S4. Arid5a promotes IL-17–induced IκBζ expression.
    • Fig. S5. Arid5a promotes IL-17 responses through distinct mechanisms.
    • Fig. S6. Arid5a binds directly to target mRNA transcripts.
    • Fig. S7. Arid5a promotes translation of IL-17–induced mRNAs.
    • Fig. S8. Arid5a promotes mRNA stability of IL-17–induced genes.

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