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

Abstract

Interleukin-17A (IL-17A) not only stimulates immunity to fungal pathogens but also contributes to autoimmune pathology. IL-17 is only a modest activator of transcription in experimental tissue culture settings. However, IL-17 controls posttranscriptional events that enhance the expression of target mRNAs. Here, we showed that the RNA binding protein (RBP) Arid5a (AT-rich interactive domain-containing protein 5a) integrated multiple IL-17-driven signaling pathways through posttranscriptional control of mRNA. IL-17 induced expression of Arid5a, which was recruited to the adaptor TRAF2. Arid5a stabilized IL-17-induced cytokine transcripts by binding to their 3' untranslated regions and also counteracted mRNA degradation mediated by the endoribonuclease MCPIP1 (Regnase-1). Arid5a inducibly associated with the eukaryotic translation initiation complex and facilitated the translation of the transcription factors (TFs) IκBζ (Nfkbiz ) and C/EBPβ (Cebpb). These TFs in turn transactivated IL-17-dependent promoters. Together, these data indicated that Arid5a orchestrates a feed-forward amplification loop, which promoted IL-17 signaling by controlling mRNA stability and translation.

Figure 1.. IL-17 increases the abundance of Arid5a, which inducibly associates with TRAF2.

(A) qRT-PCR analysis of Arid5a mRNA expression in the tongue tissue of WT or Il17ra^−/− mice at 24hrs after oral exposure to PBS (sham) or C. albicans (CAF2-1). Fold-change data are means ± SEM of at least 8 mice/group from 2 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 3 independent experiments. Right: Western blot analysis of Arid5a on lysates from ST2 cells treated with IL-17A for 4 h. Blots are representative of 3 independent experiments. Quantified band intensity values are means ± SEM from all experiments. (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 2 independent experiments (fig. S1B). (D) Co-immunoprecipitation 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 antibody against Myc. Blots are representative of 3 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 ANOVA with post hoc Tukey’s test (A), Dunnett’s test (B, left), or paired Student t-test (B, right).

Figure 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 or scrambled control and treated with IL-17 for 3 h. Fold-change data are means ± SEM from 3 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 h. Fold-change data are means ± SEM from 3 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 3 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 h. Fold-change data are means ± SEM from 3 independent experiments. (E) qRT-PCR analysis of Il6 or Cxcll mRNA in ST2 cells transfected with pooled siRNAs targeting Arid5a or scrambled control, pretreated with TNFα for 3 h, then treated with Actinomycin D and IL-17 for the indicated times. Remaining mRNA compared to time=0 data are means of ± SEM representative of 3 independent experiments. (F) Luciferase assay of Il6 3’UTR activity in HEK293T cells at 24 hours after transfect with a luciferase reporter and empty vector (EV), Flag/Myc-Arid5a, Act1-Myc, or MCPIP1 and analyzed after 24 h. *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 (t_½) were determined using equations that defined decay kinetics as shown by colored lines in the graph, as described (42) (E).

Figure 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 h. Fold-change data are means ± SEM from 3-5 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 3 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 h. Fold change data are means ± SEM from 3 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 h. Blots are representative of 3 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 IgG or eIF4G immunoprecipitates from cytoplasmic extracts of from ST2 cells after transfection with siRNAs targeting Arid5a or control siRNA and treatment with IL-17 for 3 h. Inset: Western blot analysis of eIF4G in cytoplasmic fractions immunoprecipitated with IgG or eIF4G. Data are fold-change means ± SEM representative of 3 independent experiments. (F) Co-immunoprecipitation 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 antibody against eIF4G. Blots are representative of 3 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, C to F) or Dunnett’s test (B).

Figure 4.. Arid5a mediates translation of IκBξ.

(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 3 independent experiments. (B) Western blot analysis of IκBξ in nuclear extracts from ST2 cells treated with IL-17A for indicated times. Blots are representative of 3 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 2 independent experiments. Right: Western blot analysis of IκBξ in nuclear extracts from ST2 cells transfected with siRNAs targeting Arid5a or control siRNA and treated with IL-17A for 4 h. Blots are representative of 4 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 h. Fold-change data are means ± SEM from 3 independent experiments. Right: Western blot analysis of IκBξ in whole cell lysates from primary MEFs transfected with siRNAs targeting Arid5a or control siRNA and treated with IL-17A for 24 h. Blots are representative of 3 independent experiments. (E) Western blot analysis of IκBξ and Myc-tagged Arid5a in lysates from HEK293T cells transfected with empty vector (EV), IκBξ or Flag/Myc-Arid5a. Blots are representative of 3 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 h, and then treated with Actinomycin D and IL-17 for the indicated times. Remaining mRNA compared to time=0 data are means of ± SEM representative of 2 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 h. Data are fold-change means ± SEM representative of 3 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, B); half-lives (t½) were assessed as described (42) (E).

Figure 5.. Arid5a binds directly to target mRNA transcripts.

(A to B) qRT-PCR analysis of mRNAs from ST2 cytoplasmic extracts transfected with Flag/Myc-Arid5a, treated with IL-17 for 3 h, and subjected to RIP with IgG2a or Myc. Data are fold-change means ± SEM representative of 3 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 3 independent experiments (for Il6) and 2 independent experiments for Cxcll and Csf2. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 by ANOVA with post hoc Tukey’s test.

Figure 6.. Arid5a promotes responses to IL-17 in primary MEFs and human keratinocytes.

(A) qRT-PCR analysis of primary MEFs transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 24 h. Fold change data are means ± SEM from 3 independent experiments. (B) ELISA analysis of conditioned supernatants from MEFs transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 24 h. Fold change data are means ± SEM representative of 2 independent experiments. (C) qRT-PCR analysis of human keratinocyte (N/TERT2G) cells transfected with pooled siRNAs targeting Arid5a or scrambled control and treated with IL-17 for 5 h. Fold change data are means ± SEM from 3 independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 by ANOVA with post hoc Tukey’s test.

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

Upon IL-17 stimulation, Arid5a expression is increased and this RNP is recruited to TRAF2. Arid5a promotes the mRNA stability of multiple genes, including cytokines and chemokines as well as the transcription factor Nfkbiz (IκBξ). Additionally, Arid5a enhances the translation of Nfkbiz and Cebpb, transcription factors that in turn regulate downstream genes such as Lcn2, which are not intrinsically unstable. Thus, Arid5a is a central player in the post-transcriptional IL-17 signaling cascade.