14-3-3ζ-TRAF5 axis governs interleukin-17A signaling

Abstract

IL-17A is a therapeutic target in many autoimmune diseases. Most nonhematopoietic cells express IL-17A receptors and respond to extracellular IL-17A by inducing proinflammatory cytokines. The IL-17A signal transduction triggers two broad, TRAF6- and TRAF5-dependent, intracellular signaling pathways to produce representative cytokines (IL-6) and chemokines (CXCL-1), respectively. Our limited understanding of the cross-talk between these two branches has generated a crucial gap of knowledge, leading to therapeutics indiscriminately blocking IL-17A and global inhibition of its target genes. In previous work, we discovered an elevated expression of 14-3-3 proteins in inflammatory aortic disease, a rare human autoimmune disorder with increased levels of IL-17A. Here we report that 14-3-3ζ is essential for IL-17 signaling by differentially regulating the signal-induced IL-6 and CXCL-1. Using genetically manipulated human and mouse cells, and ex vivo and in vivo rat models, we uncovered a function of 14-3-3ζ. As a part of the molecular mechanism, we show that 14-3-3ζ interacts with several TRAF proteins; in particular, its interaction with TRAF5 and TRAF6 is increased in the presence of IL-17A. In contrast to TRAF6, we found TRAF5 to be an endogenous suppressor of IL-17A-induced IL-6 production, an effect countered by 14-3-3ζ. Furthermore, we observed that 14-3-3ζ interaction with TRAF proteins is required for the IL-17A-induced IL-6 levels. Together, our results show that 14-3-3ζ is an essential component of IL-17A signaling and IL-6 production, an effect that is suppressed by TRAF5. To the best of our knowledge, this report of the 14-3-3ζ-TRAF5 axis, which differentially regulates IL-17A-induced IL-6 and CXCL-1 production, is unique.

Keywords: 14-3-3ζ; IL-17A; TRAF.

Fig. 1.

14-3-3ζ is required for IL-17A–mediated gene induction. (A) ARPE-19 cells, treated with hIL-17A in the presence of 14-3-3 pan inhibitor (BV02), were analyzed for IL6 mRNA levels by qRT-PCR. (B) CRISPR/Cas9-mediated 14-3-3ζ^KO ARPE-19 cells were analyzed for the endogenous levels of additional 14-3-3 isoforms, as indicated, using qRT-PCR (Upper) or immunoblot (Lower). (C and D) The WT and 14-3-3ζ^KO ARPE-19 cells, treated with hIL-17A, were analyzed for IL6 (C) or IL8 (D) mRNA levels using qRT-PCR. (E) The WT and 14-3-3ζ^KO ARPE-19 cells, treated with hIL-17A, were analyzed for IL-6 protein levels using ELISA. (F and G) The WT and 14-3-3ζ^KO MEFs, treated with mIL-17A, were analyzed for Il6 (F) or Il8 (G) mRNA levels using qRT-PCR. The loss of 14-3-3ζ protein in the KO MEFs is also shown as an immunoblot. (H and I) Rescuing Flag-tagged 14-3-3ζ (shown by immunoblot) by lentiviral transduction of 14-3-3ζ^KO ARPE-19 cells support IL-17A–induced IL6 (H) and IL8 (I) mRNA levels, analyzed by qRT-PCR. All results are representative of at least three experiments, *P < 0.05, ***P < 0.0005, and ****P < 0.0001; ns, not significant.

Fig. 2.

14-3-3ζ is required for IL-17A–mediated NF-κB activation. (A) Dual-luciferase activity was measured in NSC or 14-3-3ζ^KD HeLa cells, transiently coexpressing NF-κB, thymidine kinase (TK), and HA-tagged 14-3-3ζ, were treated with hIL-17A for 20 h. (B) WT or 14-3-3ζ^KO ARPE-19 cells, treated with hIL-17A for 1 h, were immunostained for endogenous p65 and analyzed by confocal microscopy. (Scale bar: 10 μm.) (C) WT or 14-3-3ζ^KO MEFs, treated with mIL-17A for 1 h, were immunostained for endogenous p65 and analyzed by confocal microscopy and the nuclear p65-expressing cells were quantified from at least 100 cells. (D and E) Nuclear fractions from WT and 14-3-3ζ^KO MEFs (D) or ARPE-19 cells (E), treated with IL-17A for 1 h, were analyzed for p65 and HDAC1 by immunoblots (shown in SI Appendix, Fig. S3), which were quantified using imageJ software. The results are representative of at least three experiments, *P < 0.05, **P < 0.005, and ***P < 0.0005.

Fig. 3.

14-3-3ζ is required for IL-17A–mediated pERK. (A–C) The WT and 14-3-3ζ^KO ARPE-19 (A and C) treated with hIL-17A, or MEFs treated with mIL-17A (B), for the indicated times and doses, were analyzed for pERK by immunoblot. (D) The 14-3-3ζ (Flag-tagged) restored 14-3-3ζ^KD HeLa cells, treated with IL-17A, were analyzed for pERK by immunoblot. EV, empty vector. All results are representative of at least three experiments, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Fig. 4.

14-3-3ζ forms a complex with TRAF proteins in cells and tissues. (A) HEK293T cells were cotransfected with HA–14-3-3ζ and Flag-TRAFs, as indicated. The cell lysates were immunoprecipitated with anti-HA, and the IPs were analyzed by immunoblot. The expression of the proteins is shown in the input. (B) Rat aortic lysates were immunoprecipitated with anti–14-3-3ζ or a control IgG, and the IPs were analyzed by immunoblot, as indicated. (C and D) ARPE-19 cells, treated with IL-17A, were immunoprecipitated with anti–14-3-3ζ or TRAF6, and the IPs were analyzed by immunoblot, as indicated. (E) Confocal analyses of TRAF6 and TRAF5 colocalization with 14-3-3ζ in hIL-17–treated ARPE-19 cells. (Scale bar, 10 µm.) (F and G) HA–14-3-3ζ or Flag-TRAF proteins were ectopically expressed in HEK293T cells, and isolated to near purity, and were subjected to cell-free interaction assay (SI Appendix, Fig. S5), and the complex was analyzed by co-IP.

Fig. 5.

TRAF5 suppresses IL-17A–stimulated 14-3-3ζ–dependent IL6 induction. (A and B) ARPE-19 cells, expressing shRNA against TRAF5 or a nonsilencing control (NSC) sequence, were analyzed for hIL-17A–induced IL6 by qRT-PCR (A) or ELISA (B). The KD of TRAF5 is shown by immunoblot (A). (C) The qRT-PCR analyses of CXCL1 mRNA in TRAF5^KD ARPE-19 cells when compared to control cells, upon IL-17A treatment. (D) The qRT-PCR analyses of Cxcl1 mRNA induction in WT and 14-3-3ζ^KO MEFs, stimulated with mIL-17A. (E) IL-17A–stimulated pERK was analyzed in control (NSC) or TRAF5^KD ARPE-19 cells by immunoblot. (F) The qRT-PCR analyses of IL6 induction in control (NSC) or TRAF5^KD ARPE-19 cells in the presence or the absence of 30 µM of U0126 (MEK inhibitor), upon IL-17A. (G) The ARPE-19–derived cells, as indicated, were analyzed for IL6 by qRT-PCR and the protein expression was validated in the immunoblot. All results are representative of at least three experiments, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Fig. 6.

The interaction with TRAF is required for 14-3-3ζ–mediated IL6 induction by IL-17A. (A) Two putative TRAF5 binding motifs (in red) of 14-3-3ζ and the mutants (M1 and M2) are shown. (B) The modeled structures of the TRAF-domain of TRAF5 (4GJH.pdb, pink color) complex with the dimeric 14-3-3ζ (5D2D.pdb, gray color) with restricted interaction to either of the two TBMs, SNEE or AYQE, are shown. The TBMs of 14-3-3ζ are shown in yellow color. (C) HEK293T cells, coexpressing V5-TRAF5 or Flag–14-3-3ζ, as indicated, were immunorecipitated with anti-Flag and elutes were analyzed by immunoblot. (D) HEK293T cells, coexpressing Flag-TRAF5 and HA–14-3-3ζ (WT or M2), were analyzed by PLA followed by confocal microscopy. (Scale bar, 5 µm.) (E) ARPE-19 cells, ectopically expressing 14-3-3ζ (WT, M1 or M2), were analyzed for IL-17A–induced IL6 by qRT-PCR. The expression of Flag-tagged 14-3-3ζ is shown by immunoblot. All results are representative of at least three experiments, ****P < 0.005.

Fig. 7.

Functional significance of 14-3-3ζ participation in IL-17A signaling. (A) Culture supernatants from either untreated or IL-17A–treated WT or 14-3-3ζ^KO MEFs were analyzed for the killing of C. albicans. (B) WT or 14-3-3ζ^KO MEFs, treated with mIL-17A, were analyzed for Defb3 by qRT-PCR. (C) The schematic design of 14-3-3ζ KO Lewis rat generation is shown; the exon 3 of rat 14-3-3ζ was targeted by the gRNAs, as indicated. A representative genotyping result is shown from the tail DNA isolated from the indicated rat strains (^+/+, ^+/−, and ^−/−). An immunoblot of 14-3-3ζ from the tail biopsy of the indicated strains is shown. (D and E) The duodenum of WT or heterozygous 14-3-3ζ Lewis rats were either untreated or treated with rat (r) IL-17A for 20 h, and the Il6 (D) and Cxcl1 (E) mRNA levels were analyzed by qRT-PCR. (F and G) The WT and 14-3-3ζ^KO rats (n = 8) were injected with rIL-17A intraperitoneally for the indicated times, and cytokine (Il-6 and Cxcl-1) levels in the sera were analyzed by ELISA. The results are representative of at least three experiments, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Fig. 8.

A proposed model for the identified 14-3-3ζ–TRAF5 axis in regulating IL-17A signaling pathways. IL-17A binding triggers a receptor complex (IL-17RA–ACT1–TRAF) activation that transduces the signal to generate transcriptional output (IL-6/IL-8/CXCL-1). Our results show that 14-3-3ζ is a TRAF-binding component that is needed for IL-17A-stimulated pERK, NF-κB activation, and IL-6/IL-8 production, but not for CXCL-1. Importantly, we uncovered a role of TRAF5 in suppressing the IL-17A–stimulated IL-6 production. The IL-17A stimulation increases the 14-3-3ζ–TRAF5 interaction that potentially inhibits the TRAF5 function and supports the IL-6 production. Overall, our model suggests that IL-17A stimulation increases 14-3-3ζ interaction to sequester TRAF5, which results in increased IL-6 levels. These results have broad implications covering IL-17A’s role in antifungal immunity to regulation of inflammation.