14-3-3ζ–TRAF5 axis governs interleukin-17A signaling Jenna McGowana,1, Cara Petera,1, Joshua Kima, Sonam Poplib, Brent Veermana, Jessica Saul-McBethc, Heather Contic, Shondra M. Pruett-Millerd, Saurabh Chattopadhyayb, and Ritu Chakravartia,2 aDepartment of Physiology & Pharmacology, College of Medicine & Life Sciences, University of Toledo, Toledo, OH 43614; bDepartment of Medical Microbiology & Immunology, College of Medicine & Life Sciences, University of Toledo, Toledo, OH 43614; cDepartment of Biological Sciences, College of Natural Sciences & Mathematics, University of Toledo, Toledo, OH 43614; and dDepartment of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105 Edited by Vishva M. Dixit, Genentech, San Francisco, CA, and approved August 31, 2020 (received for review April 27, 2020) IL-17A is a therapeutic target in many autoimmune diseases. Most of target genes, such as IL6 and IL8 (18). On the other hand, the nonhematopoietic cells express IL-17A receptors and respond to recruitment of TRAF2 and TRAF5 participates in the stabiliza- extracellular IL-17A by inducing proinflammatory cytokines. The IL- tion of mRNA transcripts of CXCL1 and CXCL5 (19). The 17A signal transduction triggers two broad, TRAF6- and TRAF5- TRAF5/TRAF2-dependent mRNA stabilization pathway is inde- dependent, intracellular signaling pathways to produce representative pendent of the presence of TRAF6 (19); however, the role of cytokines (IL-6) and chemokines (CXCL-1), respectively. Our limited TRAF5/TRAF2 in the transcriptional pathway remains unknown. understanding of the cross-talk between these two branches has In addition, IL-17A activates MAP kinases, including ERK, p38, generated a crucial gap of knowledge, leading to therapeutics and JNK, that play essential roles in autoimmune diseases (20, indiscriminately blocking IL-17A and global inhibition of its target 21). Although several regulators of each transcriptional and genes. In previous work, we discovered an elevated expression of nontranscriptional branch are known (17), there is a knowledge 14-3-3 proteins in inflammatory aortic disease, a rare human au- gap in understanding of cross-talk between the two branches. toimmune disorder with increased levels of IL-17A. Here we report Identifying regulators that promote one branch over the other will that 14-3-3ζ is essential for IL-17 signaling by differentially regu- provide critical information to design targeted therapeutics. lating the signal-induced IL-6 and CXCL-1. Using genetically manip- To address this, we examined the role of 14-3-3ζ in the IL-17A ulated human and mouse cells, and ex vivo and in vivo rat models, signaling. 14-3-3ζ is one of the seven members of 14-3-3 family we uncovered a function of 14-3-3ζ. As a part of the molecular that interacts with 0.6% of the cellular proteome and performs a mechanism, we show that 14-3-3ζ interacts with several TRAF pro- range of context-dependent functions, including signaling IMMUNOLOGY AND INFLAMMATION teins; in particular, its interaction with TRAF5 and TRAF6 is increased (22–24). The 14-3-3ζ regulates the signal transduction of GM- in the presence of IL-17A. In contrast to TRAF6, we found TRAF5 to CSF, IL-3, IL-5, STAT3, Toll-like receptor (TLR)3, and AMPK, be an endogenous suppressor of IL-17A–induced IL-6 production, an among others (25–29). Additionally, it supports the production effect countered by 14-3-3ζ. Furthermore, we observed that 14-3-3ζ of several cytokines, such as IL-13, IFN-γ, and IL-17A (30, 31). – interaction with TRAF proteins is required for the IL-17A induced IL- We previously identified increased 14-3-3 protein levels in the ζ 6 levels. Together, our results show that 14-3-3 is an essential com- aortic tissues of patients with large-vessel vasculitis (32). Large- ponent of IL-17A signaling and IL-6 production, an effect that is vessel vasculitis is a presumed autoimmune disease with in- suppressed by TRAF5. To the best of our knowledge, this report creased infiltration of Th1 and Th17 cells in the affected artery, ζ – of the 14-3-3 -TRAF5 axis, which differentially regulates IL-17A induced resulting in increased levels of IL-17A and IL-6 (33–35). In the IL-6 and CXCL-1 production, is unique. Significance 14-3-3ζ | IL-17A | TRAF Blocking IL-17A signaling is an attractive therapy in treating nterleukin-17A (IL-17A) is an inflammatory cytokine that is autoimmune diseases. Current IL-17A blockers target both Iassociated with autoimmune diseases and host-defense (1, 2). signaling branches, leading to inhibition of TRAF6-based IL-6 Increased IL-17A level in the sera of patients with rheumatoid – production, and TRAF5-based CXCL-1 production. Global sup- arthritis, psoriasis, and lupus, is well documented (3 5). The IL- pression of both branches is of concern due to the critical role 17A blocking therapy has been clinically successful to manage – IL-17A plays in the host-defense mechanisms. Our study shows psoriasis, but not other diseases (6 8). Current IL-17A blockers that 14-3-3ζ is necessary for IL-17A–stimulated IL-6 production. lack specificity and compromise the host-defense mechanisms (9, We also found that 14-3-3ζ represses TRAF5-mediated sup- 10). There is a need to design inhibitors that can target specific pression of IL-17A–stimulated IL-6 induction. Taken together, outcomes of IL-17A signaling. To achieve such a goal, we need our results show a unique cross-talk between two branches via to improve the understanding of IL-17A signal transduction and the 14-3-3ζ–TRAF5 axis that regulates the IL-17A signaling identify the regulatory mechanisms for each set of genes. output. These foundational results can lead to the design of a The most common targets of IL-17A signaling include NF- κ – unique inhibitor targeting a specific branch of IL-17A signaling B dependent cytokines, chemokines, antimicrobial peptides and obtaining the desired output. (AMP), and matrix metalloproteases (11, 12). IL-17A plays a significant role in up-regulating the production of inflammatory Author contributions: S.P., S.C., and R.C. designed research; J.M., C.P., J.K., S.P., B.V., cytokines via either transcriptional (gene expression) or post- J.S.-M., H.C., and R.C. performed research; B.V., S.M.P.-M., and R.C. contributed new transcriptional (mRNA stabilization) branches (5, 13). IL-17A reagents/analytic tools; J.M., C.P., J.K., S.P., J.S.-M., H.C., S.C., and R.C. analyzed data; signal transduction occurs via IL-17 receptors consisting of the IL- and J.M., S.C., and R.C. wrote the paper. 17RA and IL-17RC subunits (14). While IL-17RA is ubiquitously The authors declare no competing interest. expressed, IL-17RC has limited expression to nonhematopoietic This article is a PNAS Direct Submission. epithelial and mesenchymal cells (15, 16). IL-17A binding to its Published under the PNAS license. receptor results in the recruitment of the cytosolic signaling 1J.M. and C.P. contributed equally to this work. complex consisting of Act1 and several TRAF proteins [see review 2To whom correspondence may be addressed. Email: [email protected]. for further details (17)]. Recruitment of TRAF6 results in the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ activation of downstream molecules, including TAK1, eventually doi:10.1073/pnas.2008214117/-/DCSupplemental. leading to translocation of NF-κB to the nucleus and transcription www.pnas.org/cgi/doi/10.1073/pnas.2008214117 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 present study, we identified a requirement of intracellular 14-3- drives Renilla luciferase by a NF-κB promoter, and a firefly lu- 3ζ in IL-17A signal transduction. We used 14-3-3ζ knockout ciferase driven by thymidine kinase promoter in the 14-3-3ζ (KO) murine fibroblasts and human epithelial cells to examine knockdown (KD) HeLa cells, and measured luciferase activity the role of 14-3-3ζ in IL-17A signaling. Our results show that 14- after the stimulation with IL-17A (39). The ablation of 14-3-3ζ 3-3ζ is essential for IL6, IL8, and Defb3 production in IL-17A resulted in significant suppression of the IL-17A–induced NF-κB signaling; however, it has an opposite effect on CXCL1 pro- activity in a luciferase assay (lanes 2 and 4, Fig. 2A). The com- duction. To validate our finding, we engineered 14-3-3ζ global KO plementation with HA-tagged 14-3-3ζ resulted in a substantial Lewis rats using CRISPR/Cas9 and subjected them to IL-17A rescue of NF-κB activity in 14-3-3ζKD cells (lanes 4 and 6, treatment, a technique successfully used to determine the Act1 Fig. 2A). Encouraged by these results, we focused on a specific role in IL-17A signal transduction (36). Similar to cells, the 14-3- stage of NF-κB activation, the nuclear translocation of the p65 3ζKO rats show a decrease in the IL-17A–stimulated IL-6 pro- subunit of NF-κB, in IL-17A–stimulated cells. IL-17A treatment of duction but an increase in CXCL-1 level. In the absence of 14-3- ARPE-19 cells resulted in the nuclear translocation of p65, which 3ζ, IL-17A–stimulated ERK phosphorylation (pERK) and p65 was inhibited in the 14-3-3ζKO ARPE-19 cells, analyzed by confocal translocation are decreased. The 14-3-3ζ interacts with TRAFs via microscopy (Fig. 2B). Similarly, we observed IL-17A–stimulated two of its TRAF-binding motifs (TBMs), particularly TRAF5, increase in nuclear p65+ cells in WT MEFs but not in 14-3-3ζKO which bound more strongly in the presence of IL-17A.