Caini Liu

IL-17 Enhances Chemokine Gene Expression through mRNA Stabilization

IL-17 plays an important role in host defense and autoimmunity via the induction of proinflammatory gene expression, particularly in combination with TNF-α. The molecular mechanisms by which IL-17 regulates such expression are not well understood. Using the mouse chemokine CXCL1 (KC) gene as a model, we have examined the effects of IL-17 alone or in combination with TNF-α on transcriptional and posttranscriptional events. Although treatment of mouse embryonic fibroblasts with IL-17 alone only modestly increased KC expression, the combination of IL-17 with TNF-α induced a synergistic response. IL-17 treatment exerted a strong posttranscriptional effect by extending the t1/2 of the highly unstable, TNF-α-induced KC mRNA. Using a tetracycline-regulated transgene in HeLa cells, we determined that IL-17 treatment alone promoted stabilization of KC mRNA in the absence of TNF-α. IL-17 treatment exerted little effect on KC transcription or NF-κB activation, suggesting that it primarily acts posttranscriptionally. We identified a number of other mRNAs whose t1/2 are prolonged in response to IL-17, suggesting that this is a common mechanism by which IL-17 promotes enhanced gene expression. Finally, activator of NF-κB1 protein (Act1), an adaptor protein recently implicated in IL-17 signaling, was necessary for IL-17-induced stabilization, and overexpression of Act1 resulted in stabilization of KC mRNA, indicating that events downstream of Act1 are sufficient to initiate this process. Thus, the synergy between TNF-α and IL-17 reflects their independent actions on KC gene expression; TNF-α serves as a stimulus to initiate transcription through activation of NF-κB, whereas IL-17 drives mRNA stabilization through an Act1-dependent pathway.

Astrocyte-Restricted Ablation of Interleukin-17-Induced Act1-Mediated Signaling Ameliorates Autoimmune Encephalomyelitis

Interleukin-17 (IL-17) secreted by T helper 17 (Th17) cells is essential in the development of experimental autoimmune encephalomyelitis (EAE). However, it remains unclear how IL-17-mediated signaling in different cellular compartments participates in the central nervous system (CNS) inflammatory process. We examined CNS inflammation in mice with specific deletion of Act1, a critical component required for IL-17 signaling, in endothelial cells, macrophages and microglia, and neuroectoderm (neurons, astrocytes, and oligodendrocytes). In Act1-deficient mice, Th17 cells showed normal infiltration into the CNS but failed to recruit lymphocytes, neutrophils, and macrophages. Act1 deficiency in endothelial cells or in macrophages and microglia did not substantially impact the development of EAE. However, targeted Act1 deficiency in neuroectoderm-derived CNS-resident cells resulted in markedly reduced severity in EAE. Specifically, Act1-deficient astrocytes showed impaired IL-17-mediated inflammatory gene induction. Thus, astroctyes are critical in IL-17-Act1-mediated leukocyte recruitment during autoimmune-induced inflammation of the CNS.

Treatment with IL-17 prolongs the half-life of chemokine CXCL1 mRNA via the adaptor TRAF5 and the splicing-regulatory factor SF2 (ASF)

Interleukin 17 (IL-17) promotes the expression of chemokines and cytokines via the induction of gene transcription and post-transcriptional stabilization of mRNA. We show here that IL-17 enhanced the stability of chemokine CXCL1 mRNA and other mRNAs through a pathway that involved the adaptor Act1, the adaptors TRAF2 or TRAF5 and the splicing factor SF2 (also known as alternative splicing factor (ASF)). TRAF2 and TRAF5 were necessary for IL-17 to signal the stabilization of CXCL1 mRNA. Furthermore, IL-17 promoted the formation of complexes of TRAF5-TRAF2, Act1 and SF2 (ASF). Overexpression of SF2 (ASF) shortened the half-life of CXCL1 mRNA, whereas depletion of SF2 (ASF) prolonged it. SF2 (ASF) bound chemokine mRNA in unstimulated cells, whereas the SF2 (ASF)-mRNA interaction was much lower after stimulation with IL-17. Our findings define an IL-17-induced signaling pathway that links to the stabilization of selected mRNA species through Act1, TRAF2-TRAF5 and the RNA-binding protein SF2 (ASF).

Act1, a U-box E3 ubiquitin ligase for IL-17 signaling.

The adaptor protein Act1 functions as a ubiquitin ligase to mediate interleukin-17 receptor–dependent activation of nuclear factor-κB. Acting Out a New Role Interleukin-17A (IL-17A) is the founding member of a family of proinflammatory cytokines implicated in the development of autoimmunity and the fight against microbial infection. IL-17 signaling, which is mediated by a number of IL-17 receptor (IL-17R) complexes (see the Perspective by Levin), depends on TRAF6, a scaffold protein and E3 ubiquitin ligase, and activates the transcription factor NF-κB. TRAF6 is recruited to IL-17Rs through another adaptor protein, Act1. Liu et al. now show that Act1 is also a member of the U-box family of E3 enzymes and that its ubiquitin ligase activity is required for IL-17R signaling. In response to IL-17, TRAF6 was recruited to Act1 and underwent Lys63-mediated ubiquitination, a process that was required for the activation of NF-κB. A mutant form of TRAF6 lacking the residue targeted by ubiquitin failed to support IL-17–mediated activation of NF-κB in TRAF6-deficient cells. Together, these data identify TRAF6 as a substrate of Act1 and suggest that sequential ubiquitination events regulate IL-17–induced inflammation. Interleukin-17 (IL-17), a proinflammatory cytokine mainly produced by cells of the T helper 17 (TH17) lineage, is required for host defense against bacterial and fungal infections and plays a critical role in the pathogenesis of inflammatory and autoimmune diseases. Act1 is an essential adaptor molecule in IL-17–mediated signaling and is recruited to the IL-17 receptor (IL-17R) upon IL-17 stimulation through an interaction between its SEFIR domain and that of the IL-17R. Here, we report that Act1 is a U-box E3 ubiquitin ligase and that its activity is essential for IL-17–mediated signaling pathways. Through the use of the Ubc13-Uev1A E2 complex, Act1 mediated the lysine-63–linked ubiquitination of tumor necrosis factor receptor–associated factor 6 (TRAF6), a component of IL-17–mediated signaling. Deletion and point mutations of the Act1 U-box abolished Act1-mediated ubiquitination of TRAF6 and impaired the ability of Act1 to restore IL-17–dependent signaling and expression of target genes in Act1−/− mouse embryonic fibroblasts. We also showed that the lysine-124 residue of TRAF6 was critical for efficient Act1-mediated ubiquitination of TRAF6 and for the ability of TRAF6 to mediate IL-17–induced activation of nuclear factor κB. Thus, we propose that Act1 mediates IL-17–induced signaling pathways through its E3 ubiquitin ligase activity and that TRAF6 is a critical substrate of Act1, which indicates the importance of protein ubiquitination in the IL-17–dependent inflammatory response.

The Critical Role of Epithelial-Derived Act1 in IL-17- and IL-25-Mediated Pulmonary Inflammation

IL-25 initiates, promotes, and augments Th2 immune responses. In this study, we report that Act1, a key component in IL-17-mediated signaling, is an essential signaling molecule for IL-25 signaling. Although Act1-deficient mice showed reduced expression of KC (CXCL1) and neutrophil recruitment to the airway compared with wild-type mice in response to IL-17 stimulation, Act1 deficiency abolished IL-25-induced expression of IL-4, IL-5, IL-13, eotaxin-1 (CCL11), and pulmonary eosinophilia. Using a mouse model of allergic pulmonary inflammation, we observed diminished Th2 responses and lung inflammation in Act1-deficient mice compared with wild-type mice. Importantly, Act1 deficiency in epithelial cells reduced the phenotype of allergic pulmonary inflammation due to loss of IL-17-induced neutrophilia and IL-25-induced eosinophilia, respectively. These results demonstrate the essential role of epithelial-derived Act1 in allergic pulmonary inflammation through the distinct impact of the IL-17R-Act1 and IL-25R-Act1 axes. Such findings are crucial for the understanding of pathobiology of atopic diseases, including allergic asthma, which identifies Act1 as a potential therapeutic target.

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation.

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1–TRAF6–transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.

IL-17 Signaling for mRNA Stabilization Does Not Require TNF Receptor-Associated Factor 6

IL-17 alone is a relatively weak inducer of gene expression, but cooperates with other cytokines, including TNF-α, to generate a strong response in part via prolongation of mRNA t1/2. Because TNFR-associated factor 6 (TRAF6) has been reported to be essential for signaling by IL-17, we examined its involvement in IL-17-mediated mRNA stabilization. Although overexpression of TRAF6 in HeLa cells activates NF-κB, it does not stabilize transfected KC mRNA. Furthermore, a dominant-negative TRAF6 abrogates NF-κB activation, but does not block IL-17-induced chemokine mRNA stabilization. IL-17 can stabilize KC and MIP-2 mRNAs comparably in TNF-α-treated mouse embryo fibroblasts from TRAF6+/+ and TRAF6−/− mice. TRAF6 is known to couple upstream signals with activation of p38 MAPK and mitogen activated protein kinase activated protein kinase 2, both of which have been shown to be important for Toll/IL-1R-mediated mRNA stabilization in various cell types. Inhibition of p38 MAPK, however, does not block IL-17-induced KC mRNA stabilization, and IL-17 can stabilize KC mRNA equally in mouse embryo fibroblasts from both wild-type and mitogen activated protein kinase activated protein kinase 2/3 doubly-deficient mice. Finally, IL-17 can amplify the levels of multiple TNF-α-stimulated mRNAs in wild-type and TRAF6-deficient cells, but not in cells from Act1−/− mice. Collectively, these findings demonstrate the existence of a TRAF6/p38 MAPK-independent pathway that couples the IL-17R with enhanced mRNA stability. Because the most potent effects of IL-17 on gene expression are obtained in cooperation with other cytokines such as TNF-α, these findings suggest that this pathway is a major contributing mechanism for response to IL-17.

The psoriasis-associated D10N variant of the adaptor Act1 with impaired regulation by the molecular chaperone hsp90

Act1 is an essential adaptor molecule in IL-17-mediated signaling and is recruited to the IL-17 receptor upon IL-17 stimulation. Here, we report that Act1 is a client protein of the molecular chaperone, Hsp90. The Act1 variant (D10N) linked to psoriasis susceptibility is defective in its interaction with Hsp90, resulting in a global loss of Act1 function. Act1-/- mice modeled the mechanistic link between Act1 loss of function and psoriasis susceptibility. Although Act1 is necessary for IL-17-mediated inflammation, Act1-/- mice exhibited a hyper TH17 response and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17-signaling, IL-22 is the main contributor to skin inflammation, providing a molecular mechanism for the association of Act1 (D10N) with psoriasis susceptibility.

HuR Is Required for IL-17–Induced Act1-Mediated CXCL1 and CXCL5 mRNA Stabilization

IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17–induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17–induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17–induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17–induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17–induced inflammation.

Cutting Edge: TNF Receptor-Associated Factor 4 Restricts IL-17–Mediated Pathology and Signaling Processes

The effector T cell subset, Th17, plays a significant role in the pathogenesis of multiple sclerosis and of other autoimmune diseases. The signature cytokine, IL-17, engages the IL-17R and recruits the E3-ligase NF-κB activator 1 (Act1) upon stimulation. In this study, we examined the role of TNFR-associated factor (TRAF)4 in IL-17 signaling and Th17-mediated autoimmune encephalomyelitis. Primary cells from TRAF4-deficient mice displayed markedly enhanced IL-17–activated signaling pathways and induction of chemokine mRNA. Adoptive transfer of MOG35–55 specific wild-type Th17 cells into TRAF4-deficient recipient mice induced an earlier onset of disease. Mechanistically, we found that TRAF4 and TRAF6 used the same TRAF binding sites on Act1, allowing the competition of TRAF4 with TRAF6 for the interaction with Act1. Taken together, the results of this study reveal the necessity of a unique role of TRAF4 in restricting the effects of IL-17 signaling and Th17-mediated disease.