Act1 drives chemoresistance via regulation of antioxidant RNA metabolism and redox homeostasis.

The IL-17 receptor adaptor molecule Act1, an RNA-binding protein, plays a critical role in IL-17-mediated cancer progression. Here, we report a novel mechanism of how IL-17/Act1 induces chemoresistance by modulating redox homeostasis through epitranscriptomic regulation of antioxidant RNA metabolism. Transcriptome-wide mapping of direct Act1-RNA interactions revealed that Act1 binds to the 5'UTR of antioxidant mRNAs and Wilms' tumor 1-associating protein (WTAP), a key regulator in m6A methyltransferase complex. Strikingly, Act1's binding sites are located in proximity to m6A modification sites, which allows Act1 to promote the recruitment of elF3G for cap-independent translation. Loss of Act1's RNA binding activity or Wtap knockdown abolished IL-17-induced m6A modification and translation of Wtap and antioxidant mRNAs, indicating a feedforward mechanism of the Act1-WTAP loop. We then developed antisense oligonucleotides (Wtap ASO) that specifically disrupt Act1's binding to Wtap mRNA, abolishing IL-17/Act1-WTAP-mediated antioxidant protein production during chemotherapy. Wtap ASO substantially increased the antitumor efficacy of cisplatin, demonstrating a potential therapeutic strategy for chemoresistance.

Mechanisms of Corticosteroid Resistance in Type 17 Asthma.

IL-17A plays an important role in the pathogenesis of asthma, particularly the neutrophilic corticosteroid (CS)-resistant subtype of asthma. Clinical studies suggest that a subset of asthma patients, i.e., Th17/IL-17A-mediated (type 17) CS-resistant neutrophilic asthma, may improve with Th17/IL-17A pathway blockade. However, little is known about the mechanisms underlying type 17 asthma and CS response. In this article, we show that blood levels of lipocalin-2 (LCN2) and serum amyloid A (SAA) levels are positively correlated with IL-17A levels and are not inhibited by high-dose CS usage in asthma patients. In airway cell culture systems, IL-17A induces these two secreted proteins, and their induction is enhanced by CS. Furthermore, plasma LCN2 and SAA levels are increased in mice on a preclinical type 17 asthma model, correlated to IL-17A levels, and are not reduced by glucocorticoid (GC). In the mechanistic studies, we identify CEBPB as the critical transcription factor responsible for the synergistic induction of LCN2 and SAA by IL-17A and GC. IL-17A and GC collaboratively regulate CEBPB at both transcriptional and posttranscriptional levels. The posttranscriptional regulation of CEBPB is mediated in part by Act1, the adaptor and RNA binding protein in IL-17A signaling, which directly binds CEBPB mRNA and inhibits its degradation. Overall, our findings suggest that blood LCN2 and SAA levels may be associated with a type 17 asthma subtype and provide insight into the molecular mechanism of the IL-17A-Act1/CEBPB axis on these CS-resistant genes.

IL-17-induced HIF1α drives resistance to anti-PD-L1 via fibroblast-mediated immune exclusion.

Increasing evidence suggests that intratumoral inflammation has an outsized influence on antitumor immunity. Here, we report that IL-17, a proinflammatory cytokine widely associated with poor prognosis in solid tumors, drives the therapeutic failure of anti-PD-L1. By timing the deletion of IL-17 signaling specifically in cancer-associated fibroblasts (CAFs) in late-stage tumors, we show that IL-17 signaling drives immune exclusion by activating a collagen deposition program in murine models of cutaneous squamous cell carcinoma (cSCC). Ablation of IL-17 signaling in CAFs increased the infiltration of cytotoxic T cells into the tumor mass and sensitized otherwise resistant cSCC to anti-PD-L1 treatment. Mechanistically, the collagen deposition program in CAFs was driven by IL-17-induced translation of HIF1α, which was mediated by direct binding of Act1, the adaptor protein of IL-17 receptor, to a stem-loop structure in the 3' untranslated region (UTR) in Hif1α mRNA. Disruption of Act1's binding to Hif1α mRNA abolished IL-17-induced collagen deposition and enhanced anti-PD-L1-mediated tumor regression.

RNA-Binding Protein HuR Promotes Airway Inflammation in a House Dust Mite-Induced Allergic Asthma Model.

Mounting evidence indicates that interleukin 17 (IL-17) is critically involved in the pathogenesis of severe asthma. We have previously reported that upon IL-17 stimulation, Act1, an IL-17-receptor-complex adaptor, directly binds to its target mRNAs and utilizes other proteins, such as HuR, to upregulate mRNA stability and translation. HuR mRNA targets include multiple asthma-related genes. In this study, we have used house dust mite (HDM), a natural allergen, to test the role of HuR in the pathogenesis of allergic asthma. We found that HuR deletion in airway epithelium diminished HDM-induced lung inflammation, including neutrophil and eosinophil infiltration. While Th2 cytokines were not altered, the production of CXCL1, CXCL5 and CCL11 chemokines was significantly diminished. Airway smooth muscle (ASM) cells contribute to the pathogenesis of allergic asthma by orchestrating inflammatory and remodeling responses. We found that IL-17 treatment of ASM cells induced translocation of HuR from nucleus to cytoplasm, where it bound directly to Cxcl1 and Ccl11 mRNA. Deletion of HuR in ASM cells decreased their proliferation as well as CXCL1 and CCL11 production in response to IL-17. Taken together, our findings demonstrate the importance of HuR-mediated regulation of gene expression to the pathogenesis of allergic asthma, in both airway epithelial and ASM cells.

The role of interleukin-17 in tumor development and progression.

IL-17, a potent proinflammatory cytokine, has been shown to intimately contribute to the formation, growth, and metastasis of a wide range of malignancies. Recent studies implicate IL-17 as a link among inflammation, wound healing, and cancer. While IL-17-mediated production of inflammatory mediators mobilizes immune-suppressive and angiogenic myeloid cells, emerging studies reveal that IL-17 can directly act on tissue stem cells to promote tissue repair and tumorigenesis. Here, we review the pleotropic impacts of IL-17 on cancer biology, focusing how IL-17-mediated inflammatory response and mitogenic signaling are exploited to equip its cancer-promoting function and discussing the implications in therapies.

IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility.

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type-specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A-induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A-induced stress fiber formation in ASMCs and attenuated IL-17A-enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.

Neuron-Specific HuR-Deficient Mice Spontaneously Develop Motor Neuron Disease.

Human Ag R (HuR) is an RNA binding protein in the ELAVL protein family. To study the neuron-specific function of HuR, we generated inducible, neuron-specific HuR-deficient mice of both sexes. After tamoxifen-induced deletion of HuR, these mice developed a phenotype consisting of poor balance, decreased movement, and decreased strength. They performed significantly worse on the rotarod test compared with littermate control mice, indicating coordination deficiency. Using the grip-strength test, it was also determined that the forelimbs of neuron-specific HuR-deficient mice were much weaker than littermate control mice. Immunostaining of the brain and cervical spinal cord showed that HuR-deficient neurons had increased levels of cleaved caspase-3, a hallmark of cell apoptosis. Caspase-3 cleavage was especially strong in pyramidal neurons and α motor neurons of HuR-deficient mice. Genome-wide microarray and real-time PCR analysis further indicated that HuR deficiency in neurons resulted in altered expression of genes in the brain involved in cell growth, including trichoplein keratin filament-binding protein, Cdkn2c, G-protein signaling modulator 2, immediate early response 2, superoxide dismutase 1, and Bcl2. The additional enriched Gene Ontology terms in the brain tissues of neuron-specific HuR-deficient mice were largely related to inflammation, including IFN-induced genes and complement components. Importantly, some of these HuR-regulated genes were also significantly altered in the brain and spinal cord of patients with amyotrophic lateral sclerosis. Additionally, neuronal HuR deficiency resulted in the redistribution of TDP43 to cytosolic granules, which has been linked to motor neuron disease. Taken together, we propose that this neuron-specific HuR-deficient mouse strain can potentially be used as a motor neuron disease model.

IL-17-receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling.

Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.

IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

IKKα negatively regulates ASC-dependent inflammasome activation.

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IκB kinase α (IKKα) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

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.

Diversity in sequence-dependent control of GRO chemokine mRNA half-life.

Neutrophil trafficking to sites of injury or infection is regulated, in part, by the closely related GRO family of chemokines (CXCL1, -2, and -3). Expression of the GRO chemokine genes is known to be determined by transcriptional bursts in response to proinflammatory stimulation, but post-transcriptional mechanisms that regulate mRNA half-life are now recognized as important determinants. mRNA half-life is regulated via distinct sequence motifs and sequence-specific, RNA-binding proteins, whose function is subject to regulation by extracellular proinflammatory stimuli. Moreover, such mechanisms exhibit cell-type and stimulus dependency. We now present evidence that in nonmyeloid cells, GRO2 and GRO3 isoforms exhibit at least two patterns of mRNA instability that are distinguished by differential sensitivity to specific mRNA-destabilizing proteins and stimulus-mediated prolongation of mRNA half-life, respectively. Although the 3' UTR regions of GRO2 and GRO3 mRNAs contain multiple AREs, GRO2 has eight AUUUA pentamers, whereas GRO3 has seven. These confer quantitative differences in half-life and show sensitivity for TTP and KSRP but not SF2/ASF. Moreover, these AUUUA determinants do not confer instability that can be modulated in response to IL-1α. In contrast, IL-1α-sensitive instability for GRO2 and GRO3 is conferred by sequences located proximal to the 3' end of the 3'UTR that are independent of the AUUUA sequence motif. These regions are insensitive to TTP and KSRP but show reduced half-life mediated by SF2/ASF. These sequence-linked, post-transcriptional activities provide substantial mechanistic diversity in the control of GRO family chemokine gene expression.

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

Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.

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.

Diversity in post-transcriptional control of neutrophil chemoattractant cytokine gene expression.

Regulation of neutrophil chemokine gene expression represents an important feature in tissue inflammation. While chemokine gene transcription through the action of NFkappaB is recognized as an essential component of this process, it is now clear that post-transcriptional mechanisms, particularly the rates of decay of mature cytoplasmic mRNA, provides an essential component of this control. Chemokine and other cytokine mRNA half life is known to be controlled via adenine-uridine rich sequence motifs localized within 3' untranslated regions (UTRs), the most common of which contains one or more copies of the pentameric AUUUA sequence. In myeloid cells AUUUA sequences confer instability through the action of RNA binding proteins such as tristetraprolin (TTP). The resulting instability can be regulated in response to extra-cellular stimuli including Toll like receptor ligands that signal to control the function of TTP through pathways involving the activation of p38 MAP kinases. Recent findings indicate that substantial mechanistic diversity is operative in non-myeloid cells in response to alternate pro-inflammatory stimuli such as IL-17. These pathways target distinct instability sequences that do not contain the AUUUA pentamer motif, do not signal through p38 MAPK, and function independently of TTP.

IL-17 regulates CXCL1 mRNA stability via an AUUUA/tristetraprolin-independent sequence.

IL-17 contributes to inflammatory response in part by promoting enhanced expression of chemokines, such as CXCL1, by prolonging the t(1/2) of this constitutively unstable mRNA. Although IL-17 is a weak stimulus for transcription of the CXCL1 gene, it strongly potentiates message accumulation via stabilization when the mRNA is transcribed in cells stimulated with TNF. In myeloid cells, LPS-induced CXCL1 mRNA stabilization is dependent on AUUUA-containing sequence motifs that are recognized by the RNA binding protein tristetraprolin (TTP). Using deletion and site-specific mutagenesis, we report that IL-17-mediated stabilization of CXCL1 mRNA in nonmyeloid cells depends on a sequence that does not contain the AUUUA motif. Furthermore, a specific two-nucleotide mutation within this region markedly abrogates sensitivity for IL-17-mediated stabilization. Consistent with this finding, the IL-17-sensitive sequence does not exhibit increased instability in the presence of TTP, and CXCL1 mRNA remains unstable and can be stabilized in response to treatment with IL-17 in embryo fibroblasts from mice in which the TTP gene has been deleted. Whereas the RNA binding protein KSRP has been shown to participate in regulating the instability of human CXCL8 mRNA, inhibitory RNA-based reduction in KSRP does not effect the instability mediated by the IL-17-sensitive sequence motif. These findings suggest that IL-17-mediated chemokine mRNA stabilization in nonmyeloid cells uses a mechanism that is distinct from that operating to control AU-rich mRNA stability in myeloid cells.

The essential role of single Ig IL-1 receptor-related molecule/Toll IL-1R8 in regulation of Th2 immune response.

A novel cytokine IL-33, an IL-1 family member, signals via ST2 receptor and promotes Th2 responses, through the activation of NF-kappaB and MAP kinases. Previous studies reported that single Ig IL-1R-related molecule (SIGIRR)/Toll IL-1R8 acts as negative regulator for TLR-IL-1R-mediated signaling. We now found that SIGIRR formed a complex with ST2 upon IL-33 stimulation and specifically inhibited IL-33/ST2-mediated signaling in cell culture model. Furthermore, IL-33-induced Th2 response was enhanced in SIGIRR-deficient mice compared with that in wild-type control mice, suggesting a negative regulatory role of SIGIRR in IL-33/ST2 signaling in vivo. Similar to ST2, SIGIRR was highly expressed in in vitro polarized Th2 cells, but not Th1 cells. SIGIRR-deficient Th2 cells produce higher levels of Th2 cytokines, including IL-5, IL-4, and IL-13, than that in wild-type cells. Moreover, SIGIRR-deficient mice developed stronger Th2 immune response in OVA-challenged asthma model. Taken together, our results suggest that SIGIRR plays an important role in the regulation of Th2 response in vivo, possibly through its impact on IL-33-ST2-mediated signaling.

Tristetraprolin regulates CXCL1 (KC) mRNA stability.

mRNAs encoding proinflammatory chemokines are regulated posttranscriptionally via adenine-uridine-rich sequences (AREs) located in the 3' untranslated region of the message, which are recognized by sequence-specific RNA-binding proteins. One ARE binding protein, tristetraprolin (TTP), has been implicated in regulating the stability of several ARE-containing mRNAs, including those encoding TNF-alpha and GM-CSF. In the present report we examined the role of TTP in regulating the decay of the mouse chemokine KC (CXCL1) mRNA. Using tetR-regulated control of transcription in TTP-deficient HEK293 cells, KC mRNA half-life was markedly decreased in the presence of TTP. Deletion and site-specific mutagenesis were used to identify multiple AUUUA sequence determinants responsible for TTP sensitivity. Although a number of studies suggest that the destabilizing activity of TTP is subject to modulation in response to ligands of Toll/IL-1 family receptors, decay mediated by TTP in 293 cells was not sensitive to stimulation with IL-1alpha. Using primary macrophages from wild-type and TTP-deficient mice, KC mRNA instability was found to be highly dependent on TTP. Furthermore, LPS-mediated stabilization of KC mRNA is blocked by inhibition of the p38 MAPK in macrophages from wild-type but not TTP-deficient mice. These findings demonstrate that TTP is the predominant regulator of KC mRNA decay in mononuclear phagocytes acting via multiple 3'-untranslated region-localized AREs. Nevertheless, KC mRNA remains highly unstable in cells that do not express TTP, suggesting that additional determinants of instability and stimulus sensitivity may operate in cell populations where TTP is not expressed.