Cell-Penetrating Peptide TAT-HuR-HNS3 Suppresses Proinflammatory Gene Expression via Competitively Blocking Interaction of HuR with Its Partners.

Proinflammatory cytokines/chemokines are commonly regulated by RNA-binding proteins at posttranscriptional levels. Human Ag R (HuR)/embryonic lethal abnormal vision-like 1 (ELAVL1) is one of the well-characterized RNA-binding proteins that increases the stability of short-lived mRNAs, which encode proinflammatory mediators. HuR employs its nucleocytoplasmic shuttling sequence (HNS) domain, interacting with poly(ADP-ribose) polymerase 1 (PARP1), which accounts for the enhanced poly-ADP-ribosylation and cytoplasmic shuttling of HuR. Also by using its HNS domain, HuR undergoes dimerization/oligomerization, underlying the increased binding of HuR with proinflammatory cytokine/chemokine mRNAs and the disassociation of the miRNA-induced silencing complex from the targets. Therefore, competitively blocking the interactions of HuR with its partners may suppress proinflammatory mediator production. In this study, peptides derived from the sequence of the HuR-HNS domain were synthesized, and their effects on interfering HuR interacting with PARP1 and HuR itself were analyzed. Moreover, cell-penetrating TAT-HuR-HNS3 was delivered into human and mouse cells or administered into mouse lungs with or without exposure of TNF-α or LPS. mRNA levels of proinflammatory mediators as well as neutrophil infiltration were evaluated. We showed that TAT-HuR-HNS3 interrupts HuR-PARP1 interaction and therefore results in a lowered poly-ADP-ribosylation level and decreased cytoplasmic distribution of HuR. TAT-HuR-HNS3 also blocks HuR dimerization and promotes Argonaute 2-based miRNA-induced silencing complex binding to the targets. Moreover, TAT-HuR-HNS3 lowers mRNA stability of proinflammatory mediators in TNF-α-treated epithelial cells and macrophages, and it decreases TNF-α-induced inflammatory responses in lungs of experimental animals. Thus, TAT-HuR-HNS3 is a promising lead peptide for the development of inhibitors to treat inflammation-related diseases.

HuR up-regulates cell surface PD-L1 via stabilizing CMTM6 transcript in cancer.

Despite the well-established role of CMTM6 in the stabilization of cell surface PD-L1 in cancer cells, the mechanisms underlying CMTM6 expression and regulation are still largely unknown. Here we unexpectedly find a strikingly positive correlation between CMTM6 and Hu-Antigen R (HuR) expression in most types of cancer. Mechanistically, we elucidate HuR stabilizes CMTM6 mRNA via direct association with AU-rich elements (AREs) in its 3'UTR and predominantly up-regulates CMTM6, which is readily abolished by HuR-specific inhibitor, MS-444. Phenotypically, we notice abundant cell surface PD-L1 in HuR-high cancer cells, which significantly inhibits immune activation of co-cultured T cells as indicated by IL-2 production. Treatment with MS-444 completely relieves immune suppression imposed by HuR-overexpression and further stimulates immune responses. Ectopic HuR accelerates allograft tumor progression in vivo, which is greatly compromised by simultaneous administration with MS-444. Our study uncovers a novel mechanism in control of CMTM6 and therefore PD-L1 expression, and suggests the potential of combining HuR inhibitor with PD-1/PD-L1 antibodies for cancer immunotherapy.

miR-18a-3p and Its Target Protein HuR May Regulate Myogenic Differentiation in Immune-Mediated Necrotizing Myopathy.

Immune-mediated necrotizing myopathy (IMNM) is characterized by manifestation of myonecrosis and regeneration of muscle fibers; however, the underlying pathogenesis remains unclear. This study aimed to investigate the role and mechanism of miR-18a-3p and its target RNA-binding protein HuR in IMNM. HuR and miR-18a-3p levels were detected in the skeletal muscles of 18 patients with IMNM using quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) and western blotting analysis. Human myoblasts were transfected with small interfering RNA targeting HuR and miR-18a-3p mimic or inhibitor. Myogenic differentiation markers, myogenin and myosin heavy chain, were analyzed by qRT-PCR, western blotting analysis, and immunofluorescence staining. The results showed that miR-18a-3p was upregulated (p=0.0002), whereas HuR was downregulated (p=0.002) in the skeletal muscles of patients with IMNM. The expression of miR-18a-3p in patients with IMNM was negatively correlated with those of HuR (r = -0.512, p = 0.029). We also found that disease activity was positively correlated with HuR expression (r = 0.576, p = 0.012) but muscle activity was negatively correlated with miR-18a-3p expression (r = -0.550, p = 0.017). Besides, bioinformatics analysis and dual-luciferase reporter assays suggested that miR-18a-3p could directly target HuR. Cellular experiments showed that overexpression of miR-18a-3p inhibited myogenic differentiation by targeting HuR, whereas inhibition of miR-18a-3p led to opposite results. Therefore, miR-18a-3p and its target protein HuR may be responsible for modulating the myogenic process in IMNM and can thus be therapeutic targets for the same.

RNA-Binding Protein HuR Promotes Th17 Cell Differentiation and Can Be Targeted to Reduce Autoimmune Neuroinflammation.

Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help find a novel therapeutic target for treating Th17 cell-mediated diseases. In this study, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by posttranscriptionally regulating transcription factors Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation in C57BL/6J mice. Knockout of HuR altered the transcriptome of Th17 cells characterized by reducing the levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells in the spleen during experimental autoimmune encephalomyelitis. In keeping with the fact that HuR increased the abundance of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired splenic Th17 cell migration to the CNS and abolished the disease. Accordingly, targeting HuR by its inhibitor DHTS inhibited splenic Th17 cell differentiation and reduced experimental autoimmune encephalomyelitis severity. In sum, we uncovered the molecular mechanism of HuR regulating Th17 cell functions, underscoring the therapeutic value of HuR for treatment of autoimmune neuroinflammation.

Human antigen R: A potential therapeutic target for liver diseases.

Human antigen R (HuR), also known as HuA and embryonic lethal abnormal vision-like 1 (ELAVL1), is a ubiquitously expressed RNA binding protein and functions as an RNA regulator and mediates the expression of various proteins by diverse post-transcriptional mechanisms. HuR has been well characterized in the inflammatory responses and in the development of various cancers. The importance of HuR-mediated roles in cell signaling, inflammation, fibrogenesis and cancer development in the liver has attracted a great deal of attention. However, there is still a substantial gap between the current understanding of the potential roles of HuR in the progression of liver disease and whether HuR can be targeted for the treatment of liver diseases. In this review, we introduce the function and mechanistic characterization of HuR, and then focus on the physiopathological roles of HuR in the development of different liver diseases, including hepatic inflammation, alcoholic liver diseases, non-alcoholic fatty liver diseases, viral hepatitis, liver fibrosis and liver cancers. We also summarize existing approaches targeting HuR function. In conclusion, although characterizing the liver-specific HuR function and demonstrating the multi-level regulative networks of HuR in the liver are still required, emerging evidence supports the notion that HuR represents a potential therapeutic target for the treatment of chronic liver diseases.

TNF-α/calreticulin dual signaling induced NLRP3 inflammasome activation associated with HuR nucleocytoplasmic shuttling in rheumatoid arthritis.

OBJECTIVE: The present study was undertaken to validate whether TNF-α and calreticulin (CRT) serve as dual signaling to activate nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and HUVECs. The effect of human antigen R (HuR) in NLRP3 inflammasome activation was also explored in RA FLS. METHODS: Immunofluorescence was used to determine the expression of NLRP3 and adaptor protein apoptosis associated speck-like protein containing a CARD (ASC) in RA synovial tissue and HuR location in RA FLS. Western blot and quantitative real-time PCR were employed to measure the priming effect of NLRP3 inflammasome in cells and HuR expression in synovial tissue. The concentrations of IL-1ß and IL-18 were detected by enzyme linked immunosorbent assay. Immunohistochemistry was used to visualize the expression of HuR in synovial tissue. HuR knockdown in RA FLS was achieved by siRNA-mediated gene silencing. RESULTS: Higher expression of NLRP3 and ASC in RA synovial tissue than those in osteoarthritis was detected. The staining of NLRP3, ASC and cleaved IL-1ß were observed in FLS and vascular endothelial cells in RA synovium. Expression of NLRP3 and pro-IL-1ß in RA FLS and HUVECs treated with TNF-α was increased. The pro-IL-18 expression was also enhanced in HUVECs, but not in RA FLS. TNF-α/CRT dual stimulation of cells gave rise to caspase-1 p20 expression and the secretion of IL-1ß. The secreted IL-18 was also elevated in HUVECs but not in RA FLS. HuR expression was significantly elevated in RA synovial tissue. TNF-α initiated the nucleocytoplasmic shuttling of HuR in both FLS and HUVECs. The knockdown of HuR in FLS incubated with TNF-α led to reduced caspase-1 p20 protein expression and further resulted in decreased secretion of IL-1ß in the presence of CRT. CONCLUSIONS: TNF-α/CRT dual signaling induced NLRP3 inflammasome activation, which could be suppressed by HuR knockdown presumably due to the block of HuR translocating from nucleus to cytoplasma.

Divergent Innate and Epithelial Functions of the RNA-Binding Protein HuR in Intestinal Inflammation.

HuR is an abundant RNA-binding protein acting as a post-transcriptional regulator of many RNAs including mRNAs encoding inflammatory mediators, cytokines, death signalers and cell cycle regulators. In the context of intestinal pathologies, elevated HuR is considered to enhance the stability and the translation of pro-tumorigenic mRNAs providing the rationale for its pharmacological targeting. However, HuR also possesses specific regulatory functions for innate immunity and cytokine mRNA control which can oppose intestinal inflammation and tumor promotion. Here, we aim to identify contexts of intestinal inflammation where the innate immune and the epithelial functions of HuR converge or diverge. To address this, we use a disease-oriented phenotypic approach using mice lacking HuR either in intestinal epithelia or myeloid-derived immune compartments. These mice were compared for their responses to (a) Chemically induced Colitis; (b) Colitis- associated Cancer (CAC); (c) T-cell mediated enterotoxicity; (d) Citrobacter rodentium-induced colitis; and (e) TNF-driven inflammatory bowel disease. Convergent functions of epithelial and myeloid HuR included their requirement for suppressing inflammation in chemically induced colitis and their redundancies in chronic TNF-driven IBD and microbiota control. In the other contexts however, their functions diversified. Epithelial HuR was required to protect the epithelial barrier from acute inflammatory or infectious degeneration but also to promote tumor growth. In contrast, myeloid HuR was required to suppress the beneficial inflammation for pathogen clearance and tumor suppression. This cellular dichotomy in HuR's functions was validated further in mice engineered to express ubiquitously higher levels of HuR which displayed diminished pathologic and beneficial inflammatory responses, resistance to epithelial damage yet a heightened susceptibility to CAC. Our study demonstrates that epithelial and myeloid HuR affect different cellular dynamics in the intestine that need to be carefully considered for its pharmacological exploitation and points toward potential windows for harnessing HuR functions in intestinal inflammation.

Hu Antigen R Regulates Antiviral Innate Immune Responses through the Stabilization of mRNA for Polo-like Kinase 2.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I, and melanoma differentiation-associated gene 5 (MDA5) play a critical role in inducing antiviral innate immune responses by activating IFN regulatory factor 3 (IRF3) and NF-κB, which regulates the transcription of type I IFN and inflammatory cytokines. Antiviral innate immune responses are also regulated by posttranscriptional and translational mechanisms. In this study, we identified an RNA-binding protein HuR as a regulator for RLR signaling. Overexpression of HuR, but not of other Hu members, increased IFN-ß promoter activity. HuR-deficient macrophage cells exhibited decreased Ifnb1 expression after RLR stimulation, whereas they showed normal induction after stimulation with bacterial LPS or immunostimulatory DNA. Moreover, HuR-deficient cells displayed impaired nuclear translocation of IRF3 after RLR stimulation. In HuR-deficient cells, the mRNA expression of Polo-like kinase (PLK) 2 was markedly reduced. We found that HuR bound to the 3' untranslated region of Plk2 mRNA and increased its stabilization. PLK2-deficient cells also showed reduced IRF3 nuclear translocation and Ifnb mRNA expression during RLR signaling. Together, these findings suggest that HuR bolsters RLR-mediated IRF3 nuclear translocation by controlling the stability of Plk2 mRNA.

PARP1 promotes gene expression at the post-transcriptiona level by modulating the RNA-binding protein HuR.

Poly(ADP-ribosyl)ation (PARylation) is mainly catalysed by poly-ADP-ribose polymerase 1 (PARP1), whose role in gene transcription modulation has been well established. Here we show that, in response to LPS exposure, PARP1 interacts with the adenylateuridylate-rich element-binding protein embryonic lethal abnormal vision-like 1 (Elavl1)/human antigen R (HuR), resulting in its PARylation, primarily at site D226. PARP inhibition and the D226 mutation impair HuR's PARylation, nucleocytoplasmic shuttling and mRNA binding. Increases in mRNA level or stability of pro-inflammatory cytokines/chemokines are abolished by PARP1 ablation or inhibition, or blocked in D226A HuR-expressing cells. The present study demonstrates a mechanism to regulate gene expression at the post-transcriptional level, and suggests that blocking the interaction of PARP1 with HuR could be a strategy to treat inflammation-related diseases that involve increased mRNA stability.

Induction of Glucocorticoid-induced Leucine Zipper (GILZ) Contributes to Anti-inflammatory Effects of the Natural Product Curcumin in Macrophages.

GILZ (glucocorticoid-induced leucine zipper) is inducible by glucocorticoids and plays a key role in their mode of action. GILZ attenuates inflammation mainly by inhibition of NF-κB and mitogen-activated protein kinase activation but does not seem to be involved in the severe side effects observed after glucocorticoid treatment. Therefore, GILZ might be a promising target for new therapeutic approaches. The present work focuses on the natural product curcumin, which has previously been reported to inhibit NF-κB. GILZ was inducible by curcumin in macrophage cell lines, primary human monocyte-derived macrophages, and murine bone marrow-derived macrophages. The up-regulation of GILZ was neither associated with glucocorticoid receptor activation nor with transcriptional induction or mRNA or protein stabilization but was a result of enhanced translation. Because the GILZ 3'-UTR contains AU-rich elements (AREs), we analyzed the role of the mRNA-binding protein HuR, which has been shown to promote the translation of ARE-containing mRNAs. Our results suggest that curcumin treatment induces HuR expression. An RNA immunoprecipitation assay confirmed that HuR can bind GILZ mRNA. In accordance, HuR overexpression led to increased GILZ protein levels but had no effect on GILZ mRNA expression. Our data employing siRNA in LPS-activated RAW264.7 macrophages show that curcumin facilitates its anti-inflammatory action by induction of GILZ in macrophages. Experiments with LPS-activated bone marrow-derived macrophages from wild-type and GILZ knock-out mice demonstrated that curcumin inhibits the activity of inflammatory regulators, such as NF-κB or ERK, and subsequent TNF-α production via GILZ. In summary, our data indicate that HuR-dependent GILZ induction contributes to the anti-inflammatory properties of curcumin.

Transcriptomic-Wide Discovery of Direct and Indirect HuR RNA Targets in Activated CD4+ T Cells.

Due to poor correlation between steady state mRNA levels and protein product, purely transcriptomic profiling methods may miss genes posttranscriptionally regulated by RNA binding proteins (RBPs) and microRNAs (miRNAs). RNA immunoprecipitation (RIP) methods developed to identify in vivo targets of RBPs have greatly elucidated those mRNAs which may be regulated via transcript stability and translation. The RBP HuR (ELAVL1) and family members are major stabilizers of mRNA. Many labs have identified HuR mRNA targets; however, many of these analyses have been performed in cell lines and oftentimes are not independent biological replicates. Little is known about how HuR target mRNAs behave in conditional knock-out models. In the present work, we performed HuR RIP-Seq and RNA-Seq to investigate HuR direct and indirect targets using a novel conditional knock-out model of HuR genetic ablation during CD4+ T activation and Th2 differentiation. Using independent biological replicates, we generated a high coverage RIP-Seq data set (>160 million reads) that was analyzed using bioinformatics methods specifically designed to find direct mRNA targets in RIP-Seq data. Simultaneously, another set of independent biological replicates were sequenced by RNA-Seq (>425 million reads) to identify indirect HuR targets. These direct and indirect targets were combined to determine canonical pathways in CD4+ T cell activation and differentiation for which HuR plays an important role. We show that HuR may regulate genes in multiple canonical pathways involved in T cell activation especially the CD28 family signaling pathway. These data provide insights into potential HuR-regulated genes during T cell activation and immune mechanisms.