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

Genetic Dissection of the Cellular Pathways and Signaling Mechanisms in Modeled Tumor Necrosis Factor–induced Crohn's-like Inflammatory Bowel Disease

Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (TnfΔAREmouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon γ and requires the function of CD8+ T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell–derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor–deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow–derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase–deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology

Truncation of the Deubiquitinating Domain of CYLD in Myelomonocytic Cells Attenuates Inflammatory Responses

The cylindromatosis tumor suppressor (CYLD) is a deubiquitinating enzyme that has been implicated in various aspects of adaptive and innate immune responses. Nevertheless, the role of CYLD in the function of specific types of immune cells remains elusive. In this report we have used conditional gene targeting in mice to address the role of the deubiquitinating activity of CYLD in the myelomonocytic lineage. Truncation of the deubiquitinating domain of CYLD specifically in myelomonocytic cells impaired the development of lethal LPS-induced endotoxic shock and the accumulation of thioglycollate-elicited peritoneal macrophages. Our data establish CYLD as a regulator of monocyte-macrophage activation in response to inflammatory stimuli and identify it as a potential target for therapeutic intervention in relevant inflammatory disorders in humans

Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs

The RNA binding protein HuR is a gatekeeper of liver homeostasis

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is initiated by steatosis and can progress via fibrosis and cirrhosis to hepatocellular carcinoma (HCC). The RNA binding protein HuR controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte-HuR in NAFLD development and progression to fibrosis and HCC. APPROACH AND RESULTS: Hepatocyte-specific HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or a NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis and HCC development were studied by histology, flow cytometry, quantitative PCR and RNA sequencing. The liver lipidome was characterized by lipidomics analysis and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation-sequencing. Hepatocyte-specific HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition, compared to control HuR-sufficient mice. On a NAFLD-inducing diet, hepatocyte-specific HuR-deficiency resulted in exacerbated inflammation, fibrosis and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics and RNA-immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady-state, a triglyceride signature resembling that of NAFLD livers. Moreover, upregulation of Spp1 and its product osteopontin mediated, at least partially, the fibrosis development in hepatocyte-specific HuR deficiency on a NAFLD-inducing diet, as shown by experiments utilizing antibody blockade of osteopontin. CONCLUSIONS: HuR is a gatekeeper of liver homeostasis preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically

HuR controls lung branching morphogenesis and mesenchymal FGF networks

AbstractLung development is controlled by regulatory networks governing mesenchymal–epithelial interactions. Transcription factors and signaling molecules are known to participate in this process, yet little is known about the post-transcriptional regulation of these networks. Here we demonstrate that the RNA-binding protein (RBP) HuR is an essential regulator of mesenchymal responses during lung branching. Its epiblast-induced deletion blocked the morphogenesis of distal bronchial branches at the initiation of the pseudoglandular stage. The phenotype originated from defective mesenchymal responses since the conditional restriction of HuR deletion in epithelial progenitors did not affect distal branching or the completion of lung maturation. The loss of HuR resulted in the reduction of the key inducer of bud outgrowth and endodermal branching, FGF10 and one of its putative transcriptional regulators, Tbx4. Furthermore, exogenous FGF10 could rescue the branching defect of affected lung buds. HuR was found to bind and control the Fgf10 and Tbx4 mRNAs; as a result its deletion abolished their inducible post-transcriptional regulation by the mesenchymal regulator FGF9. Our data reveals HuR as the first RBP identified to play a dominant role in lung development and as a key post-transcriptional regulator of networks guiding tissue remodeling during branching morphogenesis

A transgenic macrophage-based platform to assess the efficacy and specificity of HuR modulators in inflammation

RNA-binding proteins (RBPs) are essential controllers of RNA metabolism, and their deregulation can cause various pathologies.Human Antigen R (HuR) is an RBP engaged in the nuclear and cytoplasmic regulation of critical RNAs involved in cellularhomeostasis. Conversely, its deregulation leads to tissue degeneration, cancer and immunopathology, fueling efforts for itsexploitation as a target for relevant therapeutics. An expanding list of natural and synthetic compounds has been generatedthat disrupt HuR’s affinity for RNA, block its subcellular movements or debilitate its expression. The use of such HuR-inhibitorsshows promise in the management of several cancers. However, the divergent functions of HuR on inflammatory degenerations– as revealed in animal models of HuR’s obligatory and tissue-specific ablation – raise skepticism on the use of HuRinhibitorsagainst inflammatory reactions – including those associated with tumors. In many cases, this is due to the poorintegration of appropriate cell-based assays to provide insights into applicability for controlling inflammatory reactions.Here, we present a transgenic macrophage-based platform for the functional assessment of HuR inhibitors that considersthe context of HuR activities in inflammation that fail when HuR is lost. We employ this platform to assess the specificityand efficacy of three publicly available and prototypical HuR disruptors and two anti-inflammatory compounds known toalter HuR’s subcellular localization. We demonstrate that only one HuR-disruptor possesses HuR-dependent and selectiveanti-inflammatory activities, which do not extend the pathologic side effects incurred by the complete obliteration of HuRin immune cells. Our data provide a proof-of-principle case on appropriately assessing the modulators of HuR, highlightingthe need to exploit context-specific cell-based assays for the preclinical evaluation of RBP inhibitors

Traffic Accidents in Crete (1996-2006): the Role of the Emergency Coordination Center

The general decline in traffic accidents throughout Europe is not the case for Crete, a favorite holiday destination. The extent of problem and reflections on the significant impact of the interplay of organizational, educational, & technological interventions by the Emergency Coordination Center of Crete (ECC-Crete) are presented. 10-year data from 1996-2006 have been analyzed revealing demographic, topological, and qualitative issues of traffic accidents in Grete. Primary source of data is 315000 emergency calls answered by ECC-Crete. Over this 10 year period, ECC-Crete gradually employed advanced medical technologies and electronic protocol-based handling in all phases of an emergency episode contributing to its timely and effective management. GIS/GPS technology and telemetry for biosignals in ambulances, up-to-date triage protocols combined with incidence analysis provide vital information for continuous process improvement. In 2000-2006, process improvement due to technological and organizational changes has led to increased efficiency. The mean reduction was similar to 75% in dispatch time, similar to 50% in the time at accident scene for metropolitan areas, and similar to 75% in time at the emergency ward, mainly due to medical interventions on site