Purpose. Severe injury initiates an inflammatory response that can perpetuate immunological dysfunction, uncontrolled inflammation, and subsequent multisystem organ failure. MicroRNAs (miRNAs) have recently been identified as regulators of this inflammatory response. Our study sought to identify the differential expression of unique miRNAs and their correlations with genes of the Toll-like receptor (TLR) pathways, and clinical parameters in the severely injured.Methods. Fourteen trauma patients requiring transfusion were prospectively enrolled in this institutional review board approved study. Inclusion criteria consisted of adult patients deemed clinically to be in hemorrhagic shock necessitating transfusion in the acute phase of their injury care. Peripheral blood samples were obtained after admission to the surgical intensive care unit. Expression of circulating mature miRNA from each patient, as well as from 10 healthy, age-matched controls, was determined and compared using the HiSeq 2500 sequencing system and the R software system. Gene expression of TLR signaling pathways for each patient was examined using custom gene expression polymerase chain reaction arrays. Statistical analyses were performed using general linear models and empirical Bayes methods to determine differential expression and Spearman's nonparametric correlation analysis.Results. Subjects were 21-77 years old (mean, 42), 80% male, Injury Severity Score 11-43 (mean, 26), with 11 blunt and 3 penetrating injuries. Three were intubated and 5 received blood products before arrival. Base deficit upon hospital admission was 3 to 20 (mean, 9). All patients required blood transfusion secondary to blood loss sustained during injury. Survival to discharge was 93%. Controls were 27-64 years old (mean, 40) and 60% male. Sequencing analysis revealed 69 differentially expressed miRNAs (P < .05) in the severely injured. Within the differentially expressed miRNAs, there were 12 direct and 6 indirect correlations with multiple genes involved in the TLR3 and TLR4 signaling pathways. The relationships between these same miRNAs and clinical parameters were also analyzed. We discovered 4 direct correlations with base deficit and HCO3, and 7 indirect correlations involving total fresh frozen plasma transfused, base deficit, HCO3, and PaCO2 levels.Conclusion. Differential expression and correlations between miRNAs, genes of the TLR pathways, and clinical parameters are unique findings in the severely injured and may lead to a greater understanding of the regulation of sterile inflammation after severe injury.

Abstract Posttranscriptional control by RNA binding proteins (RBPs) in CD4+ T cells is poorly understood. The RBP, HuR, regulates IL-4, IL-13 and GATA-3, by binding to AU-rich (ARE) regions in their 3’ UTRs. We hypothesized HuR is regulating CD4+ Th2 differentiation by controlling mRNA stability. We previously showed that HuR over-expression results in increased Th2 differentiation and Th2 cytokines. We generated a new model to study Th2 differentiation, distal Lck-cre ROSA HuRfl/fl, in which HuR is ablated prior to T cell activation. This resulted in profound suppression of Th2 but not Th1 cytokines, under in vitro conditions. We used the ova challenge model of airway inflammation to study antigen presentation and in vivo cytokine regulation. HuR KO mice had completely abolished lung cellular inflammation to levels similar to non-immunized mice. This further resulted in significant reductions in IL-4, IL-13 secretion, and serum IgE levels. We also investigated effects of HuR ablation in Th17 differentiation. We show HuR regulates IL-17 expression by controlling transcript stability. HuR KO T cells have greatly reduced Th17 differentiation (66% reduction) and diminished onset and severity of inflammation in an EAE model. These data indicate that HuR is required for efficient CD4+ Th2 and Th17 differentiation both in vitro and in vivo but not Th1. These findings reveal nuanced control by HuR upon different CD4+ T cell subsets.

The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell-specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuRfl/+) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuRfl/fl) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.

Abstract Interleukin 17 (IL-17) is a proinflammatory cytokine produced by activated Th17 T cells. Th17 cells are major contributors to autoimmune diseases, such as multiple sclerosis. Although the transcriptional regulation of Th17 cells is well understood, its posttranscriptional regulation is unclear. The RNA-binding protein HuR regulates the stability of many target mRNAs via binding the AU-rich elements (ARE) present in the 3’ untranslated region (UTR), including inflammatory cytokines such as IL-4, IL-13 and TNF-α. The regulation of IL-17 expression by HuR has not been established. We reported that production of mRNA and protein levels of IL-17, produced by polarized Th17 cells from HuR conditional knockout (KO) mice, were abrogated. Moreover, we demonstrated that HuR directly bound to IL-17 mRNA 3’ UTR by using immunoprecipitation and RNA precipitation (RIP) and biotin pull down assays. Th17 cells from HuR conditional KO mice had decreased IL-17 steady-state mRNA and protein levels compared to wild type cells. Mice with adoptively transferred HuR KO CD4+ T cells had delayed onset and reduced severity of experimental autoimmune encephalomyelitis (EAE) compared to wild type CD4+ T cells. Our results reveal a posttranscriptional regulatory mechanism of Th17 cells by HuR, which may provide novel therapeutic targets for treatment of IL-17- mediated autoimmune neuroinflammation.

IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17-producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3' untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4(+) Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17(+) cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3' untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4(+) T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.

Background Naive CD4+ T cells can differentiate into different subsets. Whereas transcriptional regulation of CD4+ T cells is well studied, posttranscriptional control by RNA binding proteins (RBPs) and microRNAs is poorly understood. CD4+ Th2 mediated diseases such as allergen-induced asthma, are driven by GATA-3, IL-4 and IL-13. The RBP, HuR, has been shown to posttranscriptionally regulate many early response genes, including IL-4 and IL-13. GATA-3 contains AU-rich elements (ARE) in its 3’ untranslated region (UTR) which are binding sites for HuR. We first identified GATA-3, IL-4 and IL-13 as HuR targets using RIP-Chip (RNA immunoprecipitation applied to microarrays). We hypothesized that HuR may be coordinately regulating Th2 differentiation.

Abstract Posttranscriptional control by RNA binding proteins (RBPs) is poorly understood. Th2 mediated diseases such as asthma, are driven by GATA-3, IL-4 and IL-13. The RBP, HuR, regulates IL-4 and IL-13 by increasing mRNA stability. We identified GATA-3, IL-4 and IL-13 as HuR targets and hypothesized HuR may be coordinately regulating Th2 differentiation by controlling mRNA stability and translatability. We made a HuR CD4+T transgenic mouse, and a HuR conditional knock out mouse (HuRfl/fl) to ablate HuR in T cells. HuR over-expression in transgenic CD4+ T cells stabilized GATA-3, IL-4 and IL-13 mRNAs, and increased mRNA, protein and Th2 polarization. HuR knock-down resulted in decreases in GATA-3, IL-4 and IL-13 expression. Findings were confirmed in human naïve and memory T cells. We defined HuR binding sites in GATA-3. Th2 cells with low HuR levels (26%) from HuRfl/+ mice, had decreases in IL-4, IL-13 and GATA-3 mRNA but not protein. Surprisingly, CD4+ T cells from HuRfl/fl mice (93% knockdown) showed increased IL-2, IL-4, IL-13 mRNA and protein. IL-4 transcription increased but mRNA stability remained unchanged. IL-2 and IL-13 transcription were unaltered but there were increases in mRNA stability. Polysomal gradient analysis revealed similar translation. We analyzed gene expression in KO vs. control CD4+ T cells to determine HuR targets. Ova challenge model of asthma corroborated ex vivo results. These data suggest HuR levels regulate Th2 cytokines and Th2 differentiation.

Although situational risk factors for incisional hernia formation are known, the methods used to determine who would be most susceptible to develop one are unreliable. We hypothesized that patients with recurrent incisional hernias may possess unique gene expression profiles.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate–rich elements (AREs) present in the 3′ untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3′UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.

Abstract Breast cancer is one of the most prevalent cancers worldwide. Distance metastasis is responsible for patient mortality. Therefore, understanding the mechanisms underlying tumor pathogenesis and metastasis is crucial for the development of novel therapies as well as preventive strategies for those who are prone to breast cancer. In contrast to transcriptional gene regulation, posttranscriptional control mechanisms of gene expression are poorly understood. Yet, many metastasis genes are regulated by RNA binding proteins (RBPs) at the levels of mRNA stability and translation. The paraneoplastic antigen, HuR, is an RBP shown to regulate multiple genes that are significantly related to breast cancer metastasis by stabilizing target mRNAs and facilitating translation into proteins. Using novel techniques developed in our lab of RNA immunoprecipitation applied to microarrays (RIP-Chip) we identified novel discrete HuR-associated mRNAs in triple negative breast cancer and estrogen receptor positive breast cancer. Many of these HuR-associated mRNA transcripts were metastasis related. We investigated the role of HuR in an aggressive triple-negative breast cancer metastatic cell line, LM2. The LM2 cells were retrovirally transduced with triple fusion reporter construct encoding thymidine kinase1, GFP and firefly luciferase to obtain in vivo and in vitro tracking capabilities. The cells were further transfected with plasmid containing HA-HuR or empty vector control to demonstrate the function of HuR in LM2. HuR over-expressing clones showed greater metastatic capability by in vitro matrigel invasion assay. Furthermore, athymic mice that were intravenously injected with HuR over-expressing LM2 cells had greater (335-fold more) tumor metastasis to the lungs than empty vector control injected group as measured by IVIS imaging. Mice injected with HuR over-expressing LM2 cells were more moribund and had greater mortality as compared with mice injected with empty vector control cells. These results suggest that HuR may play a role in breast cancer metastasis by stabilizing various pro-metastatic genes. The implications of this work are twofold. First, HuR RIP-Chip can be used to identify novel cancer relevant genes and second, interference with HuR function may ameliorate distant metastasis in breast cancer, potentially providing new therapeutic approaches for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A27.

Interactions between RNA binding proteins (RBPs) and genes are not well understood, especially in regulation of angiogenesis. The RBP HuR binds to the AU-rich (ARE) regions of labile mRNAs, facilitating their translation into protein and has been hypothesized to be a tumor-maintenance gene. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR controls the expression of multiple genes involved in angiogenesis including VEGFa, HIF1a, and thrombospondin 1 (TSP1). We investigated the role of HuR in estrogen receptor negative (ER-) breast cancer. MDA-MB-231 cells with higher levels of HuR have alterations in cell cycle kinetics and faster growth. Unexpectedly, HuR overexpression significantly interfered with tumor growth in orthotopic mouse models. The putative mechanism seems to be an anti-angiogenetic effect by increasing expression of TSP1 but also surprisingly, down-regulation of VEGF, a target of HuR which it normally increases. Our findings reveal that HuR may be regulating a cluster of genes involved in blood vessel formation which controls tumor angiogenesis. An approach of modulating HuR levels may overcome limitations associated with monotherapies targeting tumor vessel formation.

Posttranscriptional gene regulation controls the expression of genes implicated in a variety of processes, including those of the immune system. The RNA-binding protein HuR regulates many early response genes as well as cytokines such as IL-4 and IL-13. Therefore, HuR has been implicated in regulating Th2 polarization and cytokine production. GATA-3, a transcription factor that is essential for Th2 polarization contains a putative HuR binding site in its 3’UTR. Immunoprecipitation (IP) of ribonucleoprotein complexes with an anti-HuR Ab revealed significant enrichment for GATA-3 mRNA. HuR association with GATA-3 3’UTR was confirmed by biotin pull-downs. To understand the role of HuR in Th2 polarization in vivo we generated a transgenic HuR CD4+ T-cell mouse model. Th2-skewed cells over-expressing HuR displayed increases in both numbers of GATA3-expressing cells, and in overall GATA-3 expression. The frequency of Th2-polarized cells, but not of Th1, increased in CD4+ T cell HuR transgenic cells and the levels of secreted IL-4 and IL-13, but not IFN-γ, also increased significantly in both activated splenocytes and polarized cells. Furthermore, a knockdown of HuR in Jurkat cells decreased protein levels of GATA-3. Hence, HuR may be functioning to coordinately posttranscriptionally regulate genes essential for Th2 polarization and function. Better understanding of posttranscriptional regulation may elucidate control mechanisms of naïve CD4+ Th2 polarization.

BACKGROUND:The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).METHODS:The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.RESULTS:We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.CONCLUSION:This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.