HuR Enhances Early Restitution of the Intestinal Epithelium by Increasing Cdc42 Translation.
ABSTRACT: The mammalian intestinal mucosa exhibits a spectrum of responses after acute injury and repairs itself rapidly to restore the epithelial integrity. The RNA-binding protein HuR regulates the stability and translation of target mRNAs and is involved in many aspects of gut epithelium homeostasis, but its exact role in the regulation of mucosal repair after injury remains unknown. We show here that HuR is essential for early intestinal epithelial restitution by increasing the expression of cell division control protein 42 (Cdc42) at the posttranscriptional level. HuR bound to the Cdc42 mRNA via its 3' untranslated region, and this association specifically enhanced Cdc42 translation without an effect on the Cdc42 mRNA level. Intestinal epithelium-specific HuR knockout not only decreased Cdc42 levels in mucosal tissues, but it also inhibited repair of damaged mucosa induced by mesenteric ischemia/reperfusion in the small intestine and by dextran sulfate sodium in the colon. Furthermore, Cdc42 silencing prevented HuR-mediated stimulation of cell migration over the wounded area by altering the subcellular distribution of F-actin. These results indicate that HuR promotes early intestinal mucosal repair after injury by increasing Cdc42 translation and demonstrate the importance of HuR deficiency in the pathogenesis of delayed mucosal healing in certain pathological conditions.
Project description:Inhibition of growth of the intestinal epithelium, a rapidly self-renewing tissue, is commonly found in various critical disorders. The RNA-binding protein HuR is highly expressed in the gut mucosa and modulates the stability and translation of target mRNAs, but its exact biological function in the intestinal epithelium remains unclear. Here, we investigated the role of HuR in intestinal homeostasis using a genetic model and further defined its target mRNAs. Targeted deletion of HuR in intestinal epithelial cells caused significant mucosal atrophy in the small intestine, as indicated by decreased cell proliferation within the crypts and subsequent shrinkages of crypts and villi. In addition, the HuR-deficient intestinal epithelium also displayed decreased regenerative potential of crypt progenitors after exposure to irradiation. HuR deficiency decreased expression of the Wnt coreceptor LDL receptor-related protein 6 (LRP6) in the mucosal tissues. At the molecular level, HuR was found to bind the Lrp6 mRNA via its 3'-untranslated region and enhanced LRP6 expression by stabilizing Lrp6 mRNA and stimulating its translation. These results indicate that HuR is essential for normal mucosal growth in the small intestine by altering Wnt signals through up-regulation of LRP6 expression and highlight a novel role of HuR deficiency in the pathogenesis of intestinal mucosal atrophy under pathological conditions.
Project description:Intestinal epithelial autophagy is crucial for host defense against invasive pathogens, and defects in this process occur frequently in patients with inflammatory bowel disease (IBD) and other mucosal disorders, but the exact mechanism that activates autophagy is poorly defined. Here, we investigated the role of RNA-binding protein HuR (human antigen R) in the posttranscriptional control of autophagy-related genes (ATGs) in the intestinal epithelium. We found that targeted deletion of HuR in intestinal epithelial cells (IECs) specifically decreased the levels of ATG16L1 in the intestinal mucosa. Intestinal mucosa from patients with IBD exhibited reduced levels of both HuR and ATG16L1. HuR directly interacted with Atg16l1 mRNA via its 3' untranslated region and enhanced ATG16L1 translation, without affecting Atg16l1 mRNA stability. Circular RNA circPABPN1 blocked HuR binding to Atg16l1 mRNA and lowered ATG16L1 production. HuR silencing in cultured IECs also prevented rapamycin-induced autophagy, which was abolished by overexpressing ATG16L1. These findings indicate that HuR regulates autophagy by modulating ATG16L1 translation via interaction with circPABPN1 in the intestinal epithelium.
Project description:Intestinal ischemic injury results sloughing of the mucosal epithelium leading to host sepsis and death unless the mucosal barrier is rapidly restored. Volvulus and neonatal necrotizing enterocolitis (NEC) in infants have been associated with intestinal ischemia, sepsis and high mortality rates. We have characterized intestinal ischemia/repair using a highly translatable porcine model in which juvenile (6-8-week-old) pigs completely and efficiently restore barrier function by way of rapid epithelial restitution and tight junction re-assembly. In contrast, separate studies showed that younger neonatal (2-week-old) pigs exhibited less robust recovery of barrier function, which may model an important cause of high mortality rates in human infants with ischemic intestinal disease. Therefore, we aimed to further refine our repair model and characterize defects in neonatal barrier repair. Here we examine the defect in neonatal mucosal repair that we hypothesize is associated with hypomaturity of the epithelial and subepithelial compartments. Following jejunal ischemia in neonatal and juvenile pigs, injured mucosa was stripped from seromuscular layers and recovered ex vivo while monitoring transepithelial electrical resistance (TEER) and 3H-mannitol flux as measures of barrier function. While ischemia-injured juvenile mucosa restored TEER above control levels, reduced flux over the recovery period and showed 93±4.7% wound closure, neonates exhibited no change in TEER, increased flux, and a 11±23.3% increase in epithelial wound size. Scanning electron microscopy revealed enterocytes at the wound margins of neonates failed to assume the restituting phenotype seen in restituting enterocytes of juveniles. To attempt rescue of injured neonatal mucosa, neonatal experiments were repeated with the addition of exogenous prostaglandins during ex vivo recovery, ex vivo recovery with full thickness intestine, in vivo recovery and direct application of injured mucosal homogenate from neonates or juveniles. Neither exogenous prostaglandins, intact seromuscular intestinal layers, nor in vivo recovery enhanced TEER or restitution in ischemia-injured neonatal mucosa. However, ex vivo exogenous application of injured juvenile mucosal homogenate produced a significant increase in TEER and enhanced histological restitution to 80±4.4% epithelial coverage in injured neonatal mucosa. Thus, neonatal mucosal repair can be rescued through direct contact with the cellular and non-cellular milieu of ischemia-injured mucosa from juvenile pigs. These findings support the hypothesis that a defect in mucosal repair in neonates is due to immature repair mechanisms within the mucosal compartment. Future studies to identify and rescue specific defects in neonatal intestinal repair mechanisms will drive development of novel clinical interventions to reduce mortality in infants affected by intestinal ischemic injury.
Project description:BACKGROUND & AIMS:Paneth cells secrete antimicrobial proteins including lysozyme via secretory autophagy as part of the mucosal protective response. The ELAV like RNA-binding protein 1 (ELAVL1, also called HuR) regulates stability and translation of messenger RNAs (mRNAs) and many aspects of mucosal physiology. We studied the posttranscriptional mechanisms by which HuR regulates Paneth cell function. METHODS:Intestinal mucosal tissues were collected from mice with intestinal epithelium (IE)-specific disruption of HuR (IE-HuR-/-), HuRfl/fl-Cre- mice (controls), and patients with inflammatory bowel diseases and analyzed by histology and immunohistochemistry. Paneth cell functions were determined by lysozyme-immunostaining assays. We isolated primary enterocytes from IE-HuR-/- and control mice and derived intestinal organoids. HuR and the chaperone CNPY3 were overexpressed from transgenes in intestinal epithelial cells (IECs) or knocked down with small interfering RNAs. We performed RNA pulldown assays to investigate interactions between HuR and its target mRNAs. RESULTS:Intestinal tissues from IE-HuR-/- mice had reduced numbers of Paneth cells, and Paneth cells had fewer lysozyme granules per cell, compared with tissues from control mice, but there were no effects on Goblet cells or enterocytes. Intestinal mucosa from patients with inflammatory bowel diseases had reduced levels of HuR and fewer Paneth cells. IE-HuR-/- mice did not have the apical distribution of TLR2 in the intestinal mucosa as observed in control mice. IECs from IE-HuR-/- mice expressed lower levels of CNPY3. Intestinal organoids from IE-HuR-/- mice were smaller and contained fewer buds compared with those generated from controls, and had fewer lysozyme-positive cells. In IECs, knockdown of HuR decreased levels of the autophagy proteins LC3-I and LC3-II, compared with control cells, and prevented rapamycin-induced autophagy. We found HuR to interact directly with the Cnpy3 mRNA coding region and increase levels of CNPY3 by increasing the stability and translation of Cnpy3 mRNA. CNPY3 bound TLR2, and cells with knockdown of CNPY3 or HuR lost membrane localization of TLR2, but increased cytoplasmic levels of TLR2. CONCLUSIONS:In studies of mice, IECs, and human tissues, we found HuR to increase expression of CNPY3 at the posttranscriptional level. CNPY3 is required for membrane localization of TLR2 and Paneth cell function.
Project description:The mammalian intestinal epithelium is one of the most rapidly self-renewing tissues in the body, and its integrity is preserved through strict regulation. The RNA-binding protein (RBP) ELAV-like family member 1 (CELF1), also referred to as CUG-binding protein 1 (CUGBP1), regulates the stability and translation of target mRNAs and is implicated in many aspects of cellular physiology. We show that CELF1 competes with the RBP HuR to modulate MYC translation and regulates intestinal epithelial homeostasis. Growth inhibition of the small intestinal mucosa by fasting in mice was associated with increased CELF1/Myc mRNA association and decreased MYC expression. At the molecular level, CELF1 was found to bind the 3'-untranslated region (UTR) of Myc mRNA and repressed MYC translation without affecting total Myc mRNA levels. HuR interacted with the same Myc 3'-UTR element, and increasing the levels of HuR decreased CELF1 binding to Myc mRNA. In contrast, increasing the concentrations of CELF1 inhibited formation of the [HuR/Myc mRNA] complex. Depletion of cellular polyamines also increased CELF1 and enhanced CELF1 association with Myc mRNA, thus suppressing MYC translation. Moreover, ectopic CELF1 overexpression caused G1-phase growth arrest, whereas CELF1 silencing promoted cell proliferation. These results indicate that CELF1 represses MYC translation by decreasing Myc mRNA association with HuR and provide new insight into the molecular functions of RBPs in the regulation of intestinal mucosal growth.
Project description:The mammalian gut microbiota is essential for normal intestinal development, renewal, and repair. Injury to the intestinal mucosa can occur with infection, surgical trauma, and in idiopathic inflammatory bowel disease. Repair of mucosal injury, termed restitution, as well as restoration of intestinal homeostasis involves induced and coordinated proliferation and migration of intestinal epithelial cells. N-formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that can specifically bind and induce responses to host-derived and bacterial peptides and small molecules. Here we report that specific members of the gut microbiota stimulate FPR1 on intestinal epithelial cells to generate reactive oxygen species via enterocyte NADPH oxidase 1 (NOX1), causing rapid phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase mitogen-activated protein kinase. These events stimulate migration and proliferation of enterocytes adjacent to colonic wounds. Taken together, these findings identify a novel role of FPR1 as pattern recognition receptors for perceiving the enteric microbiota that promotes repair of mucosal wounds via generation of reactive oxygen species from the enterocyte NOX1.
Project description:The regulatory mechanisms enabling the intestinal epithelium to maintain a high degree of regenerative capacity during mucosal injury remain unclear. Ex vivo survival and clonogenicity of intestinal stem cells (ISCs) strictly required growth response mediated by cell division control 42 (Cdc42) and Cdc42-deficient enteroids to undergo rapid apoptosis. Mechanistically, Cdc42 engaging with EGFR was required for EGF-stimulated, receptor-mediated endocytosis and sufficient to promote MAPK signaling. Proteomics and kinase analysis revealed that a physiologically, but nonconventionally, spliced Cdc42 variant 2 (V2) exhibited stronger MAPK-activating capability. Human CDC42-V2 is transcriptionally elevated in some colon tumor tissues. Accordingly, mice engineered to overexpress Cdc42-V2 in intestinal epithelium showed elevated MAPK signaling, enhanced regeneration, and reduced mucosal damage in response to irradiation. Overproducing Cdc42-V2 specifically in mouse ISCs enhanced intestinal regeneration following injury. Thus, the intrinsic Cdc42-MAPK program is required for intestinal epithelial regeneration, and elevating this signaling cascade is capable of initiating protection from genotoxic injury.
Project description:The mammalian intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis depends on a dynamic balance among proliferation, migration, apoptosis, and differentiation of intestinal epithelial cells (IECs). The protein phosphatase 2A (PP2A)-associated protein ?4 controls the activity and specificity of serine/threonine phosphatases and is thus implicated in many cellular processes. Here, using a genetic approach, we investigated the mechanisms whereby ?4 controls the homeostasis of the intestinal epithelium. In mice with ablated ?4, the small intestinal mucosa exhibited crypt hyperplasia, villus shrinkage, defective differentiation of Paneth cells, and reduced IEC migration along the crypt-villus axis. The ?4-deficient intestinal epithelium also displayed decreased expression of different intercellular junction proteins and abnormal epithelial permeability. In addition, ?4 deficiency decreased the levels of the RNA-binding protein HuR in the mucosal tissue. In cultured IECs, ectopic overexpression of HuR in ?4-deficient cells rescued the production of these intercellular junction proteins and restored the epithelial barrier function to a nearly normal level. Mechanistically, ?4 silencing destabilized HuR through a process involving HuR phosphorylation by I?B kinase ?, leading to ubiquitin-mediated proteolysis of HuR. These findings indicate that the critical impact of ?4 upon the barrier function and homeostasis of the intestinal epithelium depends largely on its ability to regulate the stability of HuR.
Project description:Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. Saccharomyces boulardii (Sb, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of ?2?1 integrin collagen receptors. In the present study, we demonstrated that ?2?1 integrin is not the sole cell-extracellular matrix receptor involved during Sb-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that Sb supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased ?v?5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, Sb-mediated changes in cell adhesion to vitronectin resulted in a reduction of the ?v?5signaling pathway. We used a monolayer wounding assay that mimics in vivo cell restitution to demonstrate that down-modulation of the ?v?5 integrin-vitronectin interaction is related to Sb-induced cell migration. We therefore postulated that Sb supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of ?v?5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.
Project description:Pathobiology of several chronic inflammatory disorders, including ulcerative colitis and Crohn's disease is related to intermittent, spontaneous injury/ulceration of mucosal surfaces. Disease morbidity has been associated with pathologic release of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF?). In this report, we show that TNF? promotes intestinal mucosal repair through upregulation of the GPCR platelet activating factor receptor (PAFR) in the intestinal epithelium. Platelet activating factor (PAF) was increased in healing mucosal wounds and its engagement with epithelial PAFR leads to activation of epidermal growth factor receptor, Src and Rac1 signaling to promote wound closure. Consistent with these findings, delayed colonic mucosal repair was observed after administration of a neutralizing TNF? antibody and in mice lacking PAFR. These findings suggest that in the injured mucosa, the pro-inflammatory milieu containing TNF? and PAF sets the stage for reparative events mediated by PAFR signaling.