Mucosal Ecological Network of Epithelium and Immune Cells for Gut Homeostasis and Tissue Healing.

The intestinal epithelial barrier includes columnar epithelial, Paneth, goblet, enteroendocrine, and tuft cells as well as other cell populations, all of which contribute properties essential for gastrointestinal homeostasis. The intestinal mucosa is covered by mucin, which contains antimicrobial peptides and secretory IgA and prevents luminal bacteria, fungi, and viruses from stimulating intestinal immune responses. Conversely, the transport of luminal microorganisms-mediated by M, dendritic, and goblet cells-into intestinal tissues facilitates the harmonization of active and quiescent mucosal immune responses. The bacterial population within gut-associated lymphoid tissues creates the intratissue cohabitations for harmonized mucosal immunity. Intermolecular and intercellular communication among epithelial, immune, and mesenchymal cells creates an environment conducive for epithelial regeneration and mucosal healing. This review summarizes the so-called intestinal mucosal ecological network-the complex but vital molecular and cellular interactions of epithelial mesenchymal cells, immune cells, and commensal microbiota that achieve intestinal homeostasis, regeneration, and healing.

GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

Divergent roles of estrogen receptor subtypes in regulating estrogen-modulated colonic ion transports and epithelial repair.

Although it was described previously for estrogen (E2) regulation of intestinal epithelial Cl- and HCO3- secretion in sex difference, almost nothing is known about the roles of estrogen receptor (ER) subtypes in regulating E2-modulated epithelial ion transports and epithelial restitution. Here, we aimed to investigate ERα and ERß subtypes in the regulation of E2-modulated colonic epithelial HCO3- and Cl- secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl- secretion but promoted Ca2+-dependent HCO3- secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERß attenuated HCO3- secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERß promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and ß-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERß were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3- and Cl- secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERß subtypes orchestrate functional homeostasis of normal colonic epithelial cells.

Enteric glial cell network function is required for epithelial barrier restitution following intestinal ischemic injury in the early postnatal period.

Ischemic damage to the intestinal epithelial barrier, such as in necrotizing enterocolitis or small intestinal volvulus, is associated with higher mortality rates in younger patients. We have recently reported a powerful pig model to investigate these age-dependent outcomes in which mucosal barrier restitution is strikingly absent in neonates but can be rescued by direct application of homogenized mucosa from older, juvenile pigs by a yet-undefined mechanism. Within the mucosa, a postnatally developing network of enteric glial cells (EGCs) is gaining recognition as a key regulator of the mucosal barrier. Therefore, we hypothesized that the developing EGC network may play an important role in coordinating intestinal barrier repair in neonates. Neonatal and juvenile jejunal mucosa recovering from surgically induced intestinal ischemia was visualized by scanning electron microscopy and the transcriptomic phenotypes were assessed by bulk RNA sequencing. EGC network density and glial activity were examined by Gene Set Enrichment Analysis, three-dimensional (3-D) volume imaging, and Western blot and its function in regulating epithelial restitution was assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate (FA), and in vitro by coculture assay. Here we refine and elaborate our translational model, confirming a neonatal phenotype characterized by a complete lack of coordinated reparative signaling in the mucosal microenvironment. Furthermore, we report important evidence that the subepithelial EGC network changes significantly over the early postnatal period and demonstrate that the proximity of a specific functional population of EGC to wounded intestinal epithelium contributes to intestinal barrier restitution following ischemic injury.NEW & NOTEWORTHY This study refines a powerful translational pig model, defining an age-dependent relationship between enteric glia and the intestinal epithelium during intestinal ischemic injury and confirming an important role for enteric glial cell (EGC) activity in driving mucosal barrier restitution. This study suggests that targeting the enteric glial network could lead to novel interventions to improve recovery from intestinal injury in neonatal patients.

miR-195 regulates intestinal epithelial restitution after wounding by altering actin-related protein-2 translation.

Early gut epithelial restitution reseals superficial wounds after acute injury, but the exact mechanism underlying this rapid mucosal repair remains largely unknown. MicroRNA-195 (miR-195) is highly expressed in the gut epithelium and involved in many aspects of mucosal pathobiology. Actin-related proteins (ARPs) are key components essential for stimulation of actin polymerization and regulate cell motility. Here, we reported that miR-195 modulates early intestinal epithelial restitution by altering ARP-2 expression at the translation level. miR-195 directly interacted with the ARP-2 mRNA, and ectopically expressed miR-195 decreased ARP-2 protein without effect on its mRNA content. In contrast, miR-195 silencing by transfection with anti-miR-195 oligo increased ARP-2 expression. Decreased ARP-2 levels by miR-195 overexpression were associated with an inhibition of early epithelial restitution, as indicated by a decrease in cell migration over the wounded area. Elevation of cellular ARP-2 levels by transfection with its transgene restored cell migration after wounding in cells overexpressing miR-195. Polyamines were found to decrease miR-195 abundance and enhanced ARP-2 translation, thus promoting epithelial restitution after wounding. Moreover, increasing the levels of miR-195 disrupted F-actin cytoskeleton organization, which was prevented by ARP2 overexpression. These results indicate that miR-195 inhibits early epithelial restitution by decreasing ARP-2 translation and that miR-195 expression is negatively regulated by cellular polyamines.

STIM1 promotes IPEC-J2 porcine epithelial cell restitution by TRPC1 signaling.

Intestinal epithelial restitution is partly dependent on cell migration, which reseals superficial wounding after injury. Here, we tested the hypothesis that stromal interaction molecule 1(STIM1) regulates porcine intestinal epithelial cell migration by activating transient receptor potential canonical 1 (TRPC1) signaling. Results showed that the knockdown of STIM1 repressed cell migration after wounding, reduced the protein concentration of STIM1 and TRPC1, and decreased the inositol trisphosphate (IP3) content in IPEC-J2 cells (p < 0.05). However, overexpression of STIM1 obtained opposite results (p < 0.05). The inhibition of TRPC1 activity by treatment with SKF96365 in cells overexpressing wild-type and mutant STIM1 attenuated the STIM1 overexpression-induced increase of cell migration, STIM1, TRPC1 and IP3 (p < 0.05). In addition, polyamine depletion caused by α-difluoromethylornithine (DFMO) resulted in the decrease of above-mentioned parameters, and exogenous polyamine could attenuate the negative effects of DFMO on IPEC-J2 cells (p < 0.05). Moreover, the overexpression of STIM1 could rescue cell migration, the protein level of STIM1 and TRPC1, and IP3 content in polyamine-deficient IPEC-J2 cells (p < 0.05). These results indicated that STIM1 could enhance porcine intestinal epithelial cell migration via the TRPC1 signaling pathway. Inhibition of cell migration by polyamine depletion resulted from the reduction of STIM1 activity.

RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration.

The airway epithelium's ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract's normal functions. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions. Inhibition of Rho-associated kinase, a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain 2 following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed reepithelialization. There were increased concentrations of phospho-myosin light chain 2 in day 7 naphthalene + RSV animals, which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.

A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair via an unfolded protein response.

Cholera toxin B subunit (CTB) exhibits broad-spectrum biologic activity upon mucosal administration. Here, we found that a recombinant CTB containing an endoplasmic reticulum (ER) retention motif (CTB-KDEL) induces colon epithelial wound healing in colitis via the activation of an unfolded protein response (UPR) in colon epithelial cells. In a Caco2 cell wound healing model, CTB-KDEL, but not CTB or CTB-KDE, facilitated cell migration via interaction with the KDEL receptor, localization in the ER, UPR activation, and subsequent TGF-ß signaling. Inhibition of the inositol-requiring enzyme 1/X-box binding protein 1 arm of UPR abolished the cell migration effect of CTB-KDEL, indicating that the pathway is indispensable for the activity. CTB-KDEL's capacity to induce UPR and epithelial restitution or wound healing was corroborated in a dextran sodium sulfate-induced acute colitis mouse model. Furthermore, CTB-KDEL induced a UPR, up-regulated wound healing pathways, and maintained viable crypts in colon explants from patients with inflammatory bowel disease (IBD). In summary, CTB-KDEL exhibits unique wound healing effects in the colon that are mediated by its localization to the ER and subsequent activation of UPR in epithelial cells. The results provide implications for a novel therapeutic approach for mucosal healing, a significant unmet need in IBD treatment.-Royal, J. M., Oh, Y. J., Grey, M. J., Lencer, W. I., Ronquillo, N., Galandiuk, S., Matoba, N. A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair via an unfolded protein response.

c-Jun enhances intestinal epithelial restitution after wounding by increasing phospholipase C-γ1 transcription.

c-Jun is an activating protein 1 (AP-1) transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, coordinates Ca2+ store mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun upregulates PLCγ1 expression and enhances PLCγ1-induced Ca2+ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLCγ1 expression at the transcription level, and this stimulation is mediated by directly interacting with AP-1 and CCAAT-enhancer-binding protein (C/EBP) binding sites that are located at the proximal region of the rat PLCγ1 promoter. Increased levels of PLCγ1 by c-Jun elevated cytosolic free Ca2+ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLCγ1/Ca2+ signal also plays an important role in polyamine-induced cell migration after wounding because increased c-Jun rescued Ca2+ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLCγ1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca2+ signal.

Multiple targets of carbon monoxide gas in the intestinal inflammation.

Inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn's disease are chronic relapsing and remitting inflammatory disorders of the intestinal tract. It is important to investigate the precise pathogenesis of IBD, to evaluate new anti-inflammatory agents, and to develop novel drugs. Carbon monoxide (CO) has emerged as an important regulator of acute and chronic inflammation of the gastrointestinal tract. The mechanism underlying its anti-inflammatory effects is only partially understood. Recent reports have demonstrated that CO could play a role in the functional modulation of epithelial and immunological cells in the intestine. In this short review, we have highlighted the recent findings that CO stimulates the epithelial cell restitution and FGF production from myofibroblasts. CO was also shown to regulate T cell activation and differentiation, and to activate macrophages. Finally, we have discussed the direction of translational research with respect to launching a novel agent for releasing CO in the intestine.

RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding.

Early mucosal restitution occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury. Our previous studies show that canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca(2+) channel (SOC) in intestinal epithelial cells (IECs) and plays an important role in early epithelial restitution by increasing Ca(2+) influx. Here we further reported that RhoA, a small GTP-binding protein, interacts with and regulates TRPC1, thus enhancing SOC-mediated Ca(2+) entry (SOCE) and epithelial restitution after wounding. RhoA physically associated with TRPC1 and formed the RhoA/TRPC1 complexes, and this interaction increased in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1). Inactivation of RhoA by treating IEC-TRPC1 cells with exoenzyme C3 transferase (C3) or ectopic expression of dominant negative RhoA (DNMRhoA) reduced RhoA/TRPC1 complexes and inhibited Ca(2+) influx after store depletion, which was paralleled by an inhibition of cell migration over the wounded area. In contrast, ectopic expression of wild-type (WT)-RhoA increased the levels of RhoA/TRPC1 complexes, induced Ca(2+) influx through activation of SOCE, and promoted cell migration after wounding. TRPC1 silencing by transfecting stable WT RhoA-transfected cells with siRNA targeting TRPC1 (siTRPC1) reduced SOCE and repressed epithelial restitution. Moreover, ectopic overexpression of WT-RhoA in polyamine-deficient cells rescued the inhibition of Ca(2+) influx and cell migration induced by polyamine depletion. These findings indicate that RhoA interacts with and activates TRPC1 and thus stimulates rapid epithelial restitution after injury by inducing Ca(2+) signaling.

Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activation.

Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim of the study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal epithelial cells (IECs) was increased at the wound edge after 24 h (P < 0.05), returned to normal after reepithelialization, and correlated with the inflammatory reaction in the experimental wounds (P < 0.001). cIAP2 was induced in vitro in regenerating Caco2 IECs after wound infliction (P < 0.01). Knockdown of cIAP2 caused a substantial impairment of the IEC regeneration through inhibition of migration (P < 0.005). cIAP2 overexpression lead to formation of migrating IECs and upregulation of expression of RhoA and Rac1 as well as GTP-activation of Rac1. Transforming growth factor-ß1 enhanced the expression of cIAP2 but was not upregulated in wounds in vivo and in vitro. NF-κB and MAPK pathways did not affect cIAP2 expression. cIAP2 is in conclusion a regulator of human intestinal wound healing through enhanced migration along with activation of Rac1, and the findings suggest that cIAP2 could be a future therapeutic target to improve intestinal wound healing.

Tissue distribution and expression dynamics of trefoil factor genes in the hydroid Hydractinia symbiolongicarpus.

Proteins of the trefoil factor family (TFF) participate in mucosal repair and are formed by single or tandemly repeated trefoil domains. TFFs have been extensively studied in mammals and amphibians, but they have not been functionally characterized in other animals. Here we report the identification of two genes expressed in the hydroid Hydractinia symbiolongicarpus, predicted to encode trefoil domain-containing peptides, one with four trefoil domains in tandem and the other one with a trefoil domain flanked by two ShKT domains. Differential expression analyses by qPCR after an immune challenge and an induced mechanical damage, reveal that the former gene (hysyTFF) had no significant changes in expression after the inductions. However, the latter (hysyTFF-like) was overexpressed after three hours post immune challenge and was downregulated after the first hour post epithelial damage. Immunoblot analyses using specific IgY antibodies revealed that hysyTFF is secreted as a high molecular weight complex. Finally, whole mount immunofluorescence assays showed that hysyTFF was predominantly expressed in the endoderm of stolons and polyps, and sparsely in the ectoderm of both polyps and larvae. Thus, the tissue distribution and expression dynamics of trefoil factor genes in H. symbiolongicarpus suggest that hysyTFF is part of an ancient mechanism of epithelial restitution, and the newly reported hysyTFF-like might act as an immune effector gene, perhaps encoding an antibacterial peptide.

Inhibitory effects of omega-3 fatty acids on injury-induced epidermal growth factor receptor transactivation contribute to delayed wound healing.

Epidermal growth factor receptor (EGFR)-mediated signaling is required for optimal intestinal wound healing. Since n-3 polyunsaturated fatty acids (PUFA), specifically docosahexaenoic acid (DHA), alter EGFR signaling and suppress downstream activation of key signaling pathways, we hypothesized that DHA would be detrimental to the process of intestinal wound healing. Using a mouse immortalized colonocyte model, DHA uniquely reduced EGFR ligand-induced receptor activation, whereas DHA and its metabolic precursor eicosapentaenoic acid (EPA) reduced wound-induced EGFR transactivation compared with control (no fatty acid or linoleic acid). Under wounding conditions, the suppression of EGFR activation was associated with a reduction in downstream activation of cytoskeletal remodeling proteins (PLCγ1, Rac1, and Cdc42). Subsequently, DHA and EPA reduced cell migration in response to wounding. Mice were fed a corn oil-, DHA-, or EPA-enriched diet prior to intestinal wounding (2.5% dextran sodium sulfate for 5 days followed by termination after 0, 3, or 6 days of recovery). Mortality was increased in EPA-fed mice and colonic histological injury scores were increased in EPA- and DHA-fed mice compared with corn oil-fed (control) mice. Although kinetics of colonic EGFR activation and downstream signaling (PLCγ1, Rac1, and Cdc42) were delayed by both n-3 PUFA, colonic repair was increased in EPA- relative to DHA-fed mice. These results indicate that, during the early response to intestinal wounding, DHA and EPA uniquely delay the activation of key wound-healing processes in the colon. This effect is mediated, at least in part, via suppression of EGFR-mediated signaling and downstream cytoskeletal remodeling.

Agent-based model of epithelial host-pathogen interactions in anastomotic leak.

BACKGROUND: There is a growing recognition of the significance of host-pathogen interactions (HPIs) in gut biology leading to a reassessment of the role of bacteria in intestinal anastomotic leak. Understanding the complexities of the early postsurgical gut HPI requires integrating knowledge of both epithelial and bacterial behaviors to generate hypotheses of potential mechanisms of interaction. Agent-based modeling is a computational method well suited to achieve this goal, and we use an agent-based model (ABM) to examine alterations in the HPI affecting reestablishment of the epithelial barrier that may subsequently lead to anastomotic leak. METHODS: Computational agents representing Pseudomonas aeruginosa were added to a previously validated ABM of epithelial restitution. Simulated experiments were performed examining the effect of radiation on bacterial binding to epithelial cells, plausibility of putative binding targets, and potential mechanisms of epithelial cell killing by virulent bacteria. RESULTS: Simulation experiments incorporating radiation effects on epithelial monolayers produced binding patterns akin to those seen in vitro and suggested that promotility integrin-laminin associations represent potential sites for bacterial binding and disruption of restitution. Simulations of potential mechanisms of epithelial cell killing suggested that an injected cytotoxin was the means by which virulent bacteria produced the tissue destruction needed to generate an anastomotic leak, a mechanism subsequently confirmed with genotyping of the virulent P aeruginosa strain. CONCLUSIONS: This study emphasizes the utility of ABM as an adjunct to traditional research methods and provides insights into the potentially critical role of HPI in the pathogenesis of anastomotic leak.

The enigmatic roles of epithelial gasdermin B: Recent discoveries and controversies.

Gasdermin B (GSDMB) belongs to a family of structurally related proteins [(i.e., gasdermins (GSDMs)]. It distinguishes itself from other members by the lack of autoinhibition but clear bioactivity of its full-length form, its preference to bind to phosphatidylinositol phosphates and sulfatides, and the ability to promote both lytic and nonlytic cellular functions. It is the only gasdermin that lacks a mouse ortholog, making in vivo mechanistic studies challenging to perform. GSDMB is abundantly expressed in epithelial cells lining organs that directly interface with the external environment, such as the gastrointestinal tract, with emerging evidence supporting its role in enteric infections, inflammatory bowel disease (IBD), and colorectal cancer. This review discusses the unique features of GSDMB among other gasdermin family members and controversies surrounding GSDMB-dependent mammalian inflammatory cell death (i.e., pyroptosis), including recent discoveries revealing both lytic and nonlytic functions of epithelial-derived GSDMB, particularly during gut health and disease.

Dynamics of differentiated-renal epithelial cell monolayer after calcium oxalate injury: The role of cyclooxygenase-2.

AIMS: Calcium oxalate (Oxa), constituent of most common kidney stones, damages renal tubular epithelial cells leading to kidney disease. Most in vitro studies designed to evaluate how Oxa exerts its harmful effects were performed in proliferative or confluent non-differentiated renal epithelial cultures; none of them considered physiological hyperosmolarity of renal medullary interstitium. Cyclooxygenase 2 (COX2) has been associated to Oxa deleterious actions; however, up to now, it is not clear how COX2 acts. In this work, we proposed an in vitro experimental system resembling renal differentiated-epithelial cells that compose medullary tubular structures which were grown and maintained in a physiological hyperosmolar environment and evaluated whether COX2 â†’ PGE2 axis (COX2 considered a cytoprotective protein for renal cells) induces Oxa damage or epithelial restitution. MAIN METHODS: MDCK cells were differentiated with NaCl hyperosmolar medium for 72 h where cells acquired the typical apical and basolateral membrane domains and a primary cilium. Then, cultures were treated with 1.5 mM Oxa for 24, 48, and 72 h to evaluate epithelial monolayer restitution dynamics and COX2-PGE2 effect. KEY FINDINGS: Oxa completely turned the differentiated phenotype into mesenchymal one (epithelial-mesenchymal transition). Such effect was partially and totally reverted after 48 and 72 h, respectively. Oxa damage was even deeper when COX2 was blocked by NS398. PGE2 addition restituted the differentiated-epithelial phenotype in a time and concentration dependence. SIGNIFICANCE: This work presents an experimental system that approaches in vitro to in vivo renal epithelial studies and, more important, warns about NSAIDS use in patients suffering from kidney stones.

Fibrin(ogen) Is Constitutively Expressed by Differentiated Intestinal Epithelial Cells and Mediates Wound Healing.

Fibrinogen is a large molecule synthesized in the liver and released in the blood. Circulating levels of fibrinogen are upregulated after bleeding or clotting events and support wound healing. In the context of an injury, thrombin activation drives conversion of fibrinogen to fibrin. Fibrin deposition contains tissue damage, stops blood loss, and prevents microbial infection. In most circumstances, fibrin needs to be removed to allow the resolution of inflammation and tissue repair, whereas failure of this may lead to the development of various disorders. However, the contribution of fibrinogen to tissue inflammation and repair is likely to be context-dependent. In this study, the concept that fibrin needs to be removed to allow tissue repair and to reduce inflammation is challenged by our observations that, in the intestine, fibrinogen is constitutively produced by a subset of intestinal epithelial cells and deposited at the basement membrane as fibrin where it serves as a substrate for wound healing under physiological conditions such as epithelial shedding at the tip of the small intestinal villus and surface epithelium of the colon as well as under pathological conditions that require rapid epithelial repair. The functional integrity of the intestine is ensured by the constant renewal of its simple epithelium. Superficial denuding of the epithelial cell layer occurs regularly and is rapidly corrected by a process called restitution that can be influenced by various soluble and insoluble factors. Epithelial cell interaction with the extracellular matrix greatly influences the healing process by acting on cell morphology, adhesion, and migration. The functional contribution of a fibrin(ogen) matrix in the intestine was studied under physiological and pathological contexts. Our results (immunofluorescence, immunoelectron microscopy, and quantitative PCR) show that fibrin(ogen) is a novel component of the basement membrane associated with the differentiated epithelial cell population in both the small intestine and colon. Fibrin(ogen) alone is a weak ligand for epithelial cells and behaves as an anti-adhesive molecule in the presence of type I collagen. Furthermore, the presence of fibrin(ogen) significantly shortens the time required to achieve closure of wounded epithelial cell monolayers and co-cultures in a PI3K-dependent manner. In human specimens with Crohn's disease, we observed a major accumulation of fibrin(ogen) throughout the tissue and at denuded sites. In mice in which fibrin formation was inhibited with dabigatran treatment, dextran sulfate sodium administration provoked a significant increase in the disease activity index and pathological features such as mucosal ulceration and crypt abscess formation. Taken together, these results suggest that fibrin(ogen) contributes to epithelial healing under both normal and pathological conditions.

TRPC1-mediated Ca2+ signaling enhances intestinal epithelial restitution by increasing α4 association with PP2Ac after wounding.

Gut epithelial restitution after superficial wounding is an important repair modality regulated by numerous factors including Ca2+ signaling and cellular polyamines. Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and its activation increases epithelial restitution by inducing Ca2+ influx after acute injury. α4 is a multiple functional protein and implicated in many aspects of cell functions by modulating protein phosphatase 2A (PP2A) stability and activity. Here we show that the clonal populations of IECs stably expressing TRPC1 (IEC-TRPC1) exhibited increased levels of α4 and PP2A catalytic subunit (PP2Ac) and that TRPC1 promoted intestinal epithelial restitution by increasing α4/PP2Ac association. The levels of α4 and PP2Ac proteins increased significantly in stable IEC-TRPC1 cells and this induction in α4/PP2Ac complexes was accompanied by an increase in IEC migration after wounding. α4 silencing by transfection with siRNA targeting α4 (siα4) or PP2Ac silencing destabilized α4/PP2Ac complexes in stable IEC-TRPC1 cells and repressed cell migration over the wounded area. Increasing the levels of cellular polyamines by stable transfection with the Odc gene stimulated α4 and PP2Ac expression and enhanced their association, thus also promoting epithelial restitution after wounding. In contrast, depletion of cellular polyamines by treatment with α-difluoromethylornithine reduced α4/PP2Ac complexes and repressed cell migration. Ectopic overexpression of α4 partially rescued rapid epithelial repair in polyamine-deficient cells. These results indicate that activation of TRPC1-mediated Ca2+ signaling enhances cell migration primarily by increasing α4/PP2Ac associations after wounding and this pathway is tightly regulated by cellular polyamines.

Suppression of epithelial restitution using an inhibitor against Rho-associated coiled-coil containing protein kinase aggravates colitis through reduced epithelial expression of A-kinase anchor protein 13.

In the gastrointestinal tract, the immediate healing response to mucosal damage is critical to sustain mucosal homeostasis. The migration of surrounding epithelial cells to cover the denuded area without proliferation is termed restitution, followed by early reparation of the damage. In this study, we determined the role of A-kinase anchor protein 13 (AKAP13) in mice with dextran sulphate sodium (DSS)-induced colitis upon mucosal injury and restitution, and investigated whether inhibition of Rho-associated coiled-coil containing protein kinase (ROCK), downstream effector of AKAP13, affects these mucosal responses. BALB/c mice were challenged with 4% or 2% DSS in their drinking water for up to 8 or 16days, respectively. During this period, mice received subcutaneous injections of fasudil hydrochloride hydrate (FH, 10mg/kg, twice per day), an inhibitor of phosphorylation of ROCK. In immunohistochemistry, AKAP13 was highly expressed in the mucosal epithelium prior to DSS-induced mucosal injury, and also expressed in ulcer-covering non-proliferative epithelium, which corresponded to restituted epithelial cells. Coadministration of FH increased serum amyloid A levels and histopathological scores for mucosal injury, as compared with the DSS group. The effects were associated with a decrease in gene expression of Akap13 in the mucosal tissue and the inhibition of restitution rata (the length of restituted epithelial cells per ulcer). These results suggested that AKAP13 and ROCK are involved in mucosal response at early injury and restitution during healing in DSS-induced colitis in mice.