Chloride channel ClC- 2 enhances intestinal epithelial tight junction barrier function via regulation of caveolin-1 and caveolar trafficking of occludin.

Previous studies have demonstrated that the chloride channel ClC-2 plays a critical role in intestinal epithelial tight junction (TJ) barrier function via intracellular trafficking of TJ protein occludin. To study the mechanism of ClC-2-mediated TJ barrier function and intracellular trafficking of occludin, we established ClC-2 over-expressing Caco-2 cell line (Caco-2CLCN2) by full length ClC-2 ORF transfection. ClC-2 over-expression (Caco-2CLCN2) significantly enhanced TJ barrier (increased TER by ≥2 times and reduced inulin flux by 50%) compared to control Caco-2pEZ cells. ClC-2 over-expression (Caco-2CLCN2) increased occludin protein level compared to control Caco-2pEZ cells. Surface biotinylation assay revealed reduced steady state endocytosis of occludin in Caco-2CLCN2 cells. Furthermore, ClC-2 over-expression led to reduction in caveolin-1 protein level and diminishment of caveolae assembly. Caveolae disruption increased TJ permeability in control but not ClC-2 over-expressing Caco-2CLCN2 cells. Selective ClC-2 channel blocker GaTx2 caused an increase in caveolin-1 protein level and reduced occludin level. Delivery of cell permeable caveolin-1 scaffolding domain reduced the occludin protein level. Over all, these results suggest that ClC- 2 enhances TJ barrier function in intestinal epithelial cells via regulation of caveolin-1 and caveolae-mediated trafficking of occludin.

Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion.

The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H+] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84-95%, P<0.005) in ClC-2-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.

Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation.

Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2.

Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors.

BACKGROUND: Linaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out. RESULTS: In ischemia-damaged pig jejunum, using measurements of transepithelial resistance, (3)H-mannitol fluxes, short-circuit current (Cl(-) secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 µM linaclotide and 1 µM lubiprostone both caused similar increases in short-circuit current (Cl(-) secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca(2+)]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear. CONCLUSIONS: Considering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS.

Characterization of turkey inducible nitric oxide synthase and identification of its expression in the intestinal epithelium following astrovirus infection.

The inducible nitric oxide synthase (iNOS) enzyme has long been recognized as a key mediator of innate immune responses to infectious diseases across the phyla. Its role in killing or inactivating bacterial, parasitic, and viral pathogens has been documented in numerous host systems. iNOS, and its innate immune mediator NO has also been described to have negative consequence on host tissues as well; therefore understanding the pathogenesis of any infectious agent which induces iNOS expression requires a better understanding of the role iNOS and NO play in that disease. Previous studies in our laboratory and others have demonstrated evidence for increased levels of iNOS and activity of its innate immune mediator NO in the intestine of turkeys infected with astrovirus. To begin to characterize the role iNOS plays in the innate immune response to astrovirus infection, we identified, characterized, developed tkiNOS specific reagents, and demonstrated that the intestinal epithelial cells induce expression of iNOS following astrovirus infection. These data are the first to our knowledge to describe the tkiNOS gene, and demonstrate that astrovirus infection induces intestinal epithelial cells to express iNOS, suggesting these cells play a key role in the antiviral response to enteric infections.

Chloride channel ClC-2 modulates tight junction barrier function via intracellular trafficking of occludin.

Previously, we have demonstrated that the chloride channel ClC-2 modulates intestinal mucosal barrier function. In the present study, we investigated the role of ClC-2 in epithelial barrier development and maintenance in Caco-2 cells. During early monolayer formation, silencing of ClC-2 with small interfering (si)RNA led to a significant delay in the development of transepithelial resistance (TER) and disruption of occludin localization. Proteomic analysis employing liquid chromatography-mass spectrometry /mass spectrometry revealed association of ClC-2 with key proteins involved in intracellular trafficking, including caveolin-1 and Rab5. In ClC-2 siRNA-treated cells, occludin colocalization with caveolin-1 was diffuse and in the subapical region. Subapically distributed occludin in ClC-2 siRNA-treated cells showed marked colocalization with Rab5. To study the link between ClC-2 and trafficking of occludin in confluent epithelial monolayers, a Caco-2 cell clone expressing ClC-2 short hairpin (sh)RNA was established. Disruption of caveolae with methyl-ß-cyclodextrin (MßCD) caused a marked drop in TER and profound redistribution of caveolin-1-occludin coimmunofluorescence in ClC-2 shRNA cells. In ClC-2 shRNA cells, focal aggregations of Rab5-occludin coimmunofluorescence were present within the cytoplasm. Wortmannin caused an acute fall in TER in ClC-2 shRNA cells and subapical, diffuse redistribution of Rab5-occludin coimmunofluorescence in ClC-2 shRNA cells. An endocytosis and recycling assay for occludin revealed higher basal rate of endocytosis of occludin in ClC-2 shRNA cells. Wortmannin significantly reduced the rate of recycling of occludin in ClC-2 shRNA cells. These data clearly indicate that ClC-2 plays an important role in the modulation of tight junctions by influencing caveolar trafficking of the tight junction protein occludin.

TGF-beta-activated kinase 1 signaling maintains intestinal integrity by preventing accumulation of reactive oxygen species in the intestinal epithelium.

The intestinal epithelium is constantly exposed to inducers of reactive oxygen species (ROS), such as commensal microorganisms. Levels of ROS are normally maintained at nontoxic levels, but dysregulation of ROS is involved in intestinal inflammatory diseases. In this article, we report that TGF-ß-activated kinase 1 (TAK1) is a key regulator of ROS in the intestinal epithelium. tak1 gene deletion in the mouse intestinal epithelium caused tissue damage involving enterocyte apoptosis, disruption of tight junctions, and inflammation. Disruption of TNF signaling, which is a major intestinal damage inducer, rescued the inflammatory conditions but not apoptosis or disruption of tight junctions in the TAK1-deficient intestinal epithelium, suggesting that TNF is not a primary inducer of the damage noted in TAK1-deficient intestinal epithelium. We found that TAK1 deficiency resulted in reduced expression of several antioxidant-responsive genes and reduced the protein level of a key antioxidant transcription factor NF-E2-related factor 2, which resulted in accumulation of ROS. Exogenous antioxidant treatment reduced apoptosis and disruption of tight junctions in the TAK1-deficient intestinal epithelium. Thus, TAK1 signaling regulates ROS through transcription factor NF-E2-related factor 2, which is important for intestinal epithelial integrity.

ClC-2 regulates mucosal barrier function associated with structural changes to the villus and epithelial tight junction.

We have previously shown an important role of the chloride channel ClC-2 in orchestrating repair of tight junctions in ischemia-injured mucosa. In this study, we examined the role of ClC-2 in regulating barrier function of normal murine intestinal mucosa. Ex vivo, ClC-2-/- ileal mucosa mounted in Ussing chambers had significantly higher transepithelial electrical resistance (TER) and reduced [(3)H]mannitol mucosal-to-serosal flux compared with wild-type (WT) mouse mucosa. We also noted that ileum from ClC-2-/- mice had a significantly reduced in vivo [(3)H]mannitol blood-to-lumen clearance compared with WT animals. By scanning electron microscopy, flat leaflike villi were found to have tapering, rounded apical tips in ClC-2-/- mucosa. By transmission electron microscopy, the apical intercellular tight junctions in ClC-2-/- intestine revealed lateral membranes that were less well defined but closely aligned compared with electron-dense and closely apposed tight junctions in WT mucosa. The width of apical tight junctions was significantly reduced in ClC-2-/- intestine. Such an alteration in tight junction ultrastructure was also noted in the testicular tissue from ClC-2-/- mice. The ClC-2-/- intestinal mucosa had reduced expression of phospho-myosin light chain (MLC), and inhibition of myosin light chain kinase (MLCK) in WT mucosa partially increased TER toward the TER in ClC-2-/- intestine. Contrary to our prior work on the reparative role of ClC-2 in injured mucosa, this study indicates that ClC-2 reduces barrier function in normal mucosa. The mechanisms underlying these differing roles are not entirely clear, although ultrastructural morphology of tight junctions and MLCK appear to be important to the function of ClC-2 in normal mucosa.

Astrovirus infection induces sodium malabsorption and redistributes sodium hydrogen exchanger expression.

Astroviruses are known to be a leading cause of diarrhea in infants and the immunocompromised; however, our understanding of this endemic pathogen is limited. Histological analyses of astrovirus pathogenesis demonstrate clinical disease is not associated with changes to intestinal architecture, inflammation, or cell death. Recent studies in vitro have suggested that astroviruses induce actin rearrangement leading to loss of barrier function. The current study used the type-2 turkey astrovirus (TAstV-2) and turkey poult model of astrovirus disease to examine how astrovirus infection affects the ultrastructure and electrophysiology of the intestinal epithelium. These data demonstrate that infection results in changes to the epithelial ultrastructure, rearrangement of F-actin, decreased absorption of sodium, as well as redistribution of the sodium/hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm. Collectively, these data suggest astrovirus infection induces sodium malabsorption, possibly through redistribution of specific sodium transporters, which results in the development of an osmotic diarrhea.

ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum.

BACKGROUND AND AIMS: Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2(-/-) mice. METHODS: Wild type, ClC-2 heterozygous and ClC-2(-/-) murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of (3)H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation. RESULTS: Following ischemic injury, ClC-2(-/-) mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2(-/-) mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2(-/-) mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2(-/-) mice. CONCLUSIONS: These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.

Comparison of the chloride channel activator lubiprostone and the oral laxative Polyethylene Glycol 3350 on mucosal barrier repair in ischemic-injured porcine intestine.

AIM: To investigate the effects of lubiprostone and Polyethylene Glycol 3350 (PEG) on mucosal barrier repair in ischemic-injured porcine intestine. METHODS: Ileum from 6 piglets (approximately 15 kg body weight) was subjected to ischemic conditions by occluding the local mesenteric circulation for 45 min in vivo. Ileal tissues from each pig were then harvested and mounted in Ussing chambers and bathed in oxygenated Ringer's solution in vitro. Intestinal barrier function was assessed by measuring transepithelial electrical resistance (TER) and mucosal-to-serosal fluxes of (3)H-mannitol and (14)C-inulin. Statistical analyses of data collected over a 120-min time course included 2-way ANOVA for the effects of time and treatment on indices of barrier function. RESULTS: Application of 1 micromol/L lubiprostone to the mucosal surface of ischemic-injured ileum in vitro induced significant elevations in TER compared to non-treated tissue. Lubiprostone also reduced mucosal-to-serosal fluxes of (3)H-mannitol and (14)C-inulin. Alternatively, application of a polyethylene laxative (PEG, 20 mmol/L) to the mucosal surface of ischemic tissues significantly increased flux of (3)H-mannitol and (14)C-inulin. CONCLUSION: This experiment demonstrates that lubiprostone stimulates recovery of barrier function in ischemic intestinal tissues whereas the PEG laxative had deleterious effects on mucosal repair. These results suggest that, unlike osmotic laxatives, lubiprostone stimulates repair of the injured intestinal barrier.

Mice lacking the Na+/H+ exchanger 2 have impaired recovery of intestinal barrier function.

Ischemic injury induces breakdown of the intestinal barrier. Recent studies in porcine postischemic tissues indicate that inhibition of NHE2 results in enhanced recovery of barrier function in vitro via a process involving interepithelial tight junctions. To further study this process, recovery of barrier function was assessed in wild-type (NHE2(+/+)) and NHE2(-/-) mice in vivo and wild-type mice in vitro. Mice were subjected to complete mesenteric ischemia in vivo, after which barrier function was measured by blood-to-lumen mannitol clearance over a 3-h recovery period or measurement of transepithelial electrical resistance (TER) in Ussing chambers immediately following ischemia. Tissues were assessed for expression of select junctional proteins. Compared with NHE2(+/+) mice, NHE2(-/-) mice had greater intestinal permeability during the postischemic recovery process. In contrast to prior porcine studies, pharmacological inhibition of NHE2 in postischemic tissues from wild-type mice also resulted in significant reductions in TER. Mucosa from NHE2(-/-) mice displayed a shift of occludin and claudin-1 expression to the Triton-X-soluble membrane fractions and showed disruption of occludin and claudin-1 localization patterns following injury. This was qualitatively and quantitatively recovered in NHE2(+/+) mice compared with NHE2(-/-) mice by the end of the 3-h recovery period. Serine phosphorylation of occludin and claudin-1 was downregulated in NHE2(-/-) postischemia compared with wild-type mice. These data indicate an important role for NHE2 in recovery of barrier function in mice via a mechanism involving tight junctions.

Gastrointestinal dysfunction induced by early weaning is attenuated by delayed weaning and mast cell blockade in pigs.

Our previous work has demonstrated that weaning at 19 days of age has deleterious effects on mucosal barrier function in piglet intestine that are mediated through peripheral CRF receptor signaling pathways. The objectives of the present study were to assess the impact of piglet age on weaning-associated intestinal dysfunction and to determine the role that mast cells play in weaning-induced breakdown of mucosal barrier function. Nursing Yorkshire-cross piglets were either weaned at 19 days of age (early-weaned, n = 8) or 28 days of age (late-weaned, n = 8) and housed in nursery pens. Twenty-four hours postweaning, segments of midjejunum and ascending colon from piglets within each weaning age group were harvested and mounted on Ussing chambers for measurements of transepithelial electrical resistance and serosal-to-mucosal [(3)H]mannitol fluxes. Early weaning resulted in reductions in transepithelial electrical resistance and increases in mucosal permeability to [(3)H]mannitol in the jejunum and colon (P < 0.01). In contrast, postweaning reductions in intestinal barrier function were not observed in piglets weaned at 28 days of age. Early-weaned piglet intestinal mucosa had increased expression of CRF receptor 1 protein, increased mucosal mast cell tryptase levels, and evidence of enhanced mast cell degranulation compared with late-weaned intestinal mucosa. Pretreatment of piglets with the mast cell stabilizer drug cromolyn, injected intraperitoneally 30 min prior to weaning, abolished the early-weaning-induced intestinal barrier disturbances. Our results indicate that early-weaning stress induces mucosal dysfunction mediated by intestinal mast cell activation and can be prevented by delaying weaning.

Recovery of mucosal barrier function in ischemic porcine ileum and colon is stimulated by a novel agonist of the ClC-2 chloride channel, lubiprostone.

Previous studies utilizing an ex vivo porcine model of intestinal ischemic injury demonstrated that prostaglandin (PG)E(2) stimulates repair of mucosal barrier function via a mechanism involving Cl(-) secretion and reductions in paracellular permeability. Further experiments revealed that the signaling mechanism for PGE(2)-induced mucosal recovery was mediated via type-2 Cl(-) channels (ClC-2). Therefore, the objective of the present study was to directly investigate the role of ClC-2 in mucosal repair by evaluating mucosal recovery in ischemia-injured intestinal mucosa treated with the selective ClC-2 agonist lubiprostone. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers, and short-circuit current (I(sc)) and transepithelial electrical resistance (TER) were measured in response to lubiprostone. Application of 0.01-1 microM lubiprostone to ischemia-injured mucosa induced concentration-dependent increases in TER, with 1 microM lubiprostone stimulating a twofold increase in TER (DeltaTER = 26 Omega.cm(2); P < 0.01). However, lubiprostone (1 microM) stimulated higher elevations in TER despite lower I(sc) responses compared with the nonselective secretory agonist PGE(2) (1 microM). Furthermore, lubiprostone significantly (P < 0.05) reduced mucosal-to-serosal fluxes of (3)H-labeled mannitol to levels comparable to those of normal control tissues and restored occludin localization to tight junctions. Activation of ClC-2 with the selective agonist lubiprostone stimulated elevations in TER and reductions in mannitol flux in ischemia-injured intestine associated with structural changes in tight junctions. Prostones such as lubiprostone may provide a selective and novel pharmacological mechanism of accelerating recovery of acutely injured intestine compared with the nonselective action of prostaglandins such as PGE(2).

Prostaglandin-mediated inhibition of Na+/H+ exchanger isoform 2 stimulates recovery of barrier function in ischemia-injured intestine.

Prostaglandins stimulate repair of the ischemia-injured intestinal barrier in the porcine ileum through a mechanism involving cAMP-dependent Cl- secretion and inhibition of electroneutral Na+/H+ exchanger (NHE) activity. In the present study, we focused on the role of individual NHE isoforms in the recovery of barrier function. Ischemia-injured porcine ileal mucosa was mounted on Ussing chambers. Short-circuit current (I(sc)), transepithelial electrical resistance (TER), and isotopic fluxes of 22Na were measured in response to PGE2 and selective inhibitors of epithelial NHE isoforms. Immunoassays were used to assess the expression of NHE isoforms. Forty-five minutes of intestinal ischemia resulted in a 45% reduction in TER (P < 0.01). Near-complete restitution occurred within 60 min. Inhibition of NHE2 with HOE-694 (25 microM) added to the mucosal surface of the injured ileum stimulated significant elevations in TER, independent of changes in I(sc) and histological evidence of restitution. Pharmacological inhibition of NHE3 or NHE1 with mucosal S-3226 (20 microM) or serosal cariporide (25 microM), respectively, had no effect. Ischemia-injured tissues treated with mucosal S-3226 or HOE-694 exhibited equivalent reductions in mucosal-to-serosal fluxes of 22Na+ (by approximately 35%) compared with nontreated ischemia-injured control tissues (P < 0.05). Intestinal ischemia resulted in increased expression of the cytoplasmic NHE regulatory factor EBP50 in NHE2 but not in NHE3 immunoprecipitates. Selective inhibition of NHE2, and not NHE3, induces recovery of barrier function in the ischemia-injured intestine.

Emergence of a nephropathogenic avian infectious bronchitis virus with a novel genotype in India.

We describe the emergence of a nephropathogenic avian infectious bronchitis virus (IBV) with a novel genotype in India. The Indian IBV isolate exhibited a relatively high degree of sequence divergence with reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%).