MiR-874 promotes intestinal barrier dysfunction through targeting AQP3 following intestinal ischemic injury

Intestinal ischemic injury is a significant clinical problem arising from diseases or as a complication of abdominal surgery. Our previous study showed aquaporin 3 is involved in intestinal barrier impairment. Here, we revealed that intestinal ischemia induced a time-dependent increase of miR-874 expression and a time-dependent decrease of AQP3 expression, and the level of miR-874 expression was inversely related to AQP3 protein expression. In addition, miR-874 promoted the paracellular permeability in vitro through targeting 3′UTR of AQP3. Two of the tight junction proteins, Occludin and Claudin-1, were found to be involved in miR-874-induced intestinal barrier dysfunction.     

Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation

Numb is highly expressed throughout the crypt-villus axis of intestinal mucosa and functions as cell fate determinant and integrator of cell-to-cell adhesion. Increased paracellular permeability of intestinal epithelial cells is associated with the epithelial barrier dysfunction of inflammatory bowel diseases (IBDs). The apical junctional complex (AJC) assembly and myosin light chain (MLC) phosphorylation regulate adherens junctions (AJ) and tight junctions (TJ). We determined whether and how Numb modulate the paracellular permeability of intestinal epithelial cells. Caco-2 intestinal epithelial cells and their Numb-interfered counterparts were used in the study for physiological, morphological and biological analyses. Numb, expressed in intestinal epithelial cells and located at the plasma membrane of Caco-2 cells in a basolateral to apical distribution, increased in the intestinal epithelial cells with the formation of the intestinal epithelial barrier. Numb expression decreased and accumulated in the cytoplasm of intestinal epithelial cells in a DSS-induced colitis mouse model. Numb co-localized with E-cadherin, ZO-1 and Par3 at the plasma membrane and interacted with E-cadherin and Par3. Knockdown of Numb in Caco-2 cells altered the F-actin structure during the Ca²⁺ switch assay, enhanced TNFα-/INF-γ-induced intestinal epithelial barrier dysfunction and TJ destruction, and increased the Claudin-2 protein level. Immunofluorescence experiments revealed that NMIIA and F-actin co-localized at the cell surface of Caco-2 cells. Numb knockdown in Caco-2 cells increased F-actin contraction and the abundance of phosphorylated MLC. Numb modulated the intestinal epithelial barrier in a Notch signaling-independent manner. These findings suggest that Numb modulates the paracellular permeability by affecting AJC assembly and MLC phosphorylation.     

A novel role for aquaporin-5 in enhancing microtubule organization and stability

Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function.     

The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio)

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K _u) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²⁺-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.     

The Rac1/JNK pathway is critical for EGFR-dependent barrier formation in human airway epithelial cells

The airway epithelial barrier provides defenses against inhaled antigens and pathogens, and alterations of epithelial barrier function have been proposed to play a significant role in the pathogenesis of chronic airway diseases. Although the epidermal growth factor receptor (EGFR) plays roles in various physiological and pathological processes on the airway epithelium, the role of EGFR on barrier function in the airway remains largely unknown. In the present study, we assessed the effects of EGFR activation on paracellular permeability in airway epithelial cells (AECs). EGFR activation induced by the addition of EGF increased transepithelial electrical resistance (TER) in AECs. An EGFR-blocking antibody eradicated the development of TER, paracellular influx of dextran, and spatial organization of tight junction. Moreover, the effects of EGFR activation on paracellular permeability were eradicated by knockdown of occludin. To identify the EGFR signaling pathway that regulates permeability barrier development, we investigated the effects of several MAP kinase inhibitors on permeability barrier function. Pretreatment with a JNK-specific inhibitor, but not an ERK- or p38-specific inhibitor, attenuated the development of TER induced by EGFR activation. Rac1 is one of the upstream activators for JNK in EGFR signaling. Rac1 knockdown attenuated the phosphorylation of JNK activation and EGFR-mediated TER development. These results suggest that EGFR positively regulates permeability barrier development through the Rac1/JNK-dependent pathway.     

TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia

Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein-tyrosine kinases (PTKs) and their substrates required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls). LPS disrupted barrier integrity in a dose- and time-dependent manner, and prior broad spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens proteins, VE-cadherin, gamma-catenin, and p120(ctn). Two SRC family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE-cadherin and p120(ctn) and paracellular permeability. In HMVEC-Ls, c-SRC, YES, FYN, and LYN were expressed at both mRNA and protein levels. Selective small interfering RNA-induced knockdown of c-SRC, FYN, or YES diminished LPS-induced SRC Tyr(416) phosphorylation, tyrosine phosphorylation of VE-cadherin and p120(ctn), and barrier disruption, whereas knockdown of LYN did not. For VE-cadherin phosphorylation, knockdown of either c-SRC or FYN provided total protection, whereas YES knockdown was only partially protective. For p120(ctn) phosphorylation, knockdown of FYN, c-SRC, or YES each provided comparable but partial protection. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates the SFKs, c-SRC, FYN, and YES, which, in turn, contribute to tyrosine phosphorylation of zonula adherens proteins to open the endothelial paracellular pathway.     

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.     

ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.     

src family kinases regulate renal epithelial paracellular permeability barrier through an occludin‐independent mechanism

Paracellular permeability is mediated by the epithelial cell tight junction. Studies in intestinal and other epithelia have suggested that the activity of src family kinases (SFKs) increases epithelial paracellular permeability through its action on the tight junction protein, occludin, but the involvement of SFKs and occludin in regulation of renal epithelial paracellular permeability is unclear. In this study, the role of SFKs in regulation of renal epithelial paracellular permeability and the involvement of occludin protein in this regulatory event was examined in two renal epithelial cell lines, LLC‐PK₁ (proximal tubule‐like) and MDCK (distal tubule‐like). The effect of broad spectrum SFK inhibitors on paracellular permeability of calcein and fluorescein‐dextran3000 were examined. SFK inhibitor treatment increased paracellular movement of both compounds in both renal epithelial cell lines. The SFK inhibitor effect was concentration‐dependent and, at low concentrations, was not associated with cell damage/death. Response to SFK inhibitors was acquired progressively after cell populations attained confluence suggesting maturation of the regulatory mechanism. Increased paracellular permeability was not associated with dramatic changes in total cell content of occludin protein, its partitioning between detergent‐soluble and ‐insoluble fractions, or its subcellular localization. Further, the SFK‐induced increase in paracellular permeability was unaffected by either occludin protein overexpression or occludin protein knockdown. These results demonstrate that SFK activity decreases paracellular permeability of renal epithelial cells, as opposed to its effect in intestinal epithelial cells, and that this regulation is not mediated by occludin protein. J. Cell. Physiol. 228: 1210–1220, 2013. © 2012 Wiley Periodicals, Inc.     

Regulation of adherens junctions and epithelial paracellular permeability: a novel function for polyamines

Maintenance of intestinal mucosal epithelial integrity requires polyamines that are involved in the multiple signaling pathways controlling gene expression and different epithelial cell functions. Integrity of the intestinal epithelial barrier depends on a complex of proteins composing different intercellular junctions, including tight junctions, adherens junctions, and desmosomes. E-cadherin is primarily found at the adherens junctions and plays a critical role in cell-cell adhesions that are fundamental to formation of the intestinal epithelial barrier. The current study determined whether polyamines regulate intestinal epithelial barrier function by altering E-cadherin expression. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced intracellular free Ca2+ concentration ([Ca2+]cyt), decreased E-cadherin expression, and increased paracellular permeability in normal intestinal epithelial cells (IEC-6 line). Polyamine depletion did not alter expression of tight junction proteins such as zona occludens (ZO)-1, ZO-2, and junctional adhesion molecule (JAM)-1. Addition of exogenous polyamine spermidine reversed the effects of DFMO on [Ca2+]cyt and E-cadherin expression and restored paracellular permeability to near normal. Elevation of [Ca2+]cyt by the Ca2+ ionophore ionomycin increased E-cadherin expression in polyamine-deficient cells. In contrast, reduction of [Ca2+]cyt by polyamine depletion or removal of extracellular Ca2+ not only inhibited expression of E-cadherin mRNA but also decreased the half-life of E-cadherin protein. These results indicate that polyamines regulate intestinal epithelial paracellular barrier function by altering E-cadherin expression and that polyamines are essential for E-cadherin expression at least partially through [Ca2+]cyt.     

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.     

Water channels and their roles in some ocular tissues

Water is a major component of the eye, and water channels (aquaporins) are ubiquitous in ocular tissues, and quite abundant at their different locations. AQP1 is expressed in corneal endothelium, lens epithelium, ciliary epithelium, and retinal pigment epithelium. AQP3 is expressed in corneal epithelium, and in conjunctival epithelium. AQP4 is expressed in ciliary epithelium and retinal Muller cells. AQP5 is expressed in corneal epithelium, and conjunctival epithelium. AQP0 is expressed in lens fiber cells. It is known that five ocular tissues transport fluid, namely: (1) Corneal endothelium; (2) Conjunctival epithelium; (3) Lens epithelium; (4) Ciliary epithelium; (5) Retinal pigment epithelium. For the corneal endothelium, aquaporins are not the main route for trans-tissue water movement, which is paracellular. Instead, we propose that aquaporins allow fast osmotic equilibration of the cell, which is necessary to maintain optimal rates of fluid movement since the cyclic paracellular water transfer mechanism operates separately and tends to create periodic osmotic imbalances (τ～5s).     

Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment

Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function.     

Heregulin activation of ErbB2/ErbB3 signaling potentiates the integrity of airway epithelial barrier

Background

Members of the ErbB family of the receptor protein tyrosine kinase superfamily mediate heregulin (HRG)-induced cell responses. Here we investigated HRG activation of ErbB receptors, and the role of this activation in the development of the permeability barrier in airway epithelial cells (AECs).

Methods

Two airway epithelial-like cell lines, Calu-3 and 16HBE were exposed to HRG or no stimulus and were evaluated with respect to their paracellular permeability as determined by transepithelial electric resistance (TER) and fluorescein isothiocyanate (FITC)-dextran flux. Tight junctions (TJs) were assessed by immunocytochemical localization of occludin and zonula occludens-1.

Results

HRG promoted the development of the permeability barrier and TJ formation by monolayers of Calu-3 and 16HBE cells. Calu-3 cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4, on their surface. ErbB3 knockdown by small interference RNA (siRNA) blunted the effects of HRG on the permeability barrier. ErbB3 is known as a kinase-dead receptor and relies on other members of the family for its phosphorylation. To identify its heterodimerization partner, we knocked down the expression of other ErbB family receptors. We found that HRG's effect on the permeability barrier could be significantly attenuated by transfecting cells with ErbB2 siRNA but not with EGFR siRNA.

Conclusion

These results indicate that HRG activation of ErbB2/ErbB3 heterodimers is essential for regulation of the permeability barrier in AECs.     

Myosin-X functions in polarized epithelial cells

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein-Myo10 localizes to lateral membrane cell-cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.     

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A(2) (PLA(2)) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA(2) activity, AA release, COX-2 expression, prostaglandin E(2) (PGE(2)) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA(2) activity and COX-2 expression and, consequently, a decrease in AA release and PGE(2) synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE(2) to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA(2), COX-2 expression, and subsequent PGE(2) release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.     

Claudin-8 expression in Madin-Darby canine kidney cells augments the paracellular barrier to cation permeation

Claudins are a family of integral membrane proteins of the tight junction that are thought to participate in the permeation of solutes across epithelia via the paracellular pathway. Claudin-8 is expressed in the distal renal tubule, which has a characteristically low passive permeability to monovalent cations. To test the hypothesis that claudin-8 plays a role in forming a tight paracellular barrier to cations, stably transfected Madin-Darby canine kidney II cell lines with inducible expression of claudin-8 were generated. Induction of claudin-8 expression was associated with down-regulation of endogenous claudin-2 protein. Other tight junction proteins were expressed and targeted normally, and the number of junctional strands was minimally altered. By Ussing chamber and radiotracer flux studies, claudin-8 expression was found to reduce paracellular permeability to monovalent inorganic and organic cations and to divalent cations but not to anions or neutral solutes. The size selectivity, charge dependence, and activation energy of paracellular cation permeation were all unchanged. These observations are consistent with a model in which claudin-2 encodes a highly cation-permeable channel, whereas claudin-8 acts primarily as a cation barrier. When exogenous claudin-8 is expressed, it replaces endogenous claudin-2, inserting in its place into existing tight junction strands, thereby reducing the apparent number of functional cation pores. Our findings suggest that claudin-8 plays an important role in the paracellular cation barrier of the distal renal tubule.     

Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation

The deleterious effects of stress on the gastrointestinal tract seem to be mainly mediated by the induction of intestinal barrier dysfunction and subsequent subtle mucosal inflammation. Cannabinoid 1 receptor (CB1R) is expressed in the mammalian gut under physiological circumstances. The aim of this investigation is to study the possible role of CB1R in the maintenance of mucosal homeostasis after stress exposure. CB1R knockout mice (CB1R(-/-)) and their wild-type (WT) counterparts were exposed to immobilization and acoustic (IA) stress for 2 h per day during 4 consecutive days. Colonic protein expression of the inducible forms of the nitric oxide synthase and cyclooxygenase (NOS2 and COX2), IgA production, permeability to (51)Cr-EDTA, and bacterial translocation to mesenteric lymph nodes were evaluated. Stress exposure induced greater expression of proinflammatory enzymes NOS2 and COX2 in colonic mucosa of CB1R(-/-) mice when compared with WT animals. These changes were related with a greater degree of colonic barrier dysfunction in CB1R(-/-) animals determined by 1) a significantly lower IgA secretion, 2) higher paracellular permeability to (51)Cr-EDTA, and 3) higher bacterial translocation, both under basal conditions and after IA stress exposure. Pharmacological antagonism with rimonabant reproduced stress-induced increase of proinflammatory enzymes in the colon described in CB1R(-/-) mice. In conclusion, CB1R exerts a protective role in the colon in vivo through the regulation of intestinal secretion of IgA and paracellular permeability. Pharmacological modulation of cannabinoid system within the gastrointestinal tract might be therapeutically useful in conditions on which intestinal inflammation and barrier dysfunction takes place after exposure to stress.     

Cytoskeletal regulation of Caco-2 intestinal monolayer paracellular permeability

An abnormal increase in intestinal paracellular permeability may be an important pathogenic factor in various intestinal diseases. The intracellular factors and processes that regulate and cause alteration of intestinal paracellular permeability are not well understood. The purpose of this study was to examine some of the intracellular processes involved in cytoskeletal regulation of intestinal epithelial paracellular permeability using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin-b and colchicine were used to disrupt the cytoskeletal elements, actin microfilaments, and microtubules. Cytochalasin-b (5 m?g/ml) and colchicine (2 × 10^?5M) at the doses used caused marked depolymerization and disruption of actin microfilaments and microtubules, respectively. Cytochalasin-b-induced disruption of actin microfilaments resulted in perturbation of tight junctions and desmosomes and an increase in Caco-2 monolayer paracellular permeability. The cytochalasin-b-induced disruption of actin microfilaments and subsequent changes in intercellular junctional complexes and paracellular permeability were not affected by inhibitors of protein synthesis (actinomycin-D or cycloheximide) or microtubule function (colchicine), but were inhibited by metabolic energy inhibitors (2,4-dinitrophenol or sodium azide). The cytochalasin-b-induced disturbance in Caco-2 actin microfilaments and intercellular junctional complexes and increase in paracellular permeability were rapidly reversed. The paracellular pathway “re-tightening” following cytochalasin-b removal was not affected by actinomycin-D, cycloheximide, or colchicine, but was inhibited by 2,4-dinitrophenol and sodium azide. The colchicine-induced disruption of microtubules did not have significant effect on actin microfilaments, intercellular junctions, or paracellular permeability. These findings suggest that cytochalasin-b-induced increase in Caco-2 monolayer paracellular permeability was due to actin microfilament mediated perturbation of intercellular junctional complexes. The re-tightening of paracellular pathways (following removal of cytochalasin-b) resulted from energy-mediated re-assembly of pre-existing actin microfilaments and intercellular junctional complexes. This re-closure process did not require protein synthesis or microtubule-mediated shuttling process. © 1995 Wiley-Liss, Inc.     

A membrane fusion protein αSNAP is a novel regulator of epithelial apical junctions

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins.