Role of iNOS in hepatocyte tight junction alteration in mouse model of experimental colitis.

A variety of hepatobiliary abnormalities occur in inflammatory bowel diseases (IBDs). The role of tight junction (TJ) in hepatobiliary complications have been well described. The purpose of this study was to investigate the role of inducible nitric oxide (NOS) in alteration of hepatocyte TJ paracellular barrier and in the rapid transcytotic vesicular pathway modification associated with intestinal inflammation. To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated iNOS wild-type (WT) mice, DNBS-treated iNOS knock out mice (iNOSKO) mice experienced a significant less rate of the extent and severity of the histological signs of colon injury. Colon levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta and interleukin-6 were also significantly reduced in iNOS-KO mice in comparison to wild-type mice. Liver histology from iNOSKO and wild-type mice iNOSWT did not show any parenchymal and portal tract inflammation at 4 days after DNBS administration. Serum total bilirubin and alanine aminotransferase, were significantly reduced in DNBS-iNOSKO mice vs DNBS-iNOSKO mice. Therefore, we found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the livers from DNBS-treated IL-10WT mice, lanthanum accumulated throughout the junctional area up to the most apical region bordering the lumen. Absence of a functional iNOS gene in iNOSKO mice resulted in a significant reduction of apical diffusion of lanthanum after DNBS-induced colitis. Immunofluorescent labeling of frozen liver sections from DNBS-iNOSWT mice showed a significant alteration of the immunolocalization for claudin-1 and zonula occludens (ZO)-1. In contrast, a significant reduced alteration in the localization of the immunosignals for claudin-1 and ZO-1 was observed in the liver from iNOSKO mice after DNBS administration. In conclusion, we suggest that the iNOS may represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in patients with IBD.     

Role of IL-10 in hepatocyte tight junction alteration in mouse model of experimental colitis

BACKGROUND: A variety of hepatobiliary abnormalities have been described in patients with chronic inflammatory bowel diseases (IBDs). The purpose of this study was to investigate the role of endogenous IL-10 in alteration of hepatocyte TJ paracellular barrier and in the rapid transcytotic vesicular pathway modification associated with intestinal inflammation. MATERIALS AND METHODS: To address this question, we used an experimental model of colitis, induced by dinitrobenzene sulfonic acid (DNBS). When compared to DNBS-treated IL-10 wild-type (IL-10WT) mice, DNBS-treated IL-10 knock-out mice (IL-10KO) mice experienced a higher rate of the extent and severity of the histological signs of colon injury. RESULTS: Colon and liver levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1 beta and interleukin-6 were also greatly enhanced in IL-10KO mice in comparison to wild-type mice. Liver histology from IL-10KO and IL-10WT did not show any parenchymal and portal tract inflammation at 4 days after DNBS administration. Serum total bilirubin and Alanine aminotransferase, were significantly increased in DNBS-IL-10KO mice vs. DNBS-IL-10KO mice. Therefore, we found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the livers from DNBS-treated IL-10WT mice; lanthanum accumulated throughout the junctional area up to the most apical region bordering the lumen. Absence of a functional IL-10 gene in IL-10KO mice resulted in a significant augmentation of apical diffusion of lanthanum after DNBS-induced colitis. Immunofluorescent labelling of frozen liver sections from DNBS-IL10KO mice, immunolocalization for and claudin-1 and ZO-1 resulted in a significant alteration in the localization of the immunosignals for claudin-1 and ZO-1 after DNBS administration in comparison with DNBS-IL10WT. CONCLUSION: In conclusion, we suggest that the absence of IL-10 may represent an important pathophysiological mechanism of hepatobiliary injuries and cholestasis observed in patients with IBD.     

Role of flavonoids in intestinal tight junction regulation

The gastrointestinal tract provides a physical barrier to the diffusion of foreign materials from the lumen into the circulatory system. Impairment of the intercellular tight junction (TJ) shield, which is the major determinant of intestinal barrier function, is associated with various diseases. Dietary flavonoids demonstrate various beneficial effects on our health; however, the information regarding their effects on TJ function is quite limited. To date, four flavonoids — epigallocatechin gallate (EGCG), genistein, myricetin and quercetin — have been reported to exhibit promotive and protective effects on intestinal TJ barrier functions. Genistein, a major soybean isoflavone, protects TJ barrier function against oxidative stress, acetaldehyde, enteric bacteria and inflammatory cytokines. Genistein blocks the tyrosine phosphorylation of the TJ proteins induced by oxidative stress and acetaldehyde, which results in the disassembly of the proteins from the junctional complex. Quercetin, a flavonol, enhances intestinal TJ barrier function through the assembly and expression of TJ proteins. The change in phosphorylation status is responsible for the quercetin-mediated assembly of TJ proteins. TJ protein induction has an additional role in this effect. This review presents the recent advances in our understanding of the flavonoid-mediated promotive and protective effects on intestinal TJ barrier function with a particular focus on intracellular molecular mechanisms.     

缝隙连接在小鼠卵泡发育过程中的作用

缝隙连接广泛分布于各组织细胞中,由其构成的胞间通道允许小分子在胞间直接传递,在胞间通讯方面起着重要的作用。缝隙连接由连接蛋白(Cx)组成,已发现Cx家族有20多个成员。在哺乳动物卵泡发育过程中,卵母细胞与周围的颗粒细胞之间形成的缝隙连接,介导胞间通讯,对原始生殖细胞迁移、卵母细胞减数分裂能力恢复、颗粒细胞分层、卵泡成腔、黄体形成、促性腺激素信号传递均有非常重要的调节作用。本文根据近年来相关的研究报道,总结了不同的缝隙连接在小鼠卵泡发育过程中的调节作用。     

Phospholipase C-gamma modulates epithelial tight junction permeability through hyperphosphorylation of tight junction proteins

Phospholipase C-gamma (PLC-gamma) is stimulated by epidermal growth factor via activation of the epidermal growth factor receptors. The PLC inhibitor, 3-nitrocoumarin (3-NC), selectively inhibited PLC-gamma in Madin-Darby canine kidney cells without affecting the activity of PLC-beta. In contrast, inhibitors of PLC-beta, hexadecylphosphocholine and, had no effect on the activity of PLC-gamma. Inhibition of PLC-gamma by 3-NC was associated with an increase in tight junction permeability across Madin-Darby canine kidney cell monolayers, as evidenced by 3-NC-induced decrease in transepithelial electrical resistance and increase in mannitol flux over a concentration range that was inhibitory to PLC-gamma. An analog of 3-NC, 7-hydroxy-3-NC (7-OH-3-NC), which was inactive as an inhibitor of PLC-gamma, also had no effect on tight junction permeability. Treatment with 3-NC caused punctate disruption in the cortical actin filaments. The PLC-gamma inhibitor, 3-NC, but not the inactive analog, 7-OH-3-NC, caused hyperphosphorylation of the tight junction proteins, occludin, ZO-1, and ZO-2. The serine/threonine kinase inhibitor, staurosporine (50-200 nm), significantly attenuated 3-NC-induced hyperphosphorylation of ZO-2. This corresponded with attenuation by staurosporine of 3-NC-induced increase in tight junction permeability, suggesting a relationship between ZO-2 phosphorylation and tight junction permeability.     

Androgen initiates Sertoli cell tight junction formation in the hypogonadal (hpg) mouse

Sertoli cell tight junctions (TJs) form at puberty as a major component of the blood-testis barrier (BTB), which is essential for spermatogenesis. This study characterized the hormonal induction of functional Sertoli cell TJ formation in vivo using the gonadotropin-deficient hypogonadal (hpg) mouse that displays prepubertal spermatogenic arrest. Androgen actions were determined in hpg mice treated for 2 or 10 days with dihydrotestosterone (DHT). Follicle-stimulating hormone (FSH) actions were studied in hpg mice expressing transgenic human FSH (hpg+tgFSH) with or without DHT treatment. TJ formation was examined by mRNA expression and immunolocalization of TJ proteins claudin-3 and claudin-11, and barrier functionality was examined by biotin tracer permeability. Immunolocalization of claudin-3 and claudin-11 was extensive at wild-type (wt) Sertoli cell TJs, which functionally excluded permeability tracer. In contrast, seminiferous tubules of hpg testes lacked claudin-3, but claudin-11 protein was present in adluminal regions of Sertoli cells. Biotin tracer permeated throughout these tubules, demonstrating dysfunctional TJs. In hpg+tgFSH testes, claudin-3 was generally absent, but claudin-11 had redistributed basally toward the TJs, where function was variable. In hpg testes, DHT treatment stimulated the redistribution of claudin-11 protein toward the basal region of Sertoli cells by Day 2, increased Cldn3 and Cldn11 mRNA expression, then induced the formation of functional TJs containing both proteins by Day 10. In hpg+tgFSH testes, TJ protein redistribution was accelerated and functional TJs formed by Day 2 of DHT treatment. We conclude that androgen stimulates initial Sertoli cell TJ formation and function in mice, whereas FSH activity is insufficient alone, but augments androgen-induced TJ function.     

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.     

Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity

Oxidative stress compromises the tight junction, but the mechanisms underlying its recovery remain unclear. We developed a model in which oxidative stress reversibly disrupts the tight junction. Exposure of Madin-Darby canine kidney cells to hydrogen peroxide markedly reduced transepithelial resistance and disrupted the staining patterns of the tight junction proteins ZO-1 and occludin. These changes were reversed by catalase. The short-term reassembly of tight junctions was not dependent on new protein synthesis, suggesting that recovery occurs through re-utilization of existing proteins. Although ATP levels were reduced, the reduction was insufficient to explain the observed changes, since a comparable reduction of ATP levels (with 2-deoxy-D-glucose) did not induce these changes. The intracellular hydrogen peroxide scavenger pyruvate protected Madin-Darby canine kidney cells from loss of transepithelial resistance as did the heavy metal scavenger N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine. Of a wide variety of agents examined, only tyrosine kinase inhibitors and protein kinase C inhibitors markedly inhibited tight junction reassembly. During reassembly, tyrosine phosphorylation in or near the lateral membrane, was detected by immunofluorescence. The tyrosine kinase inhibitors genistein and PP-2 inhibited the recovery of transepithelial resistance and perturbed the relocalization of ZO-1 and occludin to the tight junction, indicating that tyrosine kinases, possibly members of the Src family, are critical for reassembly after oxidative stress.     

Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch

Epithelial permeability is tightly regulated by intracellular messengers. Critical to maintaining barrier integrity is the formation of tight junction complexes. A number of signaling pathways have been implicated in tight junction biogenesis; however, the precise molecular mechanisms are not fully understood. A growing body of evidence suggests a role for intracellular cAMP in tight junction assembly. Using an epithelial model, we investigated the role of cAMP signal transduction in barrier recovery after Ca2+ switch. Our data demonstrate that elevation of intracellular cAMP levels significantly enhanced barrier recovery after Ca2+ switch. Parallel experiments revealed that epithelial barrier recovery is diminished by H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (protein kinase A) activity. Of the possible PKA effector proteins, the vasodilator-stimulated phosphoprotein (VASP) is an attractive candidate, since it has been implicated in actin-binding and cross-linking functions. We therefore hypothesized that VASP may play a role in the cAMP-mediated regulation of epithelial junctional reassembly after Ca2+ switch. We demonstrate here that VASP is phosphorylated via a PKA-dependent process under conditions that enhance barrier recovery. Confocal laser scanning microscopy studies revealed that VASP localizes with ZO-1 at the tight junction and at cell-cell borders and that phospho-VASP appears at the junction after Ca2+ switch. Subsequent transfection studies utilizing epithelial cells expressing truncated forms of VASP abnormal in oligomerization or actin-binding activity revealed a functional diminution of barrier recovery after Ca2+ chelation. Our present studies suggest that VASP may provide a link between cAMP signal transduction and epithelial permeability.     

Role of TNF-alpha in prenatal alterations in dams of mice under thermal stress

The possible involvement of cytokines such as tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that are suspected of causing pregnancy loss and miscarriage has been investigated in dams of mice subjected to hyperthermia. Thermal stress was induced by exposing mice dams at 40+/-2 degrees C for 4 h every day during the different phases of the gestation period whereas the normothermic animals were housed at 22+/-2 degrees C. The effect of maternal thermal stress was measured in pregnant mice at different phases of the gestation period namely, blastogenesis-implantation phase (days 0-5 postconceptionem [p.c.]), organogenesis or embryogenesis phase (days 6-15 p.c.) and fetogenesis phase (days 16-20 p.c.). Uterine examination of dams subjected to hyperthermia on days 6-15 p.c. showed maximum reduction in live fetus number, gestational index and maximum pre and postimplantation loss in comparison with dams housed in normothermic environment and dams exposed to thermal stress between days 0-5 and 16-20 p.c. Maximum resorption rate and number of non-viable fetuses were observed in dams exposed to hyperthermia during days 6-15 p.c. Elevated levels of TNF-alpha and IL-1 beta were observed in the amniotic fluid of dams subjected to hyperthermia during days 6-15 p.c. but IFN-gamma levels remained unaltered. Single intraperitoneal (i.p.) administration of recombinant mouse TNF-alpha at a dose of 1 and 0.5 ng/mice in dams on day 6 in normothermic condition resulted in a reduced number of live fetuses. Administration of anti-TNF-alpha antibody i.p. at a dose of 10 microg/dam on day 6 p.c. and subjected to thermal stress between days 6-15 p.c. increased marginally the number of fetuses but failed to attain statistical significance in comparison with days 6-15 p.c. thermally stressed dams without antibody treatment. It is concluded that the induction of TNF-alpha, in the amniotic fluid is associated with thermal stress during pregnancy and may be linked to the reproductive performances of dams. This study will help in understanding the mechanism of thermal injury in pregnant subjects.     

Role of free radicals and poly(ADP-ribose) synthetase in intestinal tight junction permeability

BACKGROUND: Small intestine permeability is frequently altered in inflammatory bowel disease and may be caused by the translocation of intestinal toxins through leaky small intestine tight junctions (TJ) and adherence (1,2). The role of hydrogen peroxide (H2O2), and nitric oxide (NO) and PARS in the permeability and structure of small intestine TJ is not clearly understood. MATERIALS AND METHODS: In vitro study, MDCK (Madin-Darby Canine Kidney) cells were exposed to H2O2 (100 microM for 2h), or zymosan (200 microl of stock solution 1 mg/ml for 4h), in the presence or absence of a treatment with poly(ADP-ribose) synthetase (PARS) inhibitor 3-aminobenzamide (3-AB: 3 mM) or with n-acetylcysteine (NAC 10 mM). In vivo study, wild-type mice (WT) and mice lacking (KO) of the inducible (or type 2) nitric oxide synthase (iNOS) were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In addition INOSWT mice were treated with 3-AB (10 mg/kg, i.p.) or with NAC (40 mg/kg, i.p.) 1 hour and 6 h after zymosan administration. RESULTS: Exposure of MDCK cells to hydrogen peroxide caused a significant impairment in mitochondrial respiration that was associated with a reduction of cells adherence as well as derangement of the junctional proteins. A significant increase of nitrate and nitrite levels, stable metabolites of nitric oxide (NO), were found in MDCK supernatant after zymosan incubation. NO production was associated with a significant reduction of cell adherence and impairment of occludin protein. Pre-treatment of the cells with 3-AB or with NAC caused a significant prevention of H2O2-mediated occludin junctional damage as well as reduced the NO-induced occludin damage. In addition, H2O2 and NO are able to induce a significant derangement of beta-catenin and Zonula Ocludence-1 (ZO-1). We found an increase of tight junctional permeability to lanthanum nitrate (molecular weight, 433) in the terminal ileal TJs in zymosan-treated iNOSWT mice compared with permeable TJ in the control animals. Zymosan-treated iNOSKO mice showed a significant increase of tight junctional permselectivity. There were no differences in strand count or strand depth in the ilea from control or treated animals. In addition, a significant disrupted immunofluorescence signal for occludin, ZO-1 and beta-catenin was observed in the terminal ilea of zymosan-treated iNOSWT mice. In ileal fragments from zymosan-treated iNOSKO mice, we found less irregular distribution patterns of occludin, ZO-1 and beta-catenin. Similarly NAC or 3-AB treatments were able to prevent zymosan-induced damage of junctional proteins in iNOSWT mice. CONCLUSION: In conclusion, this study demonstrates that the alteration of permselectivity is most likely induced by ROS and PARS activation.     

Claudins--key pieces in the tight junction puzzle

Tight junctions restrict the flow of ions and aqueous molecules between cells by forming a selective barrier to the paracellular pathway. Permeability of the tight junction barrier is determined by a class of transmembrane proteins known as claudins. The relationship between claudins and paracellular permeability is complex and determined not only by the profile of claudin expression but also by the arrangement of claudins and other proteins into tight junction strands. This review summarizes progress in understanding how claudins are assembled into tight junctions and how they interact with other tight junction proteins.     

Role of peripheral and central opioid activity in analgesia induced by restraint stress

Rats subjected to prolonged restraint showed an increase in tail flick latency which outlasted the period of restraint by 15 min. This restraint could be blocked but not reversed by 1 mg/kg of naltrexone hydrochloride given subcutaneously. Naltrexone methobromide, administered subcutaneously in doses of 10 or 25 mg/kg, did not block the analgesia indicating that peripheral opioid receptors were probably not involved. Naltrexone hydrochloride was shown to have no effect on brain tryptophan uptake in restrained rats, a neurochemical event which had previously been shown to be critical to restraint-induced analgesia.     

Role of occludin, a tight junction protein, in blastocoel formation, and in the paracellular permeability and differentiation of trophectoderm in preimplantation mouse embryos

Tight junctions (TJ) are critical for blastocoel formation in mammalian embryos. The present study aimed to examine the role of tight junctions in the differentiation of the trophectoderm (TE), and in the pluripotency of blastomeres, as well as in the formation and integrity of the blastocoel. We examined the effect of occludin antibody on blastocoel formation, blastocyst permeability, and expression of H19 and Oct-4, markers of TE differentiation and blastomere pluripotency, respectively. Eight-cell mouse embryos and morulae were cultured in the presence or absence of occludin antibody for 31 h. Occludin antibody inhibited blastocoel formation and increased permeability of the TE of nascent and expanding blastocysts to FITC-dextran (4 kDa), a permeability tracer. At the same time Oct-4 expression increased while expression of H19 became barely detectable. These observations indicate that occludin is involved in establishing the blastocoel, as well as in maintaining its impermeability, and that the development of tight junction is critical for TE formation in mouse embryos.     

A model for flow through discontinuities in the tight junction of the endothelial intercellular cleft

A mathematical model for steady flow through a discontinuity in the tight junction of an endothelial intercellular cleft is presented. Subject to plausible assumptions the problem of calculating the flow in the cleft, in either the presence or the absence of a fibre matrix, reduces to the solution of Laplace's equation in a two-dimensional domain. For an idealized geometry representing a discontinuity between two semi-infinite tight junction regions, a general analytic solution is found by means of conformal mappings. The model geometry, unlike those assumed in previous studies, allows the tight junction regions to be out of alignment with each other, and even to overlap, modelling flow through a tortuous, rather than a direct, pathway. Useful asymptotic approximations for the flow rate are derived when the discontinuity is either very small or very large. For small discontinuities, the predicted flow rate is much greater than a naïve estimate based on uniform parallel flow through the discontinuity. For the special case where the tight junction regions are aligned with each other, comparison of our results with those of an approximate treatment due to Tsayet al. [Chem. Engng Commun. 82, 67–102 (1989)] shows generally very close agreement.     

Cystine reduces tight junction permeability and intestinal inflammation induced by oxidative stress in Caco-2 cells

Intestinal oxidative stress produces pro-inflammatory cytokines, which increase tight junction (TJ) permeability, leading to intestinal and systemic inflammation. Cystine (Cys2) is a substrate of glutathione (GSH) and inhibits inflammation, however, it is unclear whether Cys2 locally improves intestinal barrier dysfunction. Thus, we investigated the local effects of Cys2 on oxidative stress-induced TJ permeability and intestinal inflammatory responses. Caco-2 cells were cultured in a Cys2-supplemented medium for 24 h and then treated with H₂O₂ for 2 h. We assessed TJ permeability by measuring transepithelial electrical resistance and the paracellular flux of fluorescein isothiocyanate–dextran 4 kDa. We measured the concentration of Cys2 and GSH after Cys2 pretreatment. The mRNA expression of pro-inflammatory cytokines was assessed. In addition, the levels of TJ proteins were assessed by measuring the expression of TJ proteins in the whole cells and the ratio of TJ proteins in the detergent-insoluble fractions to soluble fractions (IS/S ratio). Cys2 treatment reduced H₂O₂-induced TJ permeability. Cys2 did not change the expression of TJ proteins in the whole cells, however, suppressed the IS/S ratio of claudin-4. Intercellular levels of Cys2 and GSH significantly increased in cells treated with Cys2. Cys2 treatment suppressed the mRNA expression of pro-inflammatory cytokines, and the mRNA levels were significantly correlated with TJ permeability. In conclusion, Cys2 treatment locally reduced oxidative stress-induced intestinal barrier dysfunction possively due to the mitigation of claudin-4 dislocalization. Furthermore, the effect of Cys2 on the improvement of intestinal barrier function is related to the local suppression of oxidative stress-induced pro-inflammatory responses.