Diabetes-Related Changes in Rat Cerebral Occludin and Zonula Occludens-1 (ZO-1) Expression

The endothelial or epithelial tight junctions create a barrier to diffusion of solutes. Since experimental diabetes mellitus is associated with considerable alterations in the blood-brain barrier (BBB), it is possible that specific tight junction proteins may be altered in diabetes. To test this hypothesis, Western and Northern blot analysis were carried out to measure the steady-state level of occludin and zonula occludens-one (ZO-1) proteins and mRNA levels in cerebral tissue of streptozotocin-induced diabetic rats and the results were compared to insulin treated diabetic rats and vehicle injected control rats. The cerebral occludin content in diabetic rats (115.4 ± 18.6 arbitrary units) was significantly reduced compared to insulin-treated diabetic rats (649.1 ± 141.2) or control rats (552.9 ± 82.9), p < 0.001. The ZO-1 content of cerebral tissue from diabetic rats (1240.6 ± 199.7 arbitrary units) was not significantly altered compared to controls (1310.8 ± 256.9). The cerebral occludin mRNA content relative to G3PDH mRNA was 1.35 ± 0.07 and 1.34 ± 0.19 in control and diabetic rats respectively. The cerebral ZO-1 mRNA content relative to G₃PDH mRNA in diabetic and control rats was 1.135 ± 0.123 and 0.956 ± 0.038 respectively. These differences did not achieve statistical significance. It is concluded that diabetes alters the molecular anatomy of the tight junctions in cerebral tissue by altering the content of select structural proteins.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Prenatal exposure to low doses of bisphenol A alters the periductal stroma and glandular cell function in the rat ventral prostate

Environmental estrogens (xenoestrogens) are chemicals that bind to estrogen receptor, mimic estrogenic actions, and may have adverse effects on both human and wildlife health. Bisphenol A (BPA), a monomer used in the manufacture of epoxy resins and polycarbonate has estrogenic activity. In male rodents prenatal exposure to BPA resulted in modifications at the genital tract level. Our objective was to examine the effects of in utero exposure to low, environmentally relevant levels, of the xenoestrogen BPA on proliferation and differentiation of epithelial and stromal cells on the prepubertal rat ventral prostate. To characterize the periductal stromal cells phenotype the expression of vimentin and smooth muscle alpha-actin was evaluated. Androgen receptor (AR) and prostatic acid phosphatase (PAP) expression were also evaluated in epithelial and stromal compartments. Prenatal exposure to BPA increases the fibroblastic:smooth muscle cells ratio and decreases the number of AR-positive cells of periductal stroma of the ventral prostate. In contrast, no differences in AR expression were observed in epithelial cells between control and BPA-treated groups. No changes in proliferation patterns were observed in epithelial and stromal compartments; however, the expression of PAP was diminished in prostate ductal secretory cells of rats in utero exposed to BPA. Our results suggest that prenatal exposure to BPA altered the differentiation pattern of periductal stromal cells of the ventral prostate. These findings are significant in light of the data on human prostate cancers where alterations in the stroma compartment may enhance the invasive and/or malignant potential of the nascent tumor.     

Claudin-18: a dominant tight junction protein in Barrett's esophagus and likely contributor to its acid resistance

Barrett's esophagus (BE) is a specialized columnar epithelium (SCE) that develops as replacement for damaged squamous epithelium (SqE) in subjects with reflux disease, and as such it is apparently more acid resistant than SqE. How SCE resists acid injury is poorly understood; one means may involve altered tight junctions (TJs) since the TJ in SqE is an early target of attack and damage by acid in reflux disease. To assess this possibility, quantitative RT-PCR for 21 claudins was performed on endoscopic biopsies on SCE of BE and from healthy SqE from subjects without esophageal disease. In SCE, Cldn-18 was the most highly expressed at the mRNA level and this finding is paralleled by marked elevation in protein expression on immunoblots. In contrast in SqE, Cldn-18 was minimally expressed at the mRNA level and undetectable at the protein level. Immunofluorescence studies showed membrane localization of Cldn-18 and colocalization with the tight junction protein, zonula occludens-1. When Cldn-18 was overexpressed in MDCK II cells and mounted as monolayers in Ussing chambers, it raised electrical resistance and, as shown by lower dilution potentials to a NaCl gradient and lower diffusion potentials to acidic gradients, selectively reduced paracellular permeability to both Na(+) and H(+) compared with parental MDCK cells. We conclude that Cldn-18 is the dominant claudin in the TJ of SCE and propose that the change from a Cldn-18-deficient TJ in SqE to a Cldn-18-rich TJ in SCE contributes to the greater acid resistance of BE.     

Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function

Alveolar barrier function depends critically on the claudin family tight junction proteins. Of the major claudins expressed by alveolar epithelial cells, claudin (Cldn)-3 and Cldn-4 are the most closely related by amino acid homology, yet they differ dramatically in the pattern of expression. Previously published reports have shown that Cldn-3 is predominantly expressed by type II alveolar epithelial cells; Cldn-4 is expressed throughout the alveolar epithelium and is specifically upregulated in response to acute lung injury. Using primary rat alveolar epithelial cells transduced with yellow fluorescent protein-tagged claudin constructs, we have identified roles for Cldn-3 and Cldn-4 in alveolar epithelial barrier function. Surprisingly, increasing expression of Cldn-3 decreased alveolar epithelial barrier function, as assessed by transepithelial resistance and dye flux measurements. Conversely, increasing Cldn-4 expression improved alveolar epithelial transepithelial resistance compared with control cells. Other alveolar epithelial tight junction proteins were largely unaffected by increased expression of Cldn-3 and Cldn-4. Taken together, these results demonstrate that, in the context of the alveolar epithelium, Cldn-3 and Cldn-4 have different effects on paracellular permeability, despite significant homology in their extracellular loop domains.     

Reformation of gap and tight junctions in regenerating liver after cholestasis

Summary Morphometric analysis of the alterations in interhepatocyte junctions induced by bile duct ligation revealed that after 48 h, during which time the serum bilirubin increased 6 to 8 fold, the membrane area occupied by gap junctions on the apico-lateral and medio-lateral sides decreased from 3.6% in controls to 0.02% in the ligated group. The strands of the zonulae occludentes were reduced in number and showed increased discontinuities.Within 45 min of recanalization of the common bile duct, clusters of particles appeared within and adjacent to the tight junctional areas or in the lateral hepatocyte membrane. Subsequently, the particle aggregations localized in the apico-lateral membrane areas increased in number and size becoming finally indistinguishable from those of controls within 96 h after the onset of recanalization. The zonulae occludentes also rearranged and reestablished their original structure during this period. The serum bilirubin fell to normal within 24 h of recanalization. It is concluded that metabolic and ultrastructural restitution associated with the recanalization of the ligated bile duct have no strict temporal correlation to one another.These studies provide further evidence that alterations in gap and tight junctions induced by pathological processes, e.g. during bile duct ligation, are completely reversible when regeneration occurs.Summer student from Harvard Medical School, Boston (USA)     

Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees

Adhesive intercellular junctions between endothelial cells are formed by tight junctions and adherens junctions. In addition to promoting cell-to-cell adhesion, these structures regulate paracellular permeability, contact inhibition of endothelial cell growth, cell survival, and maintenance of cell polarity. Furthermore, adherens junctions are required for the correct organization of new vessels during embryo development or during tissue proliferation in the adult. Extensive research on cultured epithelial and endothelial cells has resulted in the identification of many molecular components of tight junctions and adherens junctions. Such studies have revealed the complexity of these structures, which are formed by membrane-associated adhesion proteins and a network of several intracellular signaling partners. This review focuses on the structural organization of junctional structures and their functional interactions in the endothelium of blood vessels and lymphatics. We emphasize the way that these structures regulate endothelial cell homeostasis by transferring specific intracellular signals and by modulating activation and signaling of growth factor receptors. This work was supported by the Associazione Italiana per la Ricerca sul Cancro, Association for International Cancer Research, European Community (Integrated Project Contract no. LSHG-CT-2004–503573; NoE MAIN 502935; NoE EVGN 503254; EUSTROKE consortium; Angioscaff consortium; Optistem consortium), Istituto Superiore di Sanità, Italian Ministry of Health, MIUR (COFIN prot: 2006058482_002), and Fondation Leducq Transatlantic Network of Excellence (E.D.). Additional support came from US National Institutes of Health grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute and AngelWorks Foundation (D.McD.).     

Gene expression profiling and its relevance to the blood-epididymal barrier in the human epididymis

The luminal environment along the epididymal duct is important for spermatozoal maturation. This environment is unique and created by the blood-epididymal barrier, which is formed by tight and adhering junctions. For the human epididymis, little information exists on the proteins that comprise these junctions. Our objectives were to assess the gene expression profiles in the different segments of the human epididymis and to identify the proteins that make up the blood-epididymal barrier. Using microarrays, we identified 2980 genes that were differentially expressed by at least 2-fold between the various segments. Of the many genes involved in diverse functions, were those that encoded adhesion proteins (cadherins and catenins) and tight junctional proteins (claudins [CLDN] and others). PCR analyses confirmed the microarray data. Immunolocalization of CLDNs 1, 3, 4, 8, and 10 revealed that the localization of CLDNs differed along the epididymis. In all three segments, CLDNs 1, 3, and 4 were localized to tight junctions, along the lateral margins of adjacent principal cells, and at the interface between basal and principal cells. CLDN8 was localized to tight junctions in all three segments, in addition to being localized in the caput along the lateral margins of principal cells, and in the corpus, at the interface between principal and basal cells. CLDN10, tight junction protein 1, and occludin were localized exclusively to tight junctions in all three epididymal segments. These data indicate that the epididymis displays a complex pattern of gene expression, which includes genes that are implicated in the formation of the blood-epididymal barrier, which suggests complex regulation of this barrier.     

Cerebral ischemia enhances tyrosine phosphorylation of occludin in brain capillaries

Cerebral ischemia induces disruption of the blood-brain barrier (BBB), and this disruption can initiate the development of brain injuries. Although the molecular structure of tight junctional complexes in the BBB has been identified, little is known about alterations of tight junctional proteins after cerebral ischemia. Therefore, we investigated alterations of tight junctional proteins, i.e., occludin and zonula occludens (ZO)-1, in isolated rat brain capillaries after microsphere-induced cerebral embolism. We demonstrated that the levels of occludin and ZO-1 had decreased after the embolism. The embolism also resulted in a marked increase in tyrosine phosphorylation of occludin, which was coincident with an increase in the activity of c-Src. These results suggest that a decrease in the levels of occludin and ZO-1, and an increase in tyrosine phosphorylation of occludin may play an important role in the disruption of tight junctions, which may lead to dysfunction of the BBB after cerebral ischemia.     

Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.     

Poly(ADP-ribose) polymerase cleavage during apoptosis: when and where?

On freeze-fracture replicas, gap junctions are frequently colocalized with tight junctions. In this study, to elucidate the relationship between gap- and tight-junction proteins, we investigated the localization of gap-junction proteins Cx32 and Cx26 and tight-junction proteins occludin, claudin-1, ZO-1, and ZO-2 in primary cultured rat hepatocytes, using confocal laser microscopy. In hepatocytes cultured in 2% DMSO and 10(-7) M glucagon medium, Cx32- but not Cx26-immunoreactive lines were observed on the most subapical plasma membrane at cell borders, while on the basolateral membrane both Cx32- and Cx26-positive spots were colocalized. Occludin-, claudin-1-, ZO-1-, and ZO-2-immunoreactive lines were also linearly observed on the most subapical plasma membrane and were colocalized with only Cx32-immunoreactive lines. In freeze-fracture analysis, many small gap-junction plaques were observed within a well-developed tight-junction strand network. The fence function of tight junctions in the cells, as examined by diffusion of labeled sphingomyelin, was well maintained. We also carried out Western blotting for Cx32 following immunoprecipitation with anti-occludin, anti-claudin-1, or anti-ZO-1 antibodies. Cx32 was detectable in all immunoprecipitates. These results suggest that Cx32 gap junctions, but not those with Cx26, are closely coordinated with the expression and function of tight junctions in hepatocytes and that Cx32 gap-junction formation may affect cell polarity through modification of tight-junction expression.     

Alterations in intercellular junctions of the uterine epithelium during the preimplantation phase in the rabbit

Summary In the rabbit, the pseudopregnant uterus has been used as a model for studying alterations characteristic of the preimplantation phase. Alterations in intercellular junctions of the uterine epithelium were investigated during early pseudopregnancy (day 0 to day 6) by means of the freeze-fracture technique.In the uterine epithelium of oestrous females the zonula occludens belongs to the tight type of tight junctions. During pseudopregnancy an impressive proliferation of tight junctional belts can be observed. The basal strands proliferate, forming loops perpendicular to the luminal surface, whereas the more or less parallel arrangement of the luminal strands is maintained. At day 4 of pseudopregnancy macular tight junctions begin to develop on the lower portions of the lateral plasmalemma and are extensive by day 6 post hCG.Small gap junctions are infrequent between cells of the uterine epithelium and show no significant changes during the preimplantation phase.The physiological significance of the present morphological observations is discussed in the light of changes occurring during the preimplantation period.Supported by grant Kü 210/9 from the Deutsche Forschungsgemeinschaft     

Adult rats prenatally exposed to ethanol have increased gluconeogenesis and impaired insulin response of hepatic gluconeogenic genes.

Rat offspring exposed to ethanol (EtOH rats) during pregnancy are insulin resistant, but it is unknown whether they have increased gluconeogenesis. To address this issue, we determined blood glucose and liver gluconeogenic genes, proteins, and enzyme activities before and after insulin administration in juvenile and adult EtOH rats and submitted adult EtOH rats to a pyruvate challenge. In juvenile rats, basal glucose; peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA; and phosphoenolpyruvate carboxykinase enzyme activity, protein, and mRNA were similar between groups. After insulin injection, these parameters failed to decrease in EtOH rats, but glucose decreased by 30% and gluconeogenic enzymes, proteins, and mRNAs decreased by 50-70% in control rats. In adult offspring, basal peroxisome proliferator-activated receptor-coactivator-1alpha protein and mRNA levels were 40-80% higher in EtOH rats than in controls. Similarly, basal phosphoenolpyruvate carboxykinase activity, protein, and mRNA were approximately 1.8-fold greater in EtOH rats than in controls. These parameters decreased by approximately 50% after insulin injection in control rats, but they remained unchanged in EtOH rats. After insulin injection in the adult rats, glucose decreased by 60% in controls but did not decrease significantly in EtOH rats. A subset of adult EtOH rats had fasting hyperglycemia and an exaggerated glycemic response to pyruvate compared with controls. The data indicate that, after prenatal EtOH exposure, the expression of gluconeogenic genes is exaggerated in adult rat offspring and is insulin resistant in both juvenile and adult rats, explaining increased gluconeogenesis. These alterations persist through adulthood and may contribute to the pathogenesis of Type 2 diabetes after exposure to EtOH in utero.     

Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Microinjection of fluorophore-tagged cytoskeletal proteins has been a useful tool in studies of formation of focal adhesions (FA). We used this method to study the maintenance of adherens junctions (AJ) and tight junctions (TJ) of epithelial Madin-Darby bovine kidney cells. We chose alpha-actinin and vinculin as markers, because they are present both at adherens junctions and focal adhesions and their binding partners have been well characterized. Isolated FITC-labelled chicken alpha-actinin and vinculin were injected into confluent cells where they were rapidly incorporated both in FAs and AJs. The FAs remained unchanged, whereas cell-cell contacts began to fade within an hour after injection and the cells were joined to polykaryons having 5 to 13 nuclei. Short fragments of cell membranes containing injected proteins, actin, beta-catenin, cadherin, claudin, occludin and ZO-1 were visible inside the polykaryons indicating that both AJs and TJs were disintegrated as a single complex. Microinjected FITC-labelled vinculin head domain was also incorporated to both AJs and FAs, but instead of fusions it rapidly induced the detachment of the cells from the substratum probably due to high affinity of vinculin head to talin. Vinculin tail domain had no apparent effect on the cell morphology. Since small GTPases are involved in the building up of AJs, we injected active and inactive forms of cdc42 and rac proteins together with vinculin to see their effect. Active forms reduced the formation of polykaryons presumably by strengthening AJs, whereas inactive forms had no apparent effect. We suggest that excess alpha-actinin and vinculin uncouple the cell-cell adhesion junctions from the intracellular cytoskeleton which leads to fragmentation of junctional complexes and subsequent cell fusion. The results show that cell-cell adhesion sites are more dynamic and more sensitive than FAs to an imbalance in the amount of free alpha-actinin and intact vinculin.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.