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Backgrounds & Aims Increased permeability is implicated in the pathogenesis of intestinal disease. In vitro and in vivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested in vivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. Methods Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial–specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. Results ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1–deficient colonic epithelia failed to up-regulate proliferation in response to damage in vivo or Wnt signaling in vitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. Conclusions ZO-1 makes critical, tight junction–independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

BackgroundKawasaki disease (KD) is a form of systemic febrile vasculitis that is complicated with coronary artery lesions (CAL). The tight junctions that maintain the intestinal barrier also play a role in systemic inflammatory diseases. Serum zonula occludens-1 (ZO-1) expression was found to be significantly lower in asthmatic patients, and another study reported that elevated systemic ZO-1 was positively correlated with inflammation in cirrhotic patients. A murine model of KD vasculitis demonstrated that vasculitis depended on intestinal barrier dysfunction, which is maintained by tight junctions. In this study, we aimed to investigate the role of the tight junction zonula occludens-1 (ZO-1) in the treatment response of intravenous immunoglobulin (IVIG) and the occurrence of CAL formation in KD patients.MethodsWe enrolled 40 KD patients, 12 healthy controls, and 12 febrile controls in this study. The serum levels of tight junction ZO-1 were determined by enzyme-linked immunosorbent assay.ResultsThe serum ZO-1 level was higher in the fever control group but did not reach a statistical significance. KD patients who received a second dose of IVIG treatment due to initial IVIG unresponsiveness had a higher serum levels of tight junction ZO-1, but without statistical significance (2.15 ± 0.18 vs. 2.69 ± 0.31 ng/mL,p = 0.058). KD patients who developed a CAL demonstrated a significant lower serum tight junction ZO-1 levels than KD without CAL formation (1.89 ± 0.16 vs. 2.39 ± 0.15 ng/mL,p = 0.027). After multiple logistic regression analysis, ZO-1 levels [(95% confidence interval (CI): 0.058 ~ 0.941, odds ratio (OR) = 0.235,p = 0.041)] showed as the risk factor for CAL formation.ConclusionSerum levels of tight junction ZO-1 levels were lower in KD patients than fever controls and associated with CAL formation.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a common chronic functional gastrointestinal disorder. MicroRNAs (miRNAs) have been identified to be involved in different physiological and pathological processes. In this study, the role of miRNA-29a in the potential mechanism underlying the function of the intestinal mucosal barrier in IBS-D was analyzed. Human intestinal mucosal epithelia from patients with IBS-D (diagnosed as meeting the Rome IV criteria) and healthy volunteers were collected. An IBS-D mouse model was established via induction with trinitro-benzene-sulfonic acid (TNBS), and the mice were injected with miRNA-29a inhibitor. Using transmission electron microscopy (TEM), the epithelial ultrastructure of the human intestinal mucosa was examined. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, the expression level of miRNA-29a was assessed. ELISA was used to analyze the activity of D-lactate (D-LA) and diamine oxidase (DAO). Through immunohistochemistry, RT-qPCR and western blotting, the expression of tight junction protein ZO-1 (ZO-1) and claudin-1 (CLDN1) was examined. In the human intestinal mucosal epithelia from patients with IBS-D, miRNA-29a was upregulated, ZO-1 and CLDN1 were downregulated, and the junctional complex (JC) was faint and discontinuous. In the IBS-D mouse model, treatment with miRNA-29a inhibitor downregulated D-LA and DAO activity, and increased the expression of ZO-1 and CLDN1 in the intestinal mucosal epithelium. In conclusion, the present study revealed that miRNA-29a is involved in the pathogenesis of IBS-D, probably by downregulating ZO-1 and CLDN1 expression, suggesting that miRNA-29a is likely to be an important regulator of intestinal barrier function and could be a possible therapeutic target for IBS-D.

SummaryAlthough the physiological importance of biomolecular condensates is widely recognized, how it is controlled in time and space during development is largely unknown. Here we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows and internal pressure on TE cells increases. In contrast, this dissolution was less evident in TE cells attached to the inner cell mass, as they receive weaker tensile forces. Furthermore, analyses using MDCK cells have demonstrated that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via an interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to establishing robust cell-cell adhesion during early development.

Tight junction proteins are critical in maintaining homeostatic intestinal permeability. Multiple intestinal inflammatory diseases are correlated with reduced expression of tight junction proteins. We have recently reported that oral treatment of mice with Hyaluronan 35 kDa (HA35) increases colonic expression of tight junction protein zonula occludens-1 (ZO-1). Here, we investigate whether HA35 treatment enhances ZO-1 expression by direct interaction with intestinal epithelium in vitro and have identified the HA receptor responsible for HA35-mediated ZO-1 induction in colonic epithelium in vitro and in vivo. Our results reveal that HA35 treatment increases ZO-1 expression in mouse intestinal epithelial organoids, while large HA 2000 kDa is not internalized into the cells. Our immunofluorescence data indicate that layilin, but neither toll-like receptor-4 (TLR-4) nor CD44, mediate the HA35-induced ZO-1 expression in colonic epithelium in vitro and in vivo. Additionally, using layilin null mice we have determined that layilin mediates HA35 induction of ZO-1 in healthy mice and during dextran sulfate sodium (DSS)-induced colitis. Furthermore, we find that while ZO-1 expression levels are reduced, layilin expression levels are equivalent in inflammatory bowel disease (IBD) patients and non-IBD controls. Together, our data suggest that layilin is an important HA receptor, that mediates the effect of oral HA35 treatment on intestinal epithelium. HA35 holds promise as a simple dietary supplement to strengthen gut barrier defense.

Maintaining a healthy intestinal barrier, the primary physical barrier between intestinal microbiota and the underlying lamina propria, is critical for optimal health. Epithelial integrity is essential for the prevention of the entrance of luminal contents, such as bacteria and their products, through the large intestinal barrier. In this study, we investigated the protective functions of biosynthetic, specific sized, hyaluronan around 35kDa (HA35) on intestinal epithelium in healthy mice, as well as mice infected Citrobacter rodentium, an established model that mimics infection with a serious human pathogen, enteropathogenic E. coli (EPEC). Our results reveal that treatment with HA35 protects mice from Citrobacter infection and enhances the epithelial barrier function. In particular, we have found that HA35 induces the expression of tight junction protein zonula occludens (ZO)-1 in both healthy and Citrobacter infected mice, as demonstrated by immunoflurorescence and Western blot analyses. Furthermore, we determined that HA35 treatment enhances ZO-1 expression and reduces intestinal permeability at the early stages of dextran sulfate sodium (DSS)-induced colitis in mice. Together, our data demonstrate that the expression and functionality of tight junctions, are increased by HA35 treatment, suggesting a novel mechanism for the protection from Citrobacter infection.

Tumor necrosis factor-alpha (TNF-α) plays an important role in the developing process of inflammatory bowel disease. Tight junction protein zonula occludens-1 (ZO-1), one of epithelial junctional proteins, maintains the permeability of intestinal barrier. The objective of this study was to investigate the mechanism of the protective effect of baicalin on TNF-α-induced injury and ZO-1 expression in intestinal epithelial cells (IECs). We found that baicalin pretreatment significantly improved cell viability and cell migration following TNF-α stimulation. miR-191a inhibitor increased the protective effect of baicalin on cell motility injured by TNF-α. In addition, miR-191a down-regulated the mRNA and protein level of its target gene ZO-1. TNF-α stimulation increased miR-191a expression, leading to the decline of ZO-1 mRNA and protein. Moreover, pretreatment with baicalin reversed TNF-α induced decrease of ZO-1 and increase of miR-191a, miR-191a inhibitor significantly enhanced ZO-1 protein expression restored by baicalin. These results indicate that baicalin exerts a protective effect on IEC-6 (rat small intestinal epithelial cells) cells against TNF-α-induced injury, which is at least partly via inhibiting the expression of miR-191a, thus increasing ZO-1 mRNA and protein levels.

The intestinal damage can be caused by physical, infectious, and immune-mediated injury. Rapid repair, which is essential for a return to mucosal homeostasis, depends on rapid epithelial migration, i.e., restitution, as well as epithelial proliferation and differentiation. Barrier restoration is a critical component of this repair process, but the contributions of structural proteins that form the barrier to mucosal repair have not been defined. Aim: To determine the contributions of the intestinal epithelial tight junction protein zonula occludens-1 (ZO-1) to mucosal repair. Results: Intestinal epithelial ZO-1 transcript and protein expression are reduced in biopsies from inflammatory bowel disease (IBD) patients relative to healthy controls. To determine if this ZO-1 loss contributes to disease pathogenesis, we created intestinal epithelial ZO-1-deficient Tjp1f/f x villin-Cre+ mice. When stressed, for example, with 2% DSS, ZO-1-deficient mice displayed much greater mucosal damage and weight loss relative to ZO-1-sufficient mice and failed to recover fully, even 4 weeks after DSS discontinuation. To better define the defect, mice were injected with nucleoside analogs (BrdU and EdU) to label proliferating cells 1 and 3 days after DSS discontinuation. When assessed 2 hours after labeling, numbers of labeled nuclei were significantly reduced in ZO-1-deficient mice relative to ZO-1-sufficient mice. More striking, however, was the nuclear fragmentation and staining for cleaved caspase-3 at 24 hours after labeling. In vitro studies of colonoids were used to better define the mechanisms of epithelial loss. Colonoids from ZO-1-deficient mice were smaller, had reduced numbers of buds (crypt domains) and Lgr5+ stem cells, and were nonresponsive to enhanced Wnt signaling induced by the GSK3 inhibitor CHIR99021, relative to ZO-1-sufficient colonoids. Live imaging of mitosis showed misoriented mitotic spindles in ZO-1-deficient colonoids that caused one daughter cell to lose contact with the extracellular matrix. Misoriented mitotic spindles were also present in tissues from DSS-treated ZO-1-deficient, not ZO-1-sufficient, mice. Live imaging of colonoids from mRFP1-ZO-1 transgenic mice detected transient accumulation of ZO-1 at the cleavage furrow, suggesting that ZO-1 interactions at this site required for accurate mitotic spindle orientation. Conclusion: ZO-1 has a previously unappreciated, noncanonical function in mitotic spindle orientation that is independent of barrier maintenance but central to epithelial proliferation and repair. We postulate that, in the absence of ZO-1, loss of contact with the basement membrane leads to anoikis, i.e., detachment-induced apoptosis, and an abortive proliferative response that compromises repair. We speculate that ZO-1 downregulation in IBD may similarly interfere with mucosal healing. Support: NIH (DK068271, DK061931)

Background: Epithelial-mesenchymal transition (EMT) is considered to play a critical role in cancer progression and metastasis. However, the impact of EMT on the prognosis of hepatocellular carcinoma (HCC) is still elusive. In this study, we examined the relationship between the expression of EMT markers and recurrence-free survival (RFS) and overall survival (OS) in HCC patients after hepatic resection. Summary: The mRNA expression of 15 genes related to EMT was assessed by quantitative real-time polymerase chain reaction in cancerous tissues from 72 patients who underwent hepatic resection of HCC between January 2005 and December 2010 at our hospital. The upregulation of TWIST and the downregulation of tight junction protein ZO-1 (TJP1) were significantly associated with shorter RFS as well as OS. Increased levels of TWIST and decreased levels of TJP1 should be predictive markers for poor prognosis in patients with HCC after hepatectomy; those could serve as potential biomarkers for the treatment of HCC. Key Messages: A low level of TJP1 and high level of TWIST expression were prognostic factors predicting HCC after hepatic resection.

Recently, several studies showed that gastrointestinal tract may be associated with pathophysiology of Parkinson's disease (PD). Intestine tight junction protein zonula occluden-1 (ZO-1) is an important component of intestinal barrier which can be degraded by matrix metallopeptidase 9 (MMP-9). In our previous study, a significant decline in ZO-1 was observed along with enhanced MMP-9 activity in the duodenum and distal colon of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In this study, the protective effect of simvastatin on ZO-1 was investigated using an MPTP mouse model of PD. Seven days after the end of MPTP application, the expression level of ZO-1 was evaluated by immunohistochemistry. The protein expression levels of ZO-1 and MMP9 were detected by Western blotting. Meanwhile, MMP-9 activity was analyzed by gelatin zymography. MPTP treatment led to a decrease in the expression of ZO-1, which was accompanied by elevated MMP-9 activity. Treatment with simvastatin could partly reverse the MPTP-induced changes in ZO-1 expression and reduce MMP-9 protein and activity. Taken together, these findings suggest that simvastatin administration may partially reverse the impairment of ZO-1 induced by MPTP via inhibiting the activity of MMP9, fortify the impaired intestinal barrier and limit gut-derived toxins that pass across the intestinal barrier.

To investigate the involvement of stress-activated protein kinases, JNK and p38 MAPK, in the assembly of tight junctions in keratinocytes, we treated HaCaT cells with various combinations of SP600125 (an inhibitor of JNK), SB202190 (an inhibitor of p38 MAPK) and anisomycin (an activator of both JNK and p38 MAPK) and examined the localization of ZO-1, an undercoat constitutive protein of the tight junction. Short-term (8h) incubation with SP600125, SB202190 or anisomycin induced the accumulation of ZO-1 in the cell-cell contacts, with reduced ZO-1 staining in the cytoplasm, while only long-term (24h) incubation with SP600125 induced the accumulation of ZO-1. SP600125, SB202190 or SP600125 plus SB202190 treatment induced thin linear staining for ZO-1 in the cell-cell contacts. Anisomycin treatment induced thick and irregular linear staining for ZO-1, while anisomycin plus SP600125 treatment induced zipper-like staining for ZO-1. Anisomycin plus SB202190 treatment or anisomycin plus both SP600125 and SB202190 treatment for 8h failed to lead to the accumulation of ZO-1 in cell-cell contacts, but induced thin linear staining with several gaps 16 h after removal of these agents. These results suggest that the localization of ZO-1 in cell-cell contacts is differently regulated by activation and inhibition of JNK and/or p38 MAPK depending on the incubation period.

Females have a higher prevalence of most autoimmune diseases; however, the mechanism is unknown. In this study, we examined the expression of tight junction protein zonula occludens 1 (ZO-1) and estrogen receptor (ER)-α/β in human primary gut tissues by immunohistochemistry, immunofluorescence and qPCR. The expression of ZO-1 and ER-β but not ER-α was present in both male and female gut tissues. There was no sex difference in ER-β expression, but ZO-1 expression was decreased in females compared to males. In vitro, estrogen treatment decreased ZO-1 mRNA and protein expression, ZO-1 promoter activity, IL-6 production, and NF-κB activation in human primary gut tissues or the Caco-2 cells, but increased the ER-β expression in Caco-2 cells. Consistently, plasma IL-6 levels in females were reduced relative to males in vivo. Our finding indicates that estrogen may play a role in gut tight junction expression and permeability.

FHL1C is a LIM domain protein that has been implied in transcription regulation through interacting with other proteins, such as RBP-J, the critical transcription factor of the Notch signaling pathway. The LIM domain is a protein-protein interaction interface, suggesting that FHL1C could bind other proteins to enable its functions. In order to explore the interacting proteins with FHL1C, in this study we screened FHL1C-interacting proteins by using immunoprecipitation and mass spectrometric analysis. ZO-1, a member of the Zonula occludens proteins that constitute tight junctions, was sorted out as one candidate by using these techniques. Furthermore, we confirmed the interaction between FHL1C and ZO-1 in cells by using the mammalian two-hybrid assay and the co-immunoprecipitation assay, and verified that ZO-1 could interact with FHL1C through the PDZ domains of ZO-1. Moreover, with immunofluorescence staining, we found that FHL1C could induce ZO-1 translocating into nucleus. With a breast adenocarcinoma cell line MCF7, we showed that the interaction between FHL1C and ZO-1 could contribute to the epithelial-mesenchymal transition (EMT). Taken together, our study might provide new insight into the function of FHL1C on the regulation of EMT in cancer cells.

Protein-protein interactions are thought to be the next frontier in drug discovery. However, there are several well-known challenges facing development of protein-protein interaction (PPI) inhibitors that lead to slow development in the field, including difficulty identifying PPIs and difficulty designing small molecule inhibitors of relatively flat, featureless PPIs. We address these difficulties with the development of a novel method of discovering PPI hotspots, called protein painting. This technique relies on non-covalent labeling of solvent- accessible protein surfaces using small molecular dyes optimized for protein binding. The dyes block access to trypsin cleavage sites, allowing for digestion only of undyed interface regions following denaturation of the protein complex. Interface regions can be identified using mass spectrometry and used as target sequences for drug development. Here we introduce new dye chemistries and elucidate their mechanism of protein binding for the first time. This allows for rapid identification of functionally-relevant hotspots without the need to screen many dye chemistries to optimize surface coverage of the protein complex. We applied this method to elucidate functional hotspots for immmuno-oncology targets PD-1 and PD-L1 and Hippo pathway targets YAP2 and tight junction protein ZO-1. To further functionally validate the hotspot regions identified, we focused on the case study of PD-1 and PD-L1. We discovered a hotspot of PD-1 Lys 78 in the protein-protein interface, and rationally designed a series of 8 peptide inhibitors to target this hotspot. The most active peptide YRCMISYGGADYKRITV derived from PD-L1 disrupted the PD-1/PD-L1 complex with an IC50 of 5.07 µM. The predicted binding site of this peptide on PD-1 overlaps the binding site of therapeutic anti-PD-1 antibody pembrolizumab; crystal structures of pembrolizumab and PD-1 show hydrogen bonding between the antibody and our identified hotspot Lys 78. Furthermore, we prepared a cyclized analog peptide CYRAMISYGGADYKRITC by disulfide bond stapling to increase peptide stability and found that this did not significantly reduce inhibitor potency, with an IC50 of 8.02 µM. Taken together, this data suggests a specific region of PD-1 found within the larger PD-1/PD-L1 interface that may serve as a target for development of next generation small molecule PD-1/PD-L1 inhibitors. By focusing drug discovery efforts against only the PPI hotspot regions, we may accelerate drug development against these difficult targets. Citation Format: Amanda Still, Douglass Dey, Rachel Carter, Angela Dailing, Mikell Paige, Lance Liotta, Alessandra Luchini. Functionally important hotspot interfaces between immune-oncology targets PD-1 and PD-L1 and between Hippo pathway targets YAP2 and tight junction protein ZO-1 are identified using a protein-protein interaction technique optimized with novel dye chemistries [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 982.

OCTN2 (SLC22A5) is an organic cation/carnitine transporter belonging to the solute carrier transporters (SLC) family. OCTN2 is ubiquitously expressed and its presence was shown in various brain cells, including the endothelial cells forming blood-brain barrier, where it was mainly detected at abluminal membrane and in proximity of tight junctions (TJ). Since OCTN2 contains a PDZ-binding domain, the present study was focused on a possible role of transporter interaction with a TJ-associated protein ZO-1, containing PDZ domains and detected in rat Octn2 proteome. We showed previously that activation of protein kinase C (PKC) in rat astrocytes regulates Octn2 surface presence and activity. Regulation of a wild type Octn2 and its deletion mutant without a PDZ binding motif were studied in heterologous expression system in HEK293 cells. Plasma membrane presence of overexpressed Octn2 did not depend on either PKC activation or presence of PDZ-binding motif, anyhow, as assayed in proximity ligation assay, the truncation of PDZ binding motif resulted in a strongly diminished Octn2/ZO-1 interaction and in a decreased transporter activity. The same effects on Octn2 activity were detected upon PKC activation, what correlated with ZO-1 phosphorylation. It is postulated that ZO-1, when not phosphorylated by PKC, keeps Octn2 in an active state, while elimination of this binding in ΔPDZ mutant or after ZO-1 phosphorylation leads to diminution of Octn2 activity.

We questioned how tight junctions contribute to intestinal barrier function during the cell shedding that is part of physiological cell renewal. Intravital confocal microscopy studied the jejunal villus epithelium of mice expressing a fluorescent zonula occludens 1 (ZO-1) fusion protein. Vital staining also visualized the cell nucleus (Hoechst staining) or local permeability to luminal constituents (Lucifer Yellow; LY). In a cell fated to be shed, ZO-1 redistributes from the tight junction toward the apical and then basolateral cell region. ZO-1 rearrangement occurs 15 ± 6 min ( n = 28) before movement of the cell nucleus from the epithelial layer. During cell extrusion, permeation of luminal LY extends along the lateral intercellular spaces of the shedding cell only as far as the location of ZO-1. Within 3 min after detachment from the epithelial layer, nuclear chromatin condenses. After cell loss, a residual patch of ZO-1 remains in the space previously occupied by the departed cell, and the size of the patch shrinks to 14 ± 2% ( n = 15) of the original cell space over 20 min. The duration of cell shedding measured by nucleus movement (14 ± 1 min) is much less than the total duration of ZO-1 redistribution at the same sites (45 ± 2 min). In about 15% of cell shedding cases, neighboring epithelial cells also undergo extrusion with a delay of 5–10 min. With the use of normal mice, ZO-1 immunofluorescent staining of fixed tissue confirmed ZO-1 redistribution and the presence of ZO-1 patches beneath shedding cells. Immunostaining also showed that redistribution of ZO-1 occurred without corresponding mixing of apical and basolateral membrane domains as marked by ezrin or E-cadherin. ZO-1 redistribution is the earliest cellular event yet identified as a herald of physiological cell shedding, and redistribution of tight junction function along the lateral plasma membrane sustains epithelial barrier during cell shedding.

Zonula occludens (ZO)-1 is a member of the MAGUK (membrane-associated guanylate kinase homologs) family of membrane-associated signaling molecules that binds directly to both cytosolic and transmembrane components of the tight junction and is believed to organize these proteins within the apical junctional complex. It also binds directly to F-actin, although the functional relevance of this interaction is unknown. To address this issue, we have used VSVG-tagged transgenes to dissect ZO-1 and have identified a 220 amino acid region of ZO-1 that is necessary for its association with F-actin in MDCK cell pull-down assays. A GST fusion expressing this region can bind directly to F-actin in vitro, whereas a GFP fusion expressing this domain decorates actin stress fibers when expressed in MDCK cells. These results indicate that this actin-binding region (ABR) is both necessary and sufficient for binding to F-actin in vitro and in vivo. VSVG-tagged transgenes that lack the ABR still accumulate at both early and late cell-cell contacts in MDCK cells, suggesting that the ABR is not required for tight junction localization. However, accumulation of constructs lacking the ABR is markedly reduced at tight junctions in confluent cells, suggesting that the ABR does play an important role in the localization of ZO-1 at junctions. Furthermore, the ABR is required for localization to a novel actin-rich pool of ZO-1 that accumulates in puncta at the free edge of cells before initiation of cell-cell contact. We conclude that direct interactions between ZO-1 and F-actin play a role in several different steps of junction assembly.

The tight junction is the most apical intercellular junction of epithelial cells and regulates transepithelial permeability through the paracellular pathway. To examine possible functions for the tight junction-associated protein ZO-1, C-terminally truncated mutants and a deletion mutant of ZO-1 were epitope tagged and stably expressed in corneal epithelial cell lines. Only full-length ZO-1 and one N-terminal truncation mutant targeted to cell borders; other mutants showed variable cytoplasmic distributions. None of the mutants initially disrupted the localization of endogenous ZO-1. However, long-term stable expression of two of the N-terminal mutants resulted in a dramatic change in cell shape and patterns of gene expression. An elongated fibroblast-like shape replaced characteristic epithelial cobblestone morphology. In addition, vimentin and smooth muscle actin expression were up-regulated, although variable cytokeratin expression remained, suggesting a partial transformation to a mesenchymal cell type. Concomitant with the morphological change, the expression of the integral membrane tight junction protein occludin was significantly down-regulated. The localizations of endogenous ZO-1 and another family member, ZO-2, were disrupted. These findings suggest that ZO-1 may participate in regulation of cellular differentiation.

The tight junction protein ZO-1 belongs to a family of multidomain proteins known as the membrane-associated guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to interact with the transmembrane protein occludin, a second tight junction-specific MAGUK, ZO-2, and F-actin, although the nature and functional significance of these interactions is poorly understood. To further elucidate the role of ZO-1 within the epithelial tight junction, we have introduced epitope-tagged fragments of ZO-1 into cultured MDCK cells and identified domains critical for the interaction with ZO-2, occludin, and F-actin. A combination of in vitro and in vivo binding assays indicate that both ZO-2 and occludin interact with specific domains within the N-terminal (MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal half of ZO-1 cosediments with F-actin. Consistent with these observations, we found that a construct encoding the N-terminal half of ZO-1 is specifically associated with tight junctions, whereas the unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In addition, we have identified a 244-amino acid domain within the N-terminal half of ZO-1, which is required for the stable incorporation of ZO-1 into the junctional complex of polarized MDCK cells. These observations suggest that one functional role of ZO-1 is to organize components of the tight junction and link them to the cortical actin cytoskeleton.

Increasing evidence has recognized tight junctions (TJs) as the lower epidermal inside-out diffusion barrier located in granular cell layers of the epidermis. However, little is known about the regulation of TJ components in epidermis. p38 pathway is one of the mitogen-activated protein kinase pathways, which controls cell growth, differentiation, and apoptosis. We have investigated the role of p38 signaling pathway in the regulation of selected desmosomal, adherens and TJ components in human primary keratinocytes during Ca(2+)-induced differentiation, as well as in cultured squamous cell carcinoma cell lines. p38 signaling pathway was inhibited in cultured keratinocytes and cutaneous squamous cell carcinoma cells using recombinant adenoviruses, small inhibitory RNAs (siRNA) and chemical inhibitors. Expression of intercellular junction proteins was investigated using Western analysis and indirect immunofluorescence (IIF). The results showed that inhibition of p38δ function by siRNA or adenovirally delivered dominant negative mutant led to markedly decreased levels of Zonula occludens-1 (ZO-1) protein in keratinocytes, while the expression of other junctional proteins studied was not altered. Immunolocalization of ZO-1 revealed that intercellular junction areas were depleted from ZO-1. Inhibition of ZO-1 by siRNA silencing did not however result in an altered expression or subcellular localization of other TJ components studied. The expression of ZO-1 in carcinoma cells was also regulated by p38. The results indicate that ZO-1 is regulated by p38δ while the other junction proteins studied are not. Since ZO-1 is an integral component of functional TJs, various pathological processes affecting signaling via p38δ may also interfere with epithelial maturation and the formation and function of TJs.

Tight junctions (TJ) regulate the paracellular movement of ions, macromolecules and immune cells across epithelia. Zonula Occludens (ZO)-1 is a multi-domain polypeptide required for the assembly of TJs. MDCK II cells lacking ZO-1, and its homolog ZO-2, have three distinct phenotypes: Reduced localization of occludin and some claudins to the TJ, increased epithelial permeability, and expansion of the apical actomyosin contractile array found at the apical junction complex (AJC). However, it is unclear exactly which ZO-1 binding domains are required to coordinate these activities. We addressed this question by examining the ability of ZO-1 domain-deletion transgenes to reverse the effects of ZO-depletion. We found that the SH3 domain and the U5 motif are required to recruit ZO-1 to the AJC and that localization is a prerequisite for normal TJ and cytoskeletal organization. The PDZ2 domain is not required for localization of ZO-1 to the AJC, but is necessary to establish the characteristic continuous circumferential band of ZO-1, occludin and claudin-2. PDZ2 is also required to establish normal permeability, but is not required for normal cytoskeletal organization. Finally, our results demonstrate that PDZ1 is critical for the normal organization of both the TJ and the AJC cytoskeleton. Our results establish that ZO-1 acts as a true scaffolding protein and that the coordinated activity of multiple domains is required for normal TJ structure and function.

Hepatocyte growth factor (HGF) is essential for epithelial restitution, a process in which epithelial cells rapidly migrate to cover desquamated epithelium after mucosal injury in the gastrointestinal tract. In this study, we aimed to elucidate the molecular mechanisms of the HGF-mediated reconstitution of gastric epithelial structures by analyzing the expression and subcellular dynamics of tight junction proteins. We treated human gastric epithelial MKN74 cells with HGF, and examined the effects of HGF on cell migration and proliferation, and the expression and subcellular dynamics of tight junction proteins; as well, we investigated the effect of HGF on paracellular permeability to macromolecules (using fluorescein isothiocyanate [FITC]-dextran). HGF significantly stimulated the migration of MKN74 cells, but not their proliferation, in a dose-dependent manner. HGF did not affect the expression of tight junction proteins, including claudin-1, -3, -4 and -7; occludin; and zonula occludens (ZO)-1. However, fluorescence immunostaining revealed that, in the cell membrane, the levels of ZO-1, but not those of occludin or claudin-4, were transiently decreased 1 h after HGF treatment. The results were further confirmed by western blotting: HGF reduced the amount of ZO-1 protein in the cell membrane fraction concomitantly with an increase in cytoplasmic ZO-1. Furthermore, HGF reduced the interaction between ZO-1 and occludin, and induced the tyrosine phosphorylation of occludin, whereas the phosphorylation status of ZO-1 was not affected by exposure to HGF. Despite a decrease in the ZO-1/occludin interaction, HGF did not affect paracellular permeability to macromolecules. HGF alters the subcellular localization of ZO-1, probably through the tyrosine phosphorylation of occludin, which may induce cell dispersion during epithelial restitution.

Tight junctions form an intercellular barrier between epithelial cells, serve to separate tissue compartments, and maintain cellular polarity. Paracellular sealing properties vary among cell types and are regulated by undefined mechanisms. Sequence of the full-length cDNA for human ZO-1, the first identified tight junction component, predicts a protein of 1736 aa. The N-terminal 793 aa are homologous to the product of the lethal(1)discs-large-1 (dlg) tumor suppressor gene of Drosophila, located in septate junctions, and to a 95-kDa protein located in the postsynaptic densities of rat brain, PSD-95. All three proteins contain both a src homology region 3 (SH3 domain), previously identified in membrane proteins involved in signal transduction, and a region homologous to guanylate kinase. ZO-1 contains an additional 943-aa C-terminal domain that is proline-rich (14.1%) and contains an alternatively spliced domain, whose expression was previously shown to correlate with variable properties of tight junctions. dlg mutations result in loss of apical-basolateral epithelial cell polarity and in neoplastic growth. These results suggest a protein family specialized for signal transduction on the cytoplasmic surface of intercellular junctions. These results also provide biochemical evidence for similarity between invertebrate septate and vertebrate tight junctions. The C-terminal domain of ZO-1, and its alternatively spliced region, appears to confer variable properties unique to tight junctions.

ZO-1 is a peripheral protein that plays a central role in the macromolecular assembly of tight junctions by interacting with integral proteins (occludin, claudins, JAMs) of the membrane of adjoining cells, with the actin cytoskeleton, and with nuclear factors. Human ZO-1 is expressed in all epithelia and some specialized endothelia as variable amounts of two related isoforms, which originate from the alternatively spliced mRNA transcripts alpha(+) and alpha(-) and whose specific differential role is still unknown. Moreover, little is known about the timing of expression of ZO-1 isoforms at the protein and mRNA level. This study shows that during growth of freshly plated Caco-2 cells, the alpha(+)/alpha(-) ratio increased as a result of simultaneous increase of alpha(+) and decrease of alpha(-). Differences in the isoform ratio also correlated with differences in epithelium differentiation. This was determined by aminopeptidase N measurements of cells grown on conventional substrates and on modified, micro/nano-patterned surfaces. A comparable shift of ZO-1 isoforms was not observed in other tumour cell lines of non-intestinal origin (A549, Calu-3). Pancreatic stem cells, propagated without exogenous differentiation stimuli, displayed a slight, stable prevalence of the alpha(-) isoform. Of the intestinal cell lines examined (Caco-2 and T84), only Caco-2 cells displayed a dramatic shift in isoform expression. This suggests that this tumour cell line retains to a higher degree a developmental programme related to the dynamic of enterocytic differentiation in vivo.

ZO-1 is a 225-kDa peripheral membrane protein present in all tight junctions. It was recently shown to consist of two isoforms that differ in the presence of an internal 80-amino acid domain termed motif-alpha. To obtain information on their distribution and potential functional significance we have localized the two isoforms in rat kidney by using antibodies that recognize either both ZO-1 isoforms or the larger, motif-alpha-containing isoform. By immunofluorescence, staining with both antibodies was demonstrated at all tight junctions of tubular epithelial cells and the epithelial cells of Bowman's capsule. In contrast, the motif-alpha-containing isoform was absent from the slit diaphragms of the glomerular epithelium and the tight junctions of glomerular and peritubular capillary endothelial cells. This restricted isoform expression was confirmed by immunoblot analysis comparing proteins from purified glomeruli with those from kidney cortex or medulla. Thus, while both isoforms are expressed in typical epithelial tight junctions, only a single isoform, lacking motif-alpha, is expressed in the highly specialized slit diaphragms, where the intercellular spaces are normally open, and in endothelial junctions, which are readily opened by physiologic signals. The differential expression of ZO-1 isoforms in structurally and functionally distinct junctions in the kidney suggests that they may contribute to defining the variable functional properties, in particular the lability of these intercellular junctions.

ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp region encoded an inframe insertion of 80 amino acids, named motif-alpha. Expression of the predicted transcripts in normal rat and human tissues and in human epithelial cell lines (Caco-2, T84, Hep G2) was shown by reverse transcription of RNA and then DNA amplification. Immunoblot analysis showed both protein isoforms were present; however, in different cell lines, their amounts differed markedly relative to each other. Immunolocalization at light and ultrastructural levels, using antibodies generated against motif-alpha or shared sequences flanking it, indicated both forms localized indistinguishably to tight junctions. These observations demonstrate the existence and variable expression of ZO-1 isoforms and raise the question whether these isoforms contribute to tight junction diversity in different epithelia.

The formation of a barrier by tight junctions is important in epithelia of various tissues. Substance P (SP) and insulin-like growth factor (IGF)-1 synergistically promote barrier function in the corneal epithelium. We have now examined the effects of SP and IGF-1 on expression of the tight-junction protein zonula occludens (ZO)-1 in A431 human epidermoid carcinoma cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analyses revealed that SP and IGF-1 increased the amounts of ZO-1 mRNA and protein in these cells in a concentration-dependent manner, with neither SP nor IGF-1 alone having such an effect. The SP- and IGF-1-induced up-regulation of ZO-1 was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and both of these effects were blocked by PD98059, an inhibitor of ERK activation. SP and IGF-1 also increased the transepithelial electrical resistance (TER) (an indicator of barrier function) of an A431 cell monolayer in a manner sensitive to PD98059. Our results thus suggest that the synergistic induction of ZO-1 expression by SP and IGF-1 may promote barrier function in skin epithelial cells.

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress.

Coeliac disease is an autoimmune enteropathy characterized by an enhanced permeability of the intestinal epithelial barrier. In epithelial cells paracellular permeability is regulated by intercellular tight junction. The cytoplasmic protein ZO-1 interacts directly with F-actin and plays a pivotal role in the structural and functional organization of tight junction. Aim. The aim of this study was to investigate the expression and localization of ZO-1 in the intestinal mucosa of coeliac patients. Patients and methods. Twenty patients with active coeliac disease, seven of whom underwent a repeat biopsy following a gluten-free diet and 27 control subjects, were studied. In all subjects, three biopsies were obtained from distal duodenum during upper gastrointestinal endoscopy. ZO-1 protein localization and levels were detected by immunofluorescence followed by confocal microscopy analysis and immunoblotting. ZO-1 mRNA expression was assessed by RT-PCR. F-actin distribution was also investigated. Results. In patients with active coeliac disease, both ZO-1 protein levels and mRNA were clearly reduced. Cytoskeletal organization was disrupted with F-actin staining concentrated at the subcortical and basal surface regions. Abnormalities in ZO-1 expression and actin organization were reversed after a gluten-free diet. Conclusions. In active coeliac disease, ZO-1 protein expression is downregulated at the transcriptional level in association with F-actin redistribution. These changes are completely reversed after a gluten-free diet and could contribute to the increased intestinal paracellular permeability observed in this disorder.

ZO-1 is a tight junction membrane protein that plays a critical role in cell-cell interaction, proliferation, and differ entiation.To localize and evaluate the expression of ZO-1 in the normal human pancreas, in pancreatic ductal adenocarcinoma (PDAC), and in chronic pancreatitis (CP).Northern and Western blot analysis revealed ZO-1 expression in all six tested pancreatic cancer cell lines. Expression of ZO-1 mRNA was increased sixfold in PDAC samples in comparison with normal samples (p = 0.04). Confocal microscopy revealed the presence of ZO-1 in the apical and apicolateral areas of ductular cells in the normal pancreas. Similarly, in CP, ZO-1 was localized at apical and apicolateral areas of small proliferating ductular cells and large metaplastic ducts. In PDAC, however, ZO-1 expression was observed irrespective of whether the cancer cells formed duct-like structures or exhibited a diffuse infiltrating pattern. Metastatic pancreatic cancer cells within lymph nodes displayed variable staining patterns, ranging from apical and apicolateral to a diffuse membranous staining.These observations suggest that ZO-1 is overexpressed in PDAC and raise the possibility that this overexpression may confer a metastatic advantage to pancreatic cancer cells.

Most of the information on the structure and function of the tight junction (TJ) has been obtained in MDCK cells. Accordingly, we have sequenced ZO-1 in this cell type, because this protein is involved in the response of the TJ to changes in Ca 21 , phosphorylation, and the cytoskeleton. ZO-1 of MDCK cells comprises 6805 bp with a predicted open reading frame of 1769 amino acids. This sequence is 92 and 87% homologous to human and mouse ZO-1, respectively. Two nuclear sorting signals located at the PDZ1 and GK domains and 17 SH3 putative binding sites at the proline-rich domain were detected. We found two new splicing regions at the proline-rich region: b had not been reported in human and mouse counterparts, and g, which was previously sequenced in human and mouse ZO-1, is now identified as a splicing region. The expression of different b and g isoforms varies according to the tissue tested. With the information provided by the sequence, Southern blot, and PCR experiments we can predict a single genomic copy of MDCK-ZO-1 that is at least 13.16 kb long. MDCK-ZO-1 mRNA is 7.4 kb long. Its expression is regulated by calcium, while the expression of MDCK-ZO-1 protein is not. © 1999 Academic Press

The olfactory system is a unique part of the central nervous system since it retains neuronal turnover and regenerative capacities in adulthood. Thus it provides an ideal model to study plasticity of membrane moities involved in cell-cell interactions. One structure particularly involved in cell-cell interaction is the tight junction, which establishes polarization of epithelial cells and creates diffusion barriers to paracellular passages. ZO-1 is a phosphoprotein peripherally associated with tight junctions. We have studied expression of ZO-1 protein in the developing and adult olfactory system of the mouse in order to get information about the localization and developmental expression of this tight junction component. ZO-1 expression has also been determined in cell cultures of olfactory bulbs. ZO-1 was present in the olfactory placode prior to formation of tight junctions. ZO-1 was localized in the developing and mature olfactory epithelium at heterotypic contacts between supporting cells and olfactory neurons as well as at homotypic contacts between both these cell types. Confocal microscopy showed quantitative differences in the ZO-1 expression among different olfactory dendrites. In the olfactory nerves ZO-1 immunolabeling was detectable between olfactory ensheathing cells. From the seventh postnatal day ZO-1 immunolabeling was detected at the mitral cell layer of the bulb on cells tentatively identified as oligodendrocytes. Myelinated tracts of the bulb were ZO-1 negative. Cell cultures of olfactory bulbs showed ZO-1 immunoreaction, mostly localized on glial fibrillary acidic protein (GFAP)-positive cells. Our results provide further evidence that ZO-1 serves functions unrelated to the tight junction complex and indicate molecular heterogeneity of these cell-cell contacts.

Hepatocyte tight junctions form the intercellular barrier between bile and blood. Cholestasis due to common bile duct ligation (CBDL) results in structural changes in the tight junction (TJ) and an overt paracellular leak, although the molecular basis for these alterations is undefined. Using the epithelial isoform of the TJ protein ZO-1 (ZO-1 alpha +) as a marker for molecular changes in hepatocyte TJs, we investigated the effects of CBDL on ZO-1 alpha + immunofluorescence (IF) localization and on ZO-1 alpha + mRNA and protein expression over 2 wk of CBDL. ZO-1 alpha + IF staining was altered after 2 days of CBDL and appeared to accumulate in pericanalicular regions after 7 and 9 days. Quantitative immunoblotting and ribonuclease protection revealed a marked increase in hepatic ZO-1 alpha + protein expression and ZO-1 alpha + mRNA levels, respectively. In contrast to changes in ZO-1 alpha + IF, which occurred throughout the lobule, and changes in mRNA and protein expression, which were maximal after 9 days of ligation, the maximal hepatocyte proliferation occurred within 2 days after CBDL and was confined to periportal regions. CBDL results in altered hepatic localization and increased expression of the TJ protein ZO-1 alpha + and appears to represent a specific response by hepatocytes to pathological junction injury independent of cell proliferation.

Background Hepatocyte growth factor (HGF) is essential for epithelial restitution, a process in which epithelial cells rapidly migrate to cover desquamated epithelium after mucosal injury in the gastrointestinal tract. In this study, we aimed to elucidate the molecular mechanisms of the HGF-mediated reconstitution of gastric epithelial structures by analyzing the expression and subcellular dynamics of tight junction proteins.

ZO-1 is a 210- to 220-kDa peripheral membrane protein associated with the cytoplasmic surface of the epithelial tight junction. Because ZO-1 may interact with other unidentified tight junction proteins, we have looked for other polypeptides that bind to ZO-1. A 160-kDa polypeptide was identified that coimmunoprecipitates with ZO-1 from detergent extracts of metabolically labeled Madin-Darby canine kidney (MDCK) cells. This polypeptide appears to be distinct from ZO-1, rather than a degradation product, by several criteria. It lacks ZO-1 epitopes recognized by both monoclonal antibodies and a polyclonal serum to ZO-1, since it is not detectable in immunoblots of either whole cell extracts or ZO-1 immunoprecipitates. Also, it exhibits a peptide map different from that of ZO-1 on one-dimensional "Cleveland gels." Moreover, because the kinetics of appearance of newly synthesized 160-kDa polypeptide in anti-ZO-1 immunoprecipitates is much slower than that of ZO-1, its presence in immunoprecipitates cannot be simply explained by degradation of ZO-1 during cell lysis. Like ZO-1, the 160-kDa polypeptide seems to be a cytoplasmic peripheral membrane protein. It cannot be labeled by two different cell surface labeling reagents. It can be extracted from the membrane by high salt concentration in the absence of detergents. As expected for a protein complex, the 160-kDa polypeptide and ZO-1 turn over with similar kinetics. We propose that the 160-kDa polypeptide is a component of the tight junction.

Material and methods: Human intestinal cells (Caco2) were grown on polarized filters. Gliadin or casein pepsyn-trypsin digests were added to the luminal culture medium for a 3-hour incubation. The intestinal barrier function was assessed by evaluation of: (1) trans-epithelial electrical resistance (TEER), (2) release of zonulin, a modulator of intercellular tight junctions (tj), measured by sandwich-ELISA, (3) luminal to serosal lactulose transport measured by high performance anion exchange chromatography and, (4) localization/migration of zonula occludens protein (ZO-1), by direct immunofluorescence.

ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS-PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."

A comparison was made of the phosphate content of the tight-junction-specific protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance, a parameter reflective of tight-junctional permeability. Analysis revealed that the ZO-1 from the low-resistance strain contained approximately twice as much phosphate as that from the high-resistance strain.

Epithelial tight junctions regulate paracellular diffusion and restrict the intermixing of apical and basolateral plasma membrane components. We now identify a Y-box transcription factor, ZONAB (ZO-1-associated nucleic acid-binding protein), that binds to the SH3 domain of ZO-1, a submembrane protein of tight junctions. ZONAB localizes to the nucleus and at tight junctions, and binds to sequences of specific promoters containing an inverted CCAAT box. In reporter assays, ZONAB and ZO-1 functionally interact in the regulation of the ErbB-2 promoter in a cell density-dependent manner. In stably transfected overexpressing cells, ZO-1 and ZONAB control expression of endogenous ErbB-2 and function in the regulation of paracellular permeability. These data indicate that tight junctions directly participate in the control of gene expression and suggest that they function in the regulation of epithelial cell differentiation.