Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis.     

Expression and distribution of symplekin regulates the assembly and function of the epithelial tight junction

Symplekin is multifunctional protein localized to both the tight junction and the nucleus with known roles in mRNA polyadenylation, proliferation, differentiation and tumorigenesis. Functions of symplekin at tight junctions have not been systematically investigated. In this study, increased expression of symplekin was observed during the formation of tight junctions in cultured HT-29 and HepG2 epithelial cells. Repression of symplekin by RNAi increased the permeability of epithelial monolayers, disrupted cellular polarity, and decreased the expression of the tight junction protein ZO-1. Moreover, symplekin was co-localized with ZO-1 at tight junctions and co-immunoprecipitated with ZO-1, indicating that ZO-1 and symplekin form complexes. In conclusion, symplekin expression regulates the assembly of tight junctions, thus helps to maintain the integrity of the epithelial monolayer and cellular polarity.     

Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.     

Polarity and morphogenesis of the eye epithelium requires the adhesion junction associated adaptor protein Traf4

ABSTRACT

During development, neuroepithelial progenitors acquire apico-basal polarity and adhere to one another via apically located tight and adherens junction complexes. This polarized neuroepithelium must continue to integrate cells arising through cell divisions and intercalation, and allow for cell movements, at the same time as undergoing morphogenesis. Cell proliferation, migration and intercalation all occur in the morphing embryonic eye. To understand how eye development might depend on dynamic epithelial adhesion, we investigated the function of a known regulator of junctional plasticity, Tumour necrosis factor receptor-associated factor 4 (Traf4). traf4a mRNA is expressed in the developing eye vesicle over the period of optic cup morphogenesis, and Traf4a loss leads to disrupted evagination and elongation of the eye vesicles, and aberrant organization and apico-basal polarity of the eye epithelium. We propose a model whereby Traf4a regulates apical junction plasticity in nascent eye epithelium, allowing for its polarization and morphogenesis.

Symbols and Abbreviations: AB: apico-basal; aPKC: atypical protein kinase-C; CRISPR: clustered regularly-interspaced short palindromic repeats; GFP: green fluorescent protein; hpf: hours post-fertilization; MO: antisense morpholino oligonucleotide; pHH3: phospho histone H3; ss: somite stage; Traf4: Tumour necrosis factor receptor-associated factor 4; ZO-1: zona occludens-1     

Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

Epithelial cell-cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell-cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin-capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics.     

Hyperoxia disrupts pulmonary epithelial barrier in newborn rats via the deterioration of occludin and ZO-1

Background

Prolonged exposure to hyperoxia in neonates can cause hyperoxic acute lung injury (HALI), which is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and maintenance of barrier properties requires intact epithelial tight junctions (TJs). However, in neonatal animals, relatively little is known about how the TJ proteins are expressed in the pulmonary epithelium, including whether expression of TJ proteins is regulated in response to hyperoxia exposure. This study determines whether changes in tight junctions play an important role in disruption of the pulmonary epithelial barrier during hyperoxic acute lung injury.

Methods

Newborn rats, randomly divided into two groups, were exposed to hyperoxia (95% oxygen) or normoxia for 1–7 days, and the severity of lung injury was assessed; location and expression of key tight junction protein occludin and ZO-1 were examined by immunofluorescence staining and immunobloting; messenger RNA in lung tissue was studied by RT-PCR; transmission electron microscopy study was performed for the detection of tight junction morphology.

Results

We found that different durations of hyperoxia exposure caused different degrees of lung injury in newborn rats. Treatment with hyperoxia for prolonged duration contributed to more serious lung injury, which was characterized by increased wet-to-dry ratio, extravascular lung water content, and bronchoalveolar lavage fluid (BALF):serum FD4 ratio. Transmission electron microscopy study demonstrated that hyperoxia destroyed the structure of tight junctions and prolonged hyperoxia exposure, enhancing the structure destruction. The results were compatible with pathohistologic findings. We found that hyperoxia markedly disrupted the membrane localization and downregulated the cytoplasm expression of the key tight junction proteins occludin and ZO-1 in the alveolar epithelium by immunofluorescence. The changes of messenger RNA and protein expression of occludin and ZO-1 in lung tissue detected by RT-PCR and immunoblotting were consistent with the degree of lung injury.

Conclusions

These data suggest that the disruption of the pulmonary epithelial barrier induced by hyperoxia is, at least in part, due to massive deterioration in the expression and localization of key TJ proteins.     

Truncation mutants of the tight junction protein ZO-1 disrupt corneal epithelial cell morphology

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.     

Enteral glutamine infusion modulates ubiquitination of heat shock proteins,Grp-75 and Apg-2, in the human duodenal mucosa

Glutamine, the most abundant amino acid in the human body, plays several important roles in the intestine. Previous studies showed that glutamine may affect protein expression by regulating ubiquitin–proteasome system. We thus aimed to evaluate the effects of gl utamine on ubiquitinated proteins in human duodenal mucosa. Five healthy male volunteers were included and received during 5 h, on two occasions and in a random order, either an enteral infusion of maltodextrins alone (0.25 g kg^?1 h^?1, control), mimicking carbohydrate-fed state, or maltodextrins with glutamine (0.117 g kg^?1 h^?1, glutamine). Endoscopic duodenal biopsies were then taken. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using anti-ubiquitin antibody. Differentially ubiquitinated proteins were then identified by liquid chromatography–electrospray ionization MS/MS. Five proteins were differentially ubiquitinated between control and glutamine conditions. Among these proteins, we identified two chaperone proteins, Grp75 and hsp74. Grp75 was less ubiquitinated after glutamine infusion compared with control. In contrast, hsp74, also called Apg-2, was more ubiquitinated after glutamine. In conclusion, we provide evidence that glutamine may regulate ubiquitination processes of specific proteins, i.e., Grp75 and Apg-2. Grp75 has protective and anti-inflammatory properties, while Apg-2 indirectly regulates stress-induced cell survival and proliferation through interaction with ZO-1. Further studies should confirm these results in stress conditions.     

Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity

Background

Probiotics are proposed to positively modulate the intestinal epithelial barrier formed by intestinal epithelial cells (IECs) and intercellular junctions. Disruption of this border alters paracellular permeability and is a key mechanism for the development of enteric infections and inflammatory bowel diseases (IBDs).

Methodology and Principal Findings

To study the in vivo effect of probiotic Escherichia coli Nissle 1917 (EcN) on the stabilization of the intestinal barrier under healthy conditions, germfree mice were colonized with EcN or K12 E. coli strain MG1655. IECs were isolated and analyzed for gene and protein expression of the tight junction molecules ZO-1 and ZO-2. Then, in order to analyze beneficial effects of EcN under inflammatory conditions, the probiotic was orally administered to BALB/c mice with acute dextran sodium sulfate (DSS) induced colitis. Colonization of gnotobiotic mice with EcN resulted in an up-regulation of ZO-1 in IECs at both mRNA and protein levels. EcN administration to DSS-treated mice reduced the loss of body weight and colon shortening. In addition, infiltration of the colon with leukocytes was ameliorated in EcN inoculated mice. Acute DSS colitis did not result in an anion secretory defect, but abrogated the sodium absorptive function of the mucosa. Additionally, intestinal barrier function was severely affected as evidenced by a strong increase in the mucosal uptake of Evans blue in vivo. Concomitant administration of EcN to DSS treated animals resulted in a significant protection against intestinal barrier dysfunction and IECs isolated from these mice exhibited a more pronounced expression of ZO-1.

Conclusion and Significance

This study convincingly demonstrates that probiotic EcN is able to mediate up-regulation of ZO-1 expression in murine IECs and confer protection from the DSS colitis-associated increase in mucosal permeability to luminal substances.     

Dimerization of the scaffolding protein ZO-1 through the second PDZ domain

The tight junction protein ZO-1 is known to link the transmembrane proteins occludin, claudins, and JAMs to many cytoplasmic proteins and the actin cytoskeleton. Although specific roles for ZO-1 at the tight junction are unknown, it is widely assumed that ZO-1, together with its homologs ZO-2 and ZO-3, serves as a platform to scaffold various transmembrane and cytoplasmic tight junction proteins. Thus the manner in which the zonula occludens (ZO) proteins multimerize has implications for the protein networks they can coordinate. The purpose of our study was to determine whether ZO-1 forms homodimers and to determine the protein interaction region. Using laser light scattering and analytical centrifugation, we show that protein sequences corresponding to the NH(2)-terminal half of ZO-1 form stable homodimers with a submicromolar equilibrium dissociation constant. Analysis of the molecular weight of different truncated forms of ZO-1 revealed that the second PDZ domain is both necessary and sufficient for dimerization. This interaction does not use the beta-finger motif described for other PDZ dimers. Furthermore, ZO-1 does not dimerize via an Src homology 3 to Guk domain interaction as was demonstrated previously for MAGUKs, like PSD-95. Results from immunoprecipitation experiments with polarized Madin-Darby canine kidney epithelial cells stably transfected with full-length GFP-ZO-1 indicate that a substantial portion of ZO-1 forms homodimers in vivo. As described previously, ZO-1 also forms heterodimers with ZO-2 and ZO-3. We conclude that the dimerization of ZO proteins is unlike that of other MAGUKs and that the previously unrecognized ZO-1 homodimers may allow formation of protein networks distinct from those of heterodimers with ZO-2 and ZO-3.     

Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier Breakdown

Background

Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs) have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins.

Methods

Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1β) for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC) were assessed by immunohistochemistry and immunoblot.

Key Results

Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC.

Conclusions & Inferences

The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.     

Differential regulation of junctional complex assembly in renal epithelial cell lines

Several signaling pathways that regulate tight junction and adherens junction assembly are being characterized. Calpeptin activates stress fiber assembly in fibroblasts by inhibiting SH2-containing phosphatase-2 (SHP-2), thereby activating Rho-GTPase signaling. Here, we have examined the effects of calpeptin on stress fiber and junctional complex assembly in Madin-Darby canine kidney (MDCK) and LLC-PK epithelial cells. Calpeptin induced disassembly of stress fibers and inhibition of Rho GTPase activity in MDCK cells. Interestingly, calpeptin augmented stress fiber formation in LLC-PK epithelial cells. Calpeptin treatment of MDCK cells resulted in a displacement of zonula occludens-1 (ZO-1) and occludin from cell-cell junctions and a loss of phosphotyrosine on ZO-1 and ZO-2, without any detectable effect on tight junction permeability. Surprisingly, calpeptin increased paracellular permeability in LLC-PK cells even though it did not affect tight junction assembly. Calpeptin also modulated adherens junction assembly in MDCK cells but not in LLC-PK cells. Calpeptin treatment of MDCK cells induced redistribution of E-cadherin and -catenin from intercellular junctions and reduced the association of p120ctn with the E-cadherin/catenin complex. Together, our studies demonstrate that calpeptin differentially regulates stress fiber and junctional complex assembly in MDCK and LLC-PK epithelial cells, indicating that these pathways may be regulated in a cell line-specific manner. calpeptin; tight junctions; adherens junctions; Rho; cadherin; p120ctn     

Differential effects of TNF (TNFSF2) and IFN-γ on intestinal epithelial cell morphogenesis and barrier function in three-dimensional culture

Background

The cytokines TNF (TNFSF2) and IFNγ are important mediators of inflammatory bowel diseases and contribute to enhanced intestinal epithelial permeability by stimulating apoptosis and/or disrupting tight junctions. Apoptosis and tight junctions are also important for epithelial tissue morphogenesis, but the effect of TNF and IFNγ on the process of intestinal epithelial morphogenesis is unknown.

Methods/Principal Findings

We have employed a three-dimensional cell culture system, reproducing in vivo-like multicellular organization of intestinal epithelial cells, to study the effect of TNF on intestinal epithelial morphogenesis and permeability. We show that human intestinal epithelial cells in three-dimensional culture assembled into luminal spheres consisting of a single layer of cells with structural, internal, and planar cell polarity. Exposure of preformed luminal spheres to TNF or IFNγ enhanced paracellular permeability, but via distinctive mechanisms. Thus, while both TNF and IFNγ, albeit in a distinguishable manner, induced the displacement of selected tight junction proteins, only TNF increased paracellular permeability via caspase-driven apoptosis and cell shedding. Infliximab and adalumimab inhibited these effects of TNF. Moreover, we demonstrate that TNF via its stimulatory effect on apoptosis fundamentally alters the process of intestinal epithelial morphogenesis, which contributes to the de novo generation of intestinal epithelial monolayers with increased permeability. Also IFNγ contributes to the de novo formation of monolayers with increased permeability, but in a manner that does not involve apoptosis.

Conclusions

Our study provides an optimized 3D model system for the integrated analysis of (real-time) intestinal epithelial paracellular permeability and morphogenesis, and reveals apoptosis as a pivotal mechanism underlying the enhanced permeability and altered morphogenesis in response to TNF, but not IFNγ.     

Interleukin-2 receptor beta subunit-dependent and -independent regulation of intestinal epithelial tight junctions

Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rbeta subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rbeta subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rbeta subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rbeta subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rbeta-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex.     

Different effects of ZO-1, ZO-2 and ZO-3 silencing on kidney collecting duct principal cell proliferation and adhesion

Coordinated cell proliferation and ability to form intercellular seals are essential features of epithelial tissue function. Tight junctions (TJs) classically act as paracellular diffusion barriers. More recently, their role in regulating epithelial cell proliferation in conjunction with scaffolding zonula occludens (ZO) proteins has come to light. The kidney collecting duct (CD) is a model of tight epithelium that displays intense proliferation during embryogenesis followed by very low cell turnover in the adult kidney. Here, we examined the influence of each ZO protein (ZO-1, -2 and -3) on CD cell proliferation. We show that all 3 ZO proteins are strongly expressed in native CD and are present at both intercellular junctions and nuclei of cultured CD principal cells (mCCD_cl1). Suppression of either ZO-1 or ZO-2 resulted in increased G₀/G₁ retention in mCCD_cl1 cells. ZO-2 suppression decreased cyclin D1 abundance while ZO-1 suppression was accompanied by increased nuclear p21 localization, the depletion of which restored cell cycle progression. Contrary to ZO-1 and ZO-2, ZO-3 expression at intercellular junctions dramatically increased with cell density and relied on the presence of ZO-1. ZO-3 depletion did not affect cell cycle progression but increased cell detachment. This latter event partly relied on increased nuclear cyclin D1 abundance and was associated with altered β1-integrin subcellular distribution and decreased occludin expression at intercellular junctions. These data reveal diverging, but interconnected, roles for each ZO protein in mCCD_cl1 proliferation. While ZO-1 and ZO-2 participate in cell cycle progression, ZO-3 is an important component of cell adhesion.