Tight junctions as regulators of tissue remodelling

Formation of tissue barriers by epithelial and endothelial cells requires neighbouring cells to interact via intercellular junctions, which includes tight junctions. Tight junctions form a semipermeable paracellular diffusion barrier and act as signalling hubs that guide cell behaviour and differentiation. Components of tight junctions are also expressed in cell types not forming tight junctions, such as cardiomyocytes, where they associate with facia adherens and/or gap junctions. This review will focus on tight junction proteins and their importance in tissue homeostasis and remodelling with a particular emphasis on what we have learned from animal models and human diseases

Functional interaction between the ZO-1-interacting transcription factor ZONAB/DbpA and the RNA processing factor symplekin

Epithelial tight junctions participate in the regulation of gene expression by controlling the activity of transcription factors that can interact with junctional components. One such protein is the Y-box transcription factor ZONAB/DbpA that binds to ZO-1, a component of the junctional plaque. Symplekin, another nuclear protein that can associate with tight junctions, functions in the regulation of polyadenylation and thereby promotes gene expression. Here, we addressed the question of whether these two proteins interact and whether this is of functional relevance. We demonstrate that ZONAB/DbpA and symplekin form a complex in kidney and intestinal epithelial cells that can be immunoprecipitated and that exists in the nucleus. The interaction between ZONAB/DbpA and symplekin can be reconstituted with recombinant proteins. In reporter gene assays in which ZONAB/DbpA functions as a repressor, symplekin functionally interacts with ZONAB/DbpA, indicating that symplekin can also promote transcriptional repression. RNAi experiments indicate that symplekin depletion reduces the nuclear accumulation and the transcriptional activity of ZONAB/DbpA in colon adenocarcinoma cells, resulting in inhibition of proliferation and reduced expression of the ZONAB/DbpA-target gene cyclin D1. Our data thus indicate that symplekin and ZONAB/DbpA cooperate in the regulation of transcription, and that they promote epithelial proliferation and cyclin D1 expression

Global cell-by-cell evaluation of endothelial viability after two methods of graft preparation in Descemet membrane endothelial keratoplasty

PURPOSE: To describe a novel method of global cell viability assessment for Descemet membrane endothelial keratoplasty (DMEK) and the comparison of two contemporary methods of donor tissue preparation. METHODS: DMEK transplants were prepared using two different methods: liquid bubble separation and manual peeling (n=8 each group). Samples were incubated with Hoechst, calcein-AM and ethidium homodimer prior to mounting on a curved imaging chamber. Z-stacked fluorescence microscopy images were combined to produce an in-focus global image capable of resolving all cell nuclei. Image processing software was used to define a calcein-positive live cell area, count all cell nuclei within this area and subtract ethidium-positive dead cells to derive the total viable endothelial cell count. Corrected global cell density was calculated by dividing the number of viable cells by the graft area, which had been corrected for imaging a curved surface. RESULTS: Corrected global cell density was lower than the central endothelial cell density in both groups: 85.5% of the pre-preparation central endothelial cell density in the peel group and 75.8% in the bubble group. Corrected global cell density was significantly lower in the liquid bubble separation group than in the peel group (p=0.04). CONCLUSIONS: Eye bank estimations of central endothelial cell density overestimate true cell density after graft preparation in DMEK. A peel method is less damaging and more consistent than a liquid bubble method. Cell loss correlated strongly with the degree of stromal hydration prior to bubble separation in the liquid bubble group

Organ culture storage of pre-prepared corneal donor material for Descemet's membrane endothelial keratoplasty

Purpose To evaluate the effect of media composition and storage method on pre-prepared Descemet's membrane endothelial keratoplasty (DMEK) grafts. Methods 50 corneas were used. Endothelial wound healing and proliferation in different media were assessed using a standard injury model. DMEK grafts were stored using three methods: peeling with free scroll storage; partial peeling with storage on the stroma and fluid bubble separation with storage on the stroma. Endothelial cell (EC) phenotype and the extent of endothelial overgrowth were examined. Global cell viability was assessed for storage methods that maintained a normal cell phenotype. Results 1 mm wounds healed within 4 days. Enhanced media did not increase EC proliferation but may have increased EC migration into the wounded area. Grafts that had been trephined showed evidence of EC overgrowth, whereas preservation of a physical barrier in the bubble group prevented this. In grafts stored in enhanced media or reapposed to the stroma after trephination, endothelial migration occurred sooner and cells underwent endothelial-mesenchymal transformation. Ongoing cell loss, with new patterns of cell death, was observed after returning grafts to storage. Grafts stored as free scrolls retained more viable ECs than grafts prepared with the fluid bubble method (74.2± 3% vs 60.3±6%, p=0.04 (n=8). Conclusion Free scroll storage is superior to liquid bubble and partial peeling techniques. Free scrolls only showed overgrowth of ECs after 4 days in organ culture, indicating a viable time window for the clinical use of pre-prepared DMEK donor material using this method. Methods for tissue preparation and storage media developed for whole corneas should not be used in pre-prepared DMEK grafts without prior evaluation

The polarized expression of Na+,K+-ATPase in epithelia depends on the association between beta-subunits located in neighboring cells

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta(1)-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta(1)-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta(1)-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta(1)-subunit forces CHO-beta cells to coexpress endogenous a-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog P,-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells

ARHGEF18/p114RhoGEF Coordinates PKA/CREB Signaling and Actomyosin Remodeling to Promote Trophoblast Cell-Cell Fusion During Placenta Morphogenesis

Coordination of cell-cell adhesion, actomyosin dynamics and gene expression is crucial for morphogenetic processes underlying tissue and organ development. Rho GTPases are main regulators of the cytoskeleton and adhesion. They are activated by guanine nucleotide exchange factors in a spatially and temporally controlled manner. However, the roles of these Rho GTPase activators during complex developmental processes are still poorly understood. ARHGEF18/p114RhoGEF is a tight junction-associated RhoA activator that forms complexes with myosin II, and regulates actomyosin contractility. Here we show that p114RhoGEF/ARHGEF18 is required for mouse syncytiotrophoblast differentiation and placenta development. In vitro and in vivo experiments identify that p114RhoGEF controls expression of AKAP12, a protein regulating protein kinase A (PKA) signaling, and is required for PKA-induced actomyosin remodeling, cAMP-responsive element binding protein (CREB)-driven gene expression of proteins required for trophoblast differentiation, and, hence, trophoblast cell-cell fusion. Our data thus indicate that p114RhoGEF links actomyosin dynamics and cell-cell junctions to PKA/CREB signaling, gene expression and cell-cell fusion

MarvelD3 regulates the c-Jun N-terminal kinase pathway during eye development in Xenopus.

Ocular morphogenesis requires several signalling pathways controlling the expression of transcription factors and cell-cycle regulators. However, despite a well-known mechanism, the dialogue between those signals and factors remains to be unveiled. Here, we identify a requirement for MarvelD3, a tight junction transmembrane protein, in eye morphogenesis in Xenopus MarvelD3 depletion led to an abnormally pigmented eye or even an eye-less phenotype, which was rescued by ectopic MarvelD3 expression. Altering MarvelD3 expression led to deregulated expression of cell-cycle regulators and transcription factors required for eye development. The eye phenotype was rescued by increased c-Jun terminal Kinase activation. Thus, MarvelD3 links tight junctions and modulation of the JNK pathway to eye morphogenesis

The tumour suppressor DLC2 ensures mitotic fidelity by coordinating spindle positioning and cell-cell adhesion.

Dividing epithelial cells need to coordinate spindle positioning with shape changes to maintain cell-cell adhesion. Microtubule interactions with the cell cortex regulate mitotic spindle positioning within the plane of division. How the spindle crosstalks with the actin cytoskeleton to ensure faithful mitosis and spindle positioning is unclear. Here we demonstrate that the tumour suppressor DLC2, a negative regulator of Cdc42, and the interacting kinesin Kif1B coordinate cell junction maintenance and planar spindle positioning by regulating microtubule growth and crosstalk with the actin cytoskeleton. Loss of DLC2 induces the mislocalization of Kif1B, increased Cdc42 activity and cortical recruitment of the Cdc42 effector mDia3, a microtubule stabilizer and promoter of actin dynamics. Accordingly, DLC2 or Kif1B depletion promotes microtubule stabilization, defective spindle positioning, chromosome misalignment and aneuploidy. The tumour suppressor DLC2 and Kif1B are thus central components of a signalling network that guides spindle positioning, cell-cell adhesion and mitotic fidelity

Small and large intestine express a truncated Dab1 isoform that assembles in cell-cell junctions and co-localizes with proteins involved in endocytosis

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junctions assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location

The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice

Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration