Homophilic interaction of junctional adhesion molecule

Junctional adhesion molecule (JAM) is an integral membrane protein that belongs to the immunoglobulin superfamily, localizes at tight junctions, and regulates both paracellular permeability and leukocyte transmigration. To investigate molecular determinants of JAM function, the extracellular domain of murine JAM was produced as a recombinant soluble protein (rsJAM) in insect cells. rsJAM consisted in large part of noncovalent homodimers, as assessed by analytical ultracentrifugation. JAM dimers were also detected at the surface of Chinese hamster ovary cells transfected with murine JAM, as evaluated by cross-linking and immunoprecipitation. Furthermore, fluid-phase rsJAM bound dose-dependently solid-phase rsJAM, and such homophilic binding was inhibited by anti-JAM Fab BV11, but not by Fab BV12. Interestingly, Fab BV11 exclusively bound rsJAM dimers (but not monomers) in solution, whereas Fab BV12 bound both dimers and monomers. Finally, we mapped the BV11 and BV12 epitopes to a largely overlapping sequence in proximity of the extracellular amino terminus of JAM. We hypothesize that rsJAM dimerization induces a BV11-positive conformation which in turn is critical for rsJAM homophilic interactions. Dimerization and homophilic binding may contribute to both adhesive function and junctional organization of JAM.     

The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability

We review here our work on the molecular and functional organization of endothelial cell-to-cell junctions. The first part of the review is dedicated to VE-cadherin, characterized by our group few years ago. This protein is a member of the large family of transmembrane adhesion proteins called cadherins. It is endothelial cell specific and plays a major role in the organization of adherens junctions. Inactivation of VE-cadherin gene or in vivo truncation of its cytoplasmic tail leads to a lethal phenotype due to the lack of correct organization of the vasculature in the embryo. We found that the defect was due to apoptosis of endothelial cells, which became unresponsive to the survival signal induced by vascular endothelial cell growth factor. Our data indicate that VE-cadherin may act as a scaffolding protein able to associate vascular endothelial cell growth factor receptor and to promote its signaling. In the second part of the review we consider another protein more recently discovered by us and called junctional adhesion molecule (JAM). This protein is a small immunoglobulin which is located at tight junctions in the endothelium and in epithelial cells. Evidence is discussed indicating that JAM takes part in the organization of tight junctions and modulates leukocyte extravasation through endothelial intercellular junctions in vitro and in vivo. The general role of tight junctions in endothelial cells is also discussed.     

Cutting edge: combined treatment of TNF-alpha and IFN-gamma causes redistribution of junctional adhesion molecule in human endothelial cells.

Proinflammatory cytokines such as TNF-alpha and IFN-gamma induce cell adhesion molecules in endothelial cells and promote transmigration of leukocytes across endothelial cells. However, when those two were administered together, leukocyte transmigration paradoxically decreased. We cloned a human and bovine homologue of the junctional adhesion molecule (JAM), a novel molecule at the tight junction, and examined the effects of proinflammatory cytokines on JAM in HUVECs. The combined treatment of TNF-alpha plus IFN-gamma caused a disappearance of JAM from intercellular junctions. However, flow cytometry, cell ELISA, and subcellular fractionation analysis demonstrated that the amount of JAM was not reduced. This suggested that JAM changed its distribution in response to proinflammatory cytokines. This redistribution of JAM might be involved in a decrease in transendothelial migration of leukocytes at inflammatory sites.     

JAM4, a junctional cell adhesion molecule interacting with a tight junction protein,MAGI-1

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.     

Cell junctions and their role in transmural diffusion in the embryonic chick heart

Summary Studies of cardiogenesis in the chick embryo focus attention upon the intercellular junctions of epicardial, myocardial, and endocardial cells, and the role they play in diffusion across the cardiac wall. Cell membranes of apposed epicardial cells approach as close together as 40 Å; those of the endocardium additionally form focal tight junctions. In the myocardium focal tight junctions are restricted to the apposed membranes of the superficial layer of cells. The majority of close appositions in all parts of the myocardium are 40 Å gap junctions. Desmosomes and fascia adherens are distributed throughout the myocardium.Diffusion of horseradish peroxidase through the epicardium and endocardium occurs primarily through the intercellular junctions. The width of the cleft between cells, 200–300 Å, also permits the diffusion between cells of the larger ferritin particles. Pinocytotic activity, responsible for ferritin transfer across mesothelial and endothelial cells in the adult, is not significant.Tracers injected into the pericardial cavity or vasculature can be observed passing through the heart in the direction of their respective diffusion gradients. Unlike the apical junctions of epithelial cells, to which they have been compared, membrane specializations of the superficial myocytes do not form a seal separating the pericardial cavity, or subepicardial space, from the extracellular spaces of the myocardium.Supported by the Medical Research Council of Canada.The author wishes to express his gratitude to Mrs. J. Blackbourn for her excellent technical assistance.     

Junctional adhesion molecule 1 regulates epithelial cell morphology through effects on beta1 integrins and Rap1 activity

Epithelial tight junctions form a selectively permeable barrier to ions and small molecules. Junctional adhesion molecule 1 (JAM1/JAM-A/F11R) is a tight junction-associated transmembrane protein that has been shown to participate in the regulation of epithelial barrier function. In a recent study, we presented evidence suggesting that JAM1 homodimer formation is critical for epithelial barrier function (Mandell, K. J., McCall, I. C., and Parkos, C. A. (2004) J. Biol. Chem. 279, 16254-16262). Here we have used small interfering RNA to investigate the effect of the loss of JAM1 expression on epithelial cell function. Consistent with our previous study, knockdown of JAM1 was observed to increase paracellular permeability in epithelial monolayers. Interestingly, knockdown of JAM1 also produced dramatic changes in cell morphology, and a similar effect was observed with expression of a JAM1 mutant lacking the putative homodimer interface. Further studies revealed that JAM1 knockdown decreased cell-matrix adhesion and spreading on matrix proteins that are ligands of beta1 integrins. These changes were characterized by a decrease in beta1 integrin protein levels and loss of beta1 integrin staining at the cell surface. Immunolabeling of cells for the small GTPase Rap1, a known activator of beta1 integrins, revealed colocalization of Rap1 with JAM1 at intercellular junctions, and knockdown of JAM1 resulted in decreased Rap1 activity. Lastly, knockdown of Rap1b resulted in diminished beta1 integrin expression and altered cell morphology analogous to that observed with knockdown of JAM1. Together, these results suggest that JAM1 regulates epithelial cell morphology and beta1 integrin expression by modulating activity of the small GTPase Rap1.     

Functional analysis of tight junctions

Epithelial and endothelial cells are joined to each other via a set of intercellular junctions that differ in their morphological appearance, composition, and function. The tight junction or zonula occludens is the intercellular junction that regulates diffusion between cells and therefore allows endothelia and epithelia to form cellular barriers that separate compartments of different composition. This intercellular gate formed by tight junctions is not only highly regulated but is size- and ion-selective and, hence, represents a semipermeable diffusion barrier. In epithelia, tight junctions form a morphological and functional border between the apical and basolateral cell surface domains. They directly contribute to the maintenance of cell surface polarity by forming a fence that prevents apical/basolateral diffusion of lipids in the outer leaflet of the plasma membrane. Here we describe a set of assays that allow the analysis of tight junctions to determine their integrity and functional state.     

JAM-C is a component of desmosomes and a ligand for CD11b/CD18-mediated neutrophil transepithelial migration

Neutrophil (PMN) transepithelial migration is dependent on the leukocyte beta(2) integrin CD11b/CD18, yet the identity of epithelial counterreceptors remain elusive. Recently, a JAM protein family member termed JAM-C was implicated in leukocyte adhesive interactions; however, its expression in epithelia and role in PMN-epithelial interactions are unknown. Here, we demonstrate that JAM-C is abundantly expressed basolaterally in intestinal epithelia and localizes to desmosomes but not tight junctions. Desmosomal localization of JAM-C was further confirmed by experiments aimed at selective disruption of tight junctions and desmosomes. In assays of PMN transepithelial migration, both JAM-C mAbs and JAM-C/Fc chimeras significantly inhibited the rate of PMN transmigration. Additional experiments revealed specific binding of JAM-C to CD11b/CD18 and provided evidence of other epithelial ligands for CD11b/CD18. These findings represent the first demonstration of direct adhesive interactions between PMN and epithelial intercellular junctions (desmosomes) that regulate PMN transepithelial migration and also suggest that JAM-C may play a role in desmosomal structure/function.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.     

Multifunctional strands in tight junctions

Tight junctions are one mode of cell-cell adhesion in epithelial and endothelial cellular sheets. They act as a primary barrier to the diffusion of solutes through the intercellular space, create a boundary between the apical and the basolateral plasma membrane domains, and recruit various cytoskeletal as well as signalling molecules at their cytoplasmic surface. New insights into the molecular architecture of tight junctions allow us to now discuss the structure and functions of this unique cell-cell adhesion apparatus in molecular terms.     

The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).

The establishment and maintenance of cellular polarity are critical for the development of multicellular organisms. PAR (partitioning-defective) proteins were identified in Caenorhabditis elegans as determinants of asymmetric cell division and polarized cell growth. Recently, vertebrate orthologues of two of these proteins, ASIP/PAR-3 and PAR-6, were found to form a signalling complex with the small GTPases Cdc42/Rac1 and with atypical protein kinase C (PKC). Here we show that ASIP/PAR-3 associates with the tight-junction-associated protein junctional adhesion molecule (JAM) in vitro and in vivo. No binding was observed with claudin-1, -4 or -5. In fibroblasts and CHO cells overexpressing JAM, endogenous ASIP is recruited to JAM at sites of cell-cell contact. Over expression of truncated JAM lacking the extracellular part disrupts ASIP/PAR-3 localization at intercellular junctions and delays ASIP/PAR-3 recruitment to newly formed cell junctions. During junction formation, JAM appears early in primordial forms of junctions. Our data suggest that the ASIP/PAR-3-aPKC complex is tethered to tight junctions via its association with JAM, indicating a potential role for JAM in the generation of cell polarity in epithelial cells.     

Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin

Junctional adhesion molecule (JAM) is an integral membrane protein that has been reported to colocalize with the tight junction molecules occludin, ZO-1, and cingulin. However, evidence for the association of JAM with these molecules is missing. Transfection of Chinese hamster ovary cells with JAM (either alone or in combination with occludin) resulted in enhanced junctional localization of both endogenous ZO-1 and cotransfected occludin. Additionally, JAM was coprecipitated with ZO-1 in the detergent-insoluble fraction of Caco-2 epithelial cells. A putative PDZ-binding motif at the cytoplasmic carboxyl terminus of JAM was required for mediating the interaction of JAM with ZO-1, as assessed by in vitro binding and coprecipitation experiments. JAM was also coprecipitated with cingulin, another cytoplasmic component of tight junctions, and this association required the amino-terminal globular head of cingulin. Taken together, these data indicate that JAM is a component of the multiprotein complex of tight junctions, which may facilitate junction assembly.     

RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin

Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity. epithelium; tight junctions; paracellular permeability; Madin-Darby canine kidney cells     

Junctional Adhesion Molecules (JAMs) are differentially expressed in fibroblasts and co-localize with ZO-1 to adherens-like junctions

Junctional Adhesion Molecules (JAMs) are components and regulators of the well-characterized epithelial and endothelial tight junction. Since the molecular components of native fibroblast adherens-like junctions remain poorly described we determined JAM expression profiles in fibroblasts. We found JAM-C on human dermal, lung, and corneal primary fibroblast cultures. Within murine lines, JAM-A was found in L-cells, JAM-C in 3T3 L1 cells, and both JAM-A and JAM-C were co-expressed in NIH 3T3 fibroblasts. In primary dermal fibroblasts, JAM-C concentrated at zipper-like junctions that formed between apposing cells. Dual immunostaining showed JAM-C co-localization with the ZO-1 intracellular scaffolding molecule at cell contacts that ranged from 7 μm to over 25 μm in length. JAM-C also labeled similar zipper-like junctions detected with N-Cadherin and Cadherin-11 antibodies. We conclude that endogenous JAM-C is an integral component of the dermal fibroblast adherens-like junction, and our data extend the expression and potential function of JAMs into mesenchymal tissues.     

Loss of Occludin and Functional Tight Junctions, but Not ZO-1, during Neural Tube Closure—Remodeling of the Neuroepithelium Prior to Neurogenesis

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappeared from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos revealed the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increased from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincided with that of N-cadherin, whose expression increased concomitantly with the disappearance of occludin. We propose that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons.     

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells

Shigella spp. are a group of Gram-negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype-specific mechanisms, which targets not only expression, but also cellular distribution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction-associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin-1 from Triton X-insoluble protein fractions upon apical exposure to T-84 cell monolayers. S. flexneri serotype 2a and 5, but not the non-invasive Escherichia coli strain F-18, share the ability to regulate expression of ZO-1, ZO-2, E-cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat-stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction-associated proteins.