N-glycosylation controls the function of junctional adhesion molecule-A

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells. JAM-A serves many roles and contributes to barrier function and cell migration and motility, and it also acts as a ligand for the leukocyte receptor LFA-1. JAM-A is reported to contain N-glycans, but the extent of this modification and its contribution to the protein’s functions are unknown. We show that human JAM-A contains a single N-glycan at N185 and that this residue is conserved across multiple mammalian species. A glycomutant lacking all N-glycans, N185Q, is able to reach the cell surface but exhibits decreased protein half-life compared with the wild- type protein. N-glycosylation of JAM-A is required for the protein’s ability to reinforce barrier function and contributes to Rap1 activity. We further show that glycosylation of N185 is required for JAM-A–mediated reduction of cell migration. Finally, we show that N-glycosylation of JAM-A regulates leukocyte adhesion and LFA-1 binding. These findings identify N-glycosylation as critical for JAM-A’s many functions.     

Abrogation of junctional adhesion molecule-A expression induces cell apoptosis and reduces breast cancer progression

Intercellular junctions promote homotypic cell to cell adhesion and transfer intracellular signals which control cell growth and apoptosis. Junctional adhesion molecule-A (JAM-A) is a transmembrane immunoglobulin located at tight junctions of normal epithelial cells of mammary ducts and glands. In the present paper we show that JAM-A acts as a survival factor for mammary carcinoma cells. JAM-A null mice expressing Polyoma Middle T under MMTV promoter develop significantly smaller mammary tumors than JAM-A positive mice. Angiogenesis and inflammatory or immune infiltrate were not statistically modified in absence of JAM-A but tumor cell apoptosis was significantly increased. Tumor cells isolated from JAM-A null mice or 4T1 cells incubated with JAM-A blocking antibodies showed reduced growth and increased apoptosis which paralleled altered junctional architecture and adhesive function. In a breast cancer clinical data set, tissue microarray data show that JAM-A expression correlates with poor prognosis. Gene expression analysis of mouse tumor samples showed a correlation between genes enriched in human G3 tumors and genes over expressed in JAM-A +/+ mammary tumors. Conversely, genes enriched in G1 human tumors correlate with genes overexpressed in JAM-A-/- tumors. We conclude that down regulation of JAM-A reduces tumor aggressive behavior by increasing cell susceptibility to apoptosis. JAM-A may be considered a negative prognostic factor and a potential therapeutic target.     

Downregulation of junctional adhesion molecule-A is involved in the progression of clear cell renal cell carcinoma

Junctional adhesion molecule-A (JAM-A) is one component of tight junctions which are involved in important processes like paracellular permeability, cell polarity, adhesion, migration, and angiogenesis. Here we describe JAM-A expression in distal convoluted tubule, connecting tubule, and in cells of the collecting duct of the healthy human kidney. In addition, JAM-A was weakly expressed in cells of the proximal tubule. Using immunofluorescence, FACS and Western blot analysis we investigated JAM-A expression in tubular cells in vitro. Interestingly, treatment of HK-2 cells with IFN-γ and TNF-α resulted in a metalloproteinase mediated downregulation of JAM-A. Importantly, in a tissue micro-array JAM-A protein expression was significantly downregulated in patients with clear cell renal cell carcinoma. Furthermore, knockdown of JAM-A with JAM-A specific siRNA induced the migration of RCC4 cells. In summary, downregulation of JAM-A is an early event in the development of renal cancer and increases the migration of renal cancer cells.     

Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells

Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.     

Evaluation of soluble junctional adhesion molecule-A as a biomarker of human brain endothelial barrier breakdown

Background

An inducible release of soluble junctional adhesion molecule-A (sJAM-A) under pro-inflammatory conditions was described in cultured non-CNS endothelial cells (EC) and increased sJAM-A serum levels were found to indicate inflammation in non-CNS vascular beds. Here we studied the regulation of JAM-A expression in cultured brain EC and evaluated sJAM-A as a serum biomarker of blood-brain barrier (BBB) function.

Methodology/Principal Findings

As previously reported in non-CNS EC types, pro-inflammatory stimulation of primary or immortalized (hCMEC/D3) human brain microvascular EC (HBMEC) induced a redistribution of cell-bound JAM-A on the cell surface away from tight junctions, along with a dissociation from the cytoskeleton. This was paralleled by reduced immunocytochemical staining of occludin and zonula occludens-1 as well as by increased paracellular permeability for dextran 3000. Both a self-developed ELISA test and Western blot analysis detected a constitutive sJAM-A release by HBMEC into culture supernatants, which importantly was unaffected by pro-inflammatory or hypoxia/reoxygenation challenge. Accordingly, serum levels of sJAM-A were unaltered in 14 patients with clinically active multiple sclerosis compared to 45 stable patients and remained unchanged in 13 patients with acute ischemic non-small vessel stroke over time.

Conclusion

Soluble JAM-A was not suited as a biomarker of BBB breakdown in our hands. The unexpected non-inducibility of sJAM-A release at the human BBB might contribute to a particular resistance of brain EC to inflammatory stimuli, protecting the CNS compartment.     

The homophilic binding of junctional adhesion molecule-C mediates tumor cell-endothelial cell interactions

The junctional adhesion molecule C (JAM-C) was recently shown to undergo a heterophilic interaction with the leukocyte beta2 integrin Mac-1, thereby mediating interactions between vascular cells in inflammatory cell recruitment. Here, the homophilic interaction of JAM-C is presented and functionally characterized to mediate tumor cell-endothelial cell interactions. Recombinant soluble JAM-C in fluid phase bound to immobilized JAM-C as assessed in a purified system; moreover, JAM-C-transfected Chinese hamster ovary (CHO) cells adhered to immobilized JAM-C. The homophilic interaction of JAM-C was mediated by the isolated amino-terminal Ig domain (D1), but not the carboxyl-terminal Ig domain (D2), of the molecule. Dimerization of JAM-A is dependent on the sequence RVE in the amino-terminal Ig domain. This motif is conserved in JAM-C (Arg64-Ile65-Glu66), and a single amino acid mutation in this motif (E66R) abolished the homophilic interaction of JAM-C. The lung carcinoma cell line NCI-H522 was found to express JAM-C. NCI-H522 cells adhered to immobilized JAM-C, as well as to JAM-C-transfected CHO cells, but not to mock-transfected CHO cells or to CHO cells transfected with the JAM-C mutant (E66R). Adhesion of NCI-H522 cells to JAM-C protein or JAM-C-transfected CHO cells was abolished in the presence of soluble JAM-C or the isolated D1. Furthermore, the adhesion of NCI-H522 cells to endothelial cells was significantly blocked by soluble JAM-C or the isolated D1. Thus, JAM-C undergoes a homophilic interaction via the Arg64-Ile65-Glu66 motif on the membrane-distal Ig domain of the molecule. The homophilic interaction of JAM-C can mediate tumor cell-endothelial cell interactions and may thereby be involved in the process of tumor cell metastasis.     

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 JAM family of junctional adhesion molecules

Junctional adhesion molecules are a family of glycoproteins characterised by two immunoglobulin folds (VH- and C2-type) in the extracellular domain. Junctional adhesion molecule proteins localise to intercellular junctions of polarised endothelial and epithelial cells but can also be expressed on circulating leukocytes and platelets. In addition, they bind several ligands, in both a homophilic and heterophilic manner, and associate with several cytoplasmic partners. All these features represent the likely determinants for the role of junctional adhesion molecule proteins in processes as diverse as junction assembly, leukocyte transmigration and platelet activation.     

Identification of regions and residues in feline junctional adhesion molecule required for feline calicivirus binding and infection

The feline junctional adhesion molecule A (fJAM-A) is a functional receptor for feline calicivirus (FCV). fJAM-A is a member of the immunoglobulin superfamily (IgSF) and consists of two Ig-like extracellular domains (D1 and D2), a membrane-spanning domain, and a short cytoplasmic tail. To identify regions of fJAM-A that interact with FCV, we purified recombinant fJAM-A ectodomain and D1 and D2 domains. We found that preincubation of FCV with the ectodomain or D1 was sufficient to inhibit FCV infection in plaque reduction assays. In enzyme-linked immunosorbent assays, FCV binding to fJAM-A ectodomain was concentration dependent and saturable; however, FCV bound D1 alone weakly and was unable to bind D2. To characterize FCV binding to surface-expressed fJAM-A, we transfected truncated and chimeric forms of fJAM-A into a nonpermissive cell line and assayed binding by flow cytometry. Only D1 was necessary for FCV binding to cells; all other domains could be replaced. Using a structure-guided mutational approach, we identified three mutants of fJAM-A within D1 (D42N, K43N, and S97A) that exhibited significantly decreased capacities to bind FCV. In contrast to our finding that D1 mediated FCV binding, we found that all domains of fJAM-A were necessary to confer susceptibility to FCV infection. Furthermore, surface expression of fJAM-A was not sufficient to permit FCV infection by all of the isolates we investigated. This indicates that (i) other cellular factors are required to permit productive FCV infection and (ii) individual FCV isolates differ in the factors they require.     

The role of junctional adhesion molecules in vascular inflammation

Junctional adhesion molecules (JAMs) of the immunoglobulin superfamily are important in the control of vascular permeability and leukocyte transmigration across endothelial-cell surfaces, by engaging in homophilic, heterophilic and lateral interactions. Through their localization on the endothelial-cell surface and expression by platelets, JAMs contribute to adhesive interactions with circulating leukocytes and platelets. Antibody-blocking studies and studies using genetically modified mice have implicated these functions of JAMs in the regulation of leukocyte recruitment to sites of inflammation and ischaemia-reperfusion injury, in growth-factor-mediated angiogenesis, atherogenesis and neointima formation. The comparison of different JAM-family members and animal models, however, shows that the picture remains rather complex. This Review summarizes recent progress and future directions in understanding the role of JAMs as 'gate keepers' in inflammation and vascular pathology.     

The role of junctional adhesion molecules in cell-cell interactions

Cell-cell-interactions are important for the regulation of tissue integrity, the generation of barriers between different tissues and body compartments thereby providing an effective defence against toxic or pathogenic agents, as well as for the regulation of inflammatory cell recruitment. Intercellular interactions are regulated by adhesion receptors on adjacent cells which upon extracellular ligand binding mediate intracellular signals. In the vasculature, neighbouring endothelial cells interact with each other through various adhesion molecules leading to the generation of junctional complexes like tight junctions (TJs) and adherens junctions (AJs) which regulate both leukocyte endothelial interactions and paracellular permeability. In this context, emerging evidence points to the importance of the family of junctional adhesion molecules (JAMs), which are localized in tight junctions of endothelial and epithelial cells and are implicated in the regulation of both leukocyte extravasation as well as junction formation and permeability.     

Structure-function analysis of reovirus binding to junctional adhesion molecule 1. Implications for the mechanism of reovirus attachment

Mammalian reoviruses are nonenveloped viruses with a long, filamentous attachment protein that dictates disease phenotypes following infection of newborn mice and is a structural homologue of the adenovirus attachment protein. Reoviruses use junctional adhesion molecule 1 (JAM1) as a serotype-independent cellular receptor. JAM1 is a broadly expressed immunoglobulin superfamily protein that forms stable homodimers and regulates tight-junction permeability and lymphocyte trafficking. We employed a series of structure-guided binding and infection experiments to define residues in human JAM1 (hJAM1) important for reovirus-receptor interactions and to gain insight into mechanisms of reovirus attachment. Binding and infection experiments using chimeric and domain deletion mutant receptor molecules indicate that the amino-terminal D1 domain of hJAM1 is required for reovirus attachment, infection, and replication. Reovirus binding to hJAM1 occurs more rapidly than homotypic hJAM1 association and is competed by excess hJAM1 in vitro and on cells. Cross-linking hJAM1 diminishes the capacity of reovirus to bind hJAM1 in vitro and on cells and negates the competitive effects of soluble hJAM1 on reovirus attachment. Finally, mutagenesis studies demonstrate that residues intimately associated with the hJAM1 dimer interface are critical for reovirus interactions with hJAM1. These results suggest that reovirus attachment disrupts hJAM1 dimers and highlight similarities between the attachment strategies of reovirus and adenovirus.     

Reovirus binding to cell surface sialic acid potentiates virus-induced apoptosis

Reovirus induces apoptosis in cultured cells and in vivo. Genetic studies indicate that the efficiency with which reovirus strains induce apoptosis is determined by the viral S1 gene, which encodes attachment protein sigma1. However, the biochemical properties of sigma1 that influence apoptosis induction are unknown. To determine whether the capacity of sigma1 to bind cell surface sialic acid determines the magnitude of the apoptotic response, we used isogenic reovirus mutants that differ in the capacity to engage sialic acid. We found that T3SA+, a virus capable of binding sialic acid, induces high levels of apoptosis in both HeLa cells and L cells. In contrast, non-sialic-acid-binding strain T3SA- induces little or no apoptosis in these cell types. Differences in the capacity of T3SA- and T3SA+ to induce apoptosis are not due to differences in viral protein synthesis or production of viral progeny. Removal of cell surface sialic acid with neuraminidase abolishes the capacity of T3SA+ to induce apoptosis. Similarly, incubation of T3SA+ with sialyllactose, a trisaccharide comprised of lactose and sialic acid, blocks apoptosis. These findings demonstrate that reovirus binding to cell surface sialic acid is a critical requirement for the efficient induction of apoptosis and suggest that virus receptor utilization plays an important role in regulating cell death.     

Runx2 deficiency in junctional epithelium of mouse molars decreases the expressions of E-cadherin and junctional adhesion molecule 1

Journal of Molecular Histology - Junctional epithelium (JE) attaching to the enamel surface seals gaps around the teeth, functioning as the first line of gingival defense. Runt-related...     

Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.     

X-ray structure of junctional adhesion molecule: structural basis for homophilic adhesion via a novel dimerization motif

Junctional adhesion molecules (JAMs) are a family of immunoglobulin-like single-span transmembrane molecules that are expressed in endothelial cells, epithelial cells, leukocytes and myocardia. JAM has been suggested to contribute to the adhesive function of tight junctions and to regulate leukocyte trans migration. We describe the crystal structure of the recombinant extracellular part of mouse JAM (rsJAM) at 2.5 A resolution. rsJAM consists of two immunoglobulin-like domains that are connected by a conformationally restrained short linker. Two rsJAM molecules form a U-shaped dimer with highly complementary interactions between the N-terminal domains. Two salt bridges are formed in a complementary manner by a novel dimerization motif, R(V,I,L)E, which is essential for the formation of rsJAM dimers in solution and common to the known members of the JAM family. Based on the crystal packing and studies with mutant rsJAM, we propose a model for homophilic adhesion of JAM. In this model, U-shaped JAM dimers are oriented in cis on the cell surface and form a two-dimensional network by trans-interactions of their N-terminal domains with JAM dimers from an opposite cell surface.