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.     

Joint detection of claudin-1 and junctional adhesion molecule-A as a therapeutic target in oral epithelial dysplasia and oral squamous cell carcinoma

Abnormal expression of claudin-1 (CLDN-1) and junctional adhesion molecule-A (JAM-A) has been described in certain malignancies but their clinical relevance is poorly understood. The present study aims to elucidate the role of CLDN-1 and JAM-A in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Changes in the expression of these proteins were identified immunohistochemically on tissue sections from patients with OED and OSCC and compared with control. A correlation between the expression level of proteins and clinicopathological features was analyzed by Pearson's correlation χ² test. The survival curve of the follow-up data was estimated by the Kaplan-Meier method followed by the log-rank test. CLDN-1 and JAM-A were highly expressed in OED and OSCC tissues when compared to control. Also, delocalization of CLDN-1 from the membrane to the cytoplasm to the nucleus was observed as the cell proceeds from normal to malignancy. Increased expression of CLDN-1 and JAM-A in both OED and OSCC were concomitant with histological grades. In addition, increased JAM-A was associated with perineural invasion of cancer cells. A positive correlation between the expression level of proteins was observed in OED (r = 0.733) and OSCC (r = 0.577). Kaplan-Meier analysis in patients with OSCC showed that the survival rate was lower in patients with high CLDN-1 and high JAM-A expression compared to low expressed patients. To conclude, the elevated level and delocalization of CLDN-1 and JAM-A suggest their use as tumor markers. A positive correlation between CLDN-1 and JAM-A suggests joint detection of these proteins as a future diagnostic tool in oral precancerous and cancerous conditions.     

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.     

Junctional adhesion molecule-A regulates cell migration and resistance to shear stress

Junctional adhesion molecule-A (JAM-A) is an adhesive protein expressed in endothelial cells, epithelial cells, platelets, and some leukocytes. JAM-A localizes to the tight junctions between contacting endothelial and epithelial cells, where it contributes to cell-cell adhesion and to the control of paracellular permeability. JAM-A also regulates cell motility, even though the quantitative biophysical features have not been characterized. In this study, we evaluated the role of JAM-A in the regulation of cell motility using JAM-A-expressing and JAM-A-deficient murine endothelial cells. We report that, in the absence of shear stress, JAM-A absence increases cell motility by increasing directional persistence but not cell speed. In addition, in the presence of shear stress, JAM-A absence increases protrusion extension in the direction of flow and increased downstream cellular displacement (while, conversely, decreasing upstream displacement). All these effects of JAM-A absence are mitigated by the microtubule-stabilizing compound taxol. A motility- and microtubule-related function, integrin-mediated adhesiveness, was only slightly reduced in JAM-A-deficient cells compared with JAM-A-expressing cells. However, overexpression of JAM-A in the JAM-A-deficient cells increased integrin adhesiveness to the same levels as those observed in taxol-treated JAM-A-deficient cells. Taken together, these data indicate that JAM-A regulates cell motility by cooperating with microtubule-stabilizing pathways.     

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.     

Development of near-infrared imaging agents for detection of junction adhesion molecule-A protein

IntroductionProstate and breast cancer are the most prevalent primary malignant human tumors globally. Prostatectomy and breast conservative surgery remain the most common definitive treatment option for the >500,000 men and women newly diagnosed with localized prostate and breast cancer each year only in the US. Morphological examination is the mainstay of diagnosis but margin under-sampling of the excised cancer tissue may lead to local recurrence. In despite of the progress of non-invasive optical imaging, there is still a clinical need for targeted optical imaging probes that could rapidly and globally visualize cancerous tissues.MethodsElevated expression of junctional adhesion molecule-A (JAM-A) on tumor cells and its multiple pro-tumorigenic activity make the JAM-A a candidate for molecular imaging. Near-infrared imaging probe, which employed anti-JAM-A monoclonal antibody (mAb) phthalocyanine dye IR700 conjugates (JAM-A mAb/IR700), was synthesized and used to identify and visualize heterotopic human prostate and breast tumor mouse xenografts in vivo.ResultsThe intravenously injected JAM-A mAb/IR700 conjugates enabled the non-invasive detection of prostate and breast cancerous tissue by fluorescence imaging. A single dose of JAM-A mAb/IR700 reduced number of mitotic cancer cells in vivo, indicating theranostic ability of this imaging agent. The JAM-A mAb/IR700 conjugates allowed us to image a specific receptor expression in prostate and breast tumors without post-image processing.ConclusionThis agent demonstrates promise as a method to image the extent of prostate and breast cancer in vivo and could assist with real-time visualization of extracapsular extension of cancerous tissue.     

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 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.     

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...