JAM-C Is Important for Lens Epithelial Cell Proliferation and Lens Fiber Maturation in Murine Lens Development.

Purpose: The underlying mechanism of congenital cataracts caused by deficiency or mutation of junctional adhesion molecule C (JAM-C) gene remains unclear. Our study aims to elucidate the abnormal developmental process in Jamc-/- lenses and reveal the genes related to lens development that JAM-C may regulate. Methods: Jamc knockout (Jamc-/-) mouse embryos and pups were generated for in vivo studies. Four key developmental stages from embryonic day (E) 12.5 to postnatal day (P) 0.5 were selected for the following experiments. Hematoxylin and eosin staining was used for histological analysis. The 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and TUNEL staining were performed to label lens epithelial cell (LEC) proliferation and apoptosis, respectively. Immunofluorescence and Western blot were used to analyze the markers of lens epithelium, cell cycle exit, and lens fiber differentiation. Results: JAM-C was expressed throughout the process of lens development. Deletion of Jamc resulted in decreased lens size and disorganized lens fibers, which arose from E16.5 and aggravated gradually. The LECs of Jamc-/- lenses showed decreased quantity and proliferation, accompanied with reduction of key transcription factor, FOXE3. The fibers in Jamc-/- lenses were disorganized. Moreover, Jamc-deficient lens fibers showed significantly altered distribution patterns of Cx46 and Cx50. The marker of fiber homeostasis, γ-crystallin, was also decreased in the inner cortex and core fibers of Jamc-/- lenses. Conclusions: Deletion of JAM-C exhibits malfunction of LEC proliferation and fiber maturation during murine lens development, which may be related to the downregulation of FOXE3 expression and abnormal localization patterns of Cx46 and Cx50.

Decreased junctional adhesion molecule 3 expression induces reactive oxygen species production and apoptosis in trophoblasts†.

Junctional adhesion molecule 3 (JAM3) is involved in epithelial cell junction, cell polarity, and motility. The molecular mechanisms underlying the role of JAM3 in placental dysfunction remain unclear. We hypothesized that JAM3 expression regulates trophoblast fusion, differentiation, proliferation, and apoptosis. Our results revealed that JAM3 was expressed in the cytotrophoblasts and syncytiotrophoblasts of first-trimester and term placental villi. JAM3 expression in cell-cell junctions decreased with the formation of syncytiotrophoblasts. Using trophoblasts as an in vitro model, we observed that forskolin and JAM3 knockdown significantly reduced JAM3 expression and increased syncytium formation. JAM3 knockdown additionally inhibited trophoblast proliferation and increased the number of trophoblasts in the sub-G1 and G2/M phases, indicating cell-cycle disturbance and apoptosis. Cell-cycle arrest was associated with the engagement of checkpoint kinase 2-cell division cycle 25C-cyclin-dependent kinase 1/cyclin B1 signaling. Increased expression of BIM, NOXA, XAF1, cytochrome c, and cleaved caspase-3 further indicated trophoblast apoptosis. Overexpression of JAM3 or recombinant JAM3 protein enhanced trophoblast adhesion and migration, which were inhibited by JAM3 knockdown. JAM3 knockdown induced reactive oxygen species and syncytin 2 expression in trophoblasts. Furthermore, H2O2-induced oxidative stress reduced JAM3 expression in trophoblasts and cell culture supernatants. H2O2 simultaneously induced trophoblast apoptosis. JAM3 expression was significantly decreased in the plasmas and placentas of patients with early-onset severe preeclampsia. Thus, our results show that JAM3 may not only be a structural component of trophoblast cell junctions but also regulates trophoblast fusion, differentiation, proliferation, apoptosis, and motility. Dysregulated trophoblast JAM3 expression is crucial in preeclampsia development.

Immunoglobulin superfamily receptor Junctional adhesion molecule 3 (Jam3) requirement for melanophore survival and patterning during formation of zebrafish stripes.

Adhesive interactions are essential for tissue patterning and morphogenesis yet difficult to study owing to functional redundancies across genes and gene families. A useful system in which to dissect roles for cell adhesion and adhesion-dependent signaling is the pattern formed by pigment cells in skin of adult zebrafish, in which stripes represent the arrangement of neural crest derived melanophores, cells homologous to melanocytes. In a forward genetic screen for adult pattern defects, we isolated the pissarro (psr) mutant, having a variegated phenotype of spots, as well as defects in adult fin and lens. We show that psr corresponds to junctional adhesion protein 3b (jam3b) encoding a zebrafish orthologue of the two immunoglobulin-like domain receptor JAM3 (JAM-C), known for roles in adhesion and signaling in other developing tissues, and for promoting metastatic behavior of human and murine melanoma cells. We found that zebrafish jam3b is expressed post-embryonically in a variety of cells including melanophores, and that jam3b mutants have defects in melanophore survival. Jam3b supported aggregation of cells in vitro and was required autonomously by melanophores for an adherent phenotype in vivo. Genetic analyses further indicated both overlapping and non-overlapping functions with the related receptor, Immunoglobulin superfamily 11 (Igsf11) and Kit receptor tyrosine kinase. These findings suggest a model for Jam3b function in zebrafish melanophores and hint at the complexity of adhesive interactions underlying pattern formation.

Junctional Adhesion Molecule-C Mediates the Recruitment of Embryonic-Endothelial Progenitor Cells to the Perivascular Niche during Tumor Angiogenesis.

: The homing of Endothelial Progenitor Cells (EPCs) to tumor angiogenic sites has been described as a multistep process, involving adhesion, migration, incorporation and sprouting, for which the underlying molecular and cellular mechanisms are yet to be fully defined. Here, we studied the expression of Junctional Adhesion Molecule-C (JAM-C) by EPCs and its role in EPC homing to tumor angiogenic vessels. For this, we used mouse embryonic-Endothelial Progenitor Cells (e-EPCs), intravital multi-fluorescence microscopy techniques and the dorsal skin-fold chamber model. JAM-C was found to be expressed by e-EPCs and endothelial cells. Blocking JAM-C did not affect adhesion of e-EPCs to endothelial monolayers in vitro but, interestingly, it did reduce their adhesion to tumor endothelium in vivo. The most striking effect of JAM-C blocking was on tube formation on matrigel in vitro and the incorporation and sprouting of e-EPCs to tumor endothelium in vivo. Our results demonstrate that JAM-C mediates e-EPC recruitment to tumor angiogenic sites, i.e., coordinated homing of EPCs to the perivascular niche, where they cluster and interact with tumor blood vessels. This suggests that JAM-C plays a critical role in the process of vascular assembly and may represent a potential therapeutic target to control tumor angiogenesis.

Junctional adhesion molecule C (JAM-C) dimerization aids cancer cell migration and metastasis.

Most cancer deaths result from metastasis, which is the dissemination of cells from a primary tumor to distant organs. Metastasis involves changes to molecules that are essential for tumor cell adhesion to the extracellular matrix and to endothelial cells. Junctional Adhesion Molecule C (JAM-C) localizes at intercellular junctions as homodimers or more affine heterodimers with JAM-B. We previously showed that the homodimerization site (E66) in JAM-C is also involved in JAM-B binding. Here we show that neoexpression of JAM-C in a JAM-C-negative carcinoma cell line induced loss of adhesive property and pro-metastatic capacities. We also identify two critical structural sites (E66 and K68) for JAM-C/JAM-B interaction by directed mutagenesis of JAM-C and studied their implication on tumor cell behavior. JAM-C mutants did not bind to JAM-B or localize correctly to junctions. Moreover, mutated JAM-C proteins increased adhesion and reduced proliferation and migration of lung carcinoma cell lines. Carcinoma cells expressing mutant JAM-C grew slower than with JAM-C WT and were not able to establish metastatic lung nodules in mice. Overall these data demonstrate that the dimerization sites E66-K68 of JAM-C affected cell adhesion, polarization and migration and are essential for tumor cell metastasis.

S-Palmitoylation of Junctional Adhesion Molecule C Regulates Its Tight Junction Localization and Cell Migration.

Junctional adhesion molecule C (JAM-C) is an immunoglobulin superfamily protein expressed in epithelial cells, endothelial cells, and leukocytes. JAM-C has been implicated in leukocyte transendothelial migration, angiogenesis, cell adhesion, cell polarity, spermatogenesis, and metastasis. Here, we show that JAM-C undergoes S-palmitoylation on two juxtamembrane cysteine residues, Cys-264 and Cys-265. We have identified DHHC7 as a JAM-C palmitoylating enzyme by screening all known palmitoyltransferases (DHHCs). Ectopic expression of DHHC7, but not a DHHC7 catalytic mutant, enhances JAM-C S-palmitoylation. Moreover, DHHC7 knockdown decreases the S-palmitoylation level of JAM-C. Palmitoylation of JAM-C promotes its localization to tight junctions and inhibits transwell migration of A549 lung cancer cells. These results suggest that S-palmitoylation of JAM-C can be potentially targeted to control cancer metastasis.

Divergent JAM-C Expression Accelerates Monocyte-Derived Cell Exit from Atherosclerotic Plaques.

Atherosclerosis, caused in part by monocytes in plaques, continues to be a disease that afflicts the modern world. Whilst significant steps have been made in treating this chronic inflammatory disease, questions remain on how to prevent monocyte and macrophage accumulation in atherosclerotic plaques. Junctional Adhesion Molecule C (JAM-C) expressed by vascular endothelium directs monocyte transendothelial migration in a unidirectional manner leading to increased inflammation. Here we show that interfering with JAM-C allows reverse-transendothelial migration of monocyte-derived cells, opening the way back out of the inflamed environment. To study the role of JAM-C in plaque regression we used a mouse model of atherosclerosis, and tested the impact of vascular JAM-C expression levels on monocyte reverse transendothelial migration using human cells. Studies in-vitro under inflammatory conditions revealed that overexpression or gene silencing of JAM-C in human endothelium exposed to flow resulted in higher rates of monocyte reverse-transendothelial migration, similar to antibody blockade. We then transplanted atherosclerotic, plaque-containing aortic arches from hyperlipidemic ApoE-/- mice into wild-type normolipidemic recipient mice. JAM-C blockade in the recipients induced greater emigration of monocyte-derived cells and further diminished the size of atherosclerotic plaques. Our findings have shown that JAM-C forms a one-way vascular barrier for leukocyte transendothelial migration only when present at homeostatic copy numbers. We have also shown that blocking JAM-C can reduce the number of atherogenic monocytes/macrophages in plaques by emigration, providing a novel therapeutic strategy for chronic inflammatory pathologies.

Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis.

Classical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.