JAM2 is a prognostic biomarker and inhibits proliferation, metastasis and epithelial-mesenchymal transition in lung adenocarcinoma.

BACKGROUND: Junctional adhesion molecule 2 (JAM2) plays a pivotal role in various biological processes, including proliferation, metastasis and angiogenesis, contributing to tumor progression. While previous studies have highlighted the polarizing functions of JAM2 in different cancer types, its specific role in lung adenocarcinoma (LUAD) remains unclear. METHODS: In this study, we harnessed multiple public databases to analyze the expression and prognostic significance of JAM2 in LUAD. Using the Linkedomics database, Matescape database and R package, we explored the associated genes, the potential biological functions and the impact of JAM2 on the tumor microenvironment. Our findings from public databases were further validated using real-time quantitative PCR, western blot and immunohistochemistry. Additionally, in vitro experiments were conducted to assess the influence of JAM2 on LUAD cell proliferation, invasion, migration, apoptosis and epithelial-mesenchymal transition. Furthermore, we established a xenograft model to investigate the in vivo effects of JAM2 on tumorigenesis. RESULTS: Our results revealed a significant downregulation of JAM2 in LUAD, and patients with low JAM2 expression exhibited unfavorable overall survival outcomes. Functional enrichment analysis indicated that JAM2 may be associated with processes such as cell adhesion, extracellular matrix, cell junctions and regulation of proliferation. Notably, increased JAM2 expression correlated with higher tumor microenvironment scores and reduced immune cell abundance. Furthermore, overexpression of JAM2 induced apoptosis, suppressed tumor proliferation and exhibited potential inhibitory effects on tumor invasion and migration through the modulation of epithelial-mesenchymal transition. Additionally, in vivo experiments confirmed that JAM2 overexpression led to a reduction in tumor growth. CONCLUSION: Overall, our study highlights the clinical significance of low JAM2 expression as a predictor of poor prognosis in LUAD patients. Moreover, JAM2 was found to exert inhibitory effects on various aspects of tumor progression. Consequently, JAM2 emerges as a promising prognostic biomarker and a potential therapeutic target for LUAD patients.

Cell fusion is differentially regulated in zebrafish post-embryonic slow and fast muscle.

Skeletal muscle fusion occurs during development, growth, and regeneration. To investigate how muscle fusion compares among different muscle cell types and developmental stages, we studied muscle cell fusion over time in wild-type, myomaker (mymk), and jam2a mutant zebrafish. Using live imaging, we show that embryonic myoblast elongation and fusion correlate tightly with slow muscle cell migration. In wild-type embryos, only fast muscle fibers are multinucleate, consistent with previous work showing that the cell fusion regulator gene mymk is specifically expressed throughout the embryonic fast muscle domain. However, by 3 weeks post-fertilization, slow muscle fibers also become multinucleate. At this late-larval stage, mymk is not expressed in muscle fibers, but is expressed in small cells near muscle fibers. Although previous work showed that both mymk and jam2a are required for embryonic fast muscle cell fusion, we observe that muscle force and function is almost normal in mymk and jam2a mutant embryos, despite the lack of fast muscle multinucleation. We show that genetic requirements change post-embryonically, with jam2a becoming much less important by late-larval stages and mymk now required for muscle fusion and growth in both fast and slow muscle cell types. Correspondingly, adult mymk mutants perform poorly in sprint and endurance tests compared to wild-type and jam2a mutants. We show that adult mymk mutant muscle contains small mononucleate myofibers with average myonuclear domain size equivalent to that in wild type adults. The mymk mutant fibers have decreased Laminin expression and increased numbers of Pax7-positive cells, suggesting that impaired fiber growth and active regeneration contribute to the muscle phenotype. Our findings identify several aspects of muscle fusion that change with time in slow and fast fibers as zebrafish develop beyond embryonic stages.

Junctional Adhesion Molecule 2 Represents a Subset of Hematopoietic Stem Cells with Enhanced Potential for T Lymphopoiesis.

The distinct lineage potential is a key feature of hematopoietic stem cell (HSC) heterogeneity, but a subset of HSCs specialized for a single lymphoid compartment has not been identified. Here we report that HSCs expressing junctional adhesion molecule 2 (Jam2) at a higher level (Jam2high HSCs) have a greater T cell reconstitution capacity. Jam2high HSCs are metabolically dormant but preferentially differentiate toward lymphocytes, especially T cell lineages. Jam2high HSCs uniquely express T cell-related genes, and the interaction with Jam1 facilitates the Notch/Delta signaling pathway. Frequency of Jam2high HSCs changes upon T cell depletion in vivo, potentially suggesting that Jam2 expression may reflect scarcity of T cells and requirement of T cell replenishment. Our findings highlight Jam2 as a potential marker for a subfraction of HSCs with an extensive lymphopoietic capacity, mainly in T lymphopoiesis.

Genetic Mutations in jamb, jamc, and myomaker Revealed Different Roles on Myoblast Fusion and Muscle Growth.

Myoblast fusion is a vital step for skeletal muscle development, growth, and regeneration. Loss of Jamb, Jamc, or Myomaker (Mymk) function impaired myoblast fusion in zebrafish embryos. In addition, mymk mutation hampered fish muscle growth. However, the effect of Jamb and Jamc deficiency on fish muscle growth is not clear. Moreover, whether jamb;jamc and jamb;mymk double mutations have stronger effects on myoblast fusion and muscle growth remains to be investigated. Here, we characterized the muscle development and growth in jamb, jamc, and mymk single and double mutants in zebrafish. We found that although myoblast fusion was compromised in jamb and jamc single or jamb;jamc double mutants, these mutant fish showed no defect in muscle cell fusion during muscle growth. The mutant fish were able to grow into adults that were indistinguishable from the wild-type sibling. In contrast, the jamb;mymk double mutants exhibited a stronger muscle phenotype compared to the jamb and jamc single and double mutants. The jamb;mymk double mutant showed reduced growth and partial lethality, similar to a mymk single mutant. Single fiber analysis of adult skeletal myofibers revealed that jamb, jamc, or jamb;jamc mutants contained mainly multinucleated myofibers, whereas jamb;mymk double mutants contained mostly mononucleated fibers. Significant intramuscular adipocyte infiltration was found in skeletal muscles of the jamb;mymk mutant. Collectively, these studies demonstrate that although Jamb, Jamc, and Mymk are all involved in myoblast fusion during early myogenesis, they have distinct roles in myoblast fusion during muscle growth. While Mymk is essential for myoblast fusion during both muscle development and growth, Jamb and Jamc are dispensable for myoblast fusion during muscle growth.

Lack of junctional adhesion molecule (JAM)-B ameliorates experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) autoaggressive CD4+ T cells cross the blood-brain barrier (BBB) and cause neuroinflammation. Therapeutic targeting of CD4+ T-cell trafficking into the CNS by blocking α4-integrins has proven beneficial for the treatment of MS but comes with associated risks, probably due to blocking CD8+ T cell mediated CNS immune surveillance. Our recent observations show that CD8+ T cells also rely on α4ß1-integrins to cross the BBB. Besides vascular cell adhesion molecule-1 (VCAM-1), we identified junctional adhesion molecule-B (JAM-B) as a novel vascular α4ß1-integrin ligand involved in CD8+ T-cell migration across the BBB. This prompted us to investigate, if JAM-B also mediates CD4+ T-cell migration across the BBB. We first ensured that encephalitogenic T cells can bind to JAM-B in vitro and next compared EAE pathogenesis in JAM-B-/- C57BL/6J mice and their wild-type littermates. Following immunization with MOGaa35-55 peptide, JAM-B-/- mice developed ameliorated EAE compared to their wild-type littermates. At the same time, we isolated higher numbers of CD45+ infiltrating immune cells from the CNS of JAM-B-/- C57BL/6J mice suffering from EAE. Immunofluorescence staining revealed that the majority of CD45+ inflammatory cells accumulated in the leptomeningeal and perivascular spaces of the CNS behind the BBB but do not gain access to the CNS parenchyma. Trapping of CNS inflammatory cells was not due to increased inflammatory cell proliferation. Neither a loss of BBB integrity or BBB polarity potentially affecting local chemokine gradients nor a lack of focal gelatinase activation required for CNS parenchymal immune cell entry across the glia limitans could be detected in JAM-B-/- mice. Lack of a role for JAM-B in the effector phase of EAE was supported by the observation that we did not detect any role for JAM-B in EAE pathogenesis, when EAE was elicited by in vitro activated MOG aa35-55-specific CD4+ effector T cells. On the other hand, we also failed to demonstrate any role of JAM-B in in vivo priming, proliferation or polarization of MOGaa35-55-specific CD4+ T cells in peripheral immune organs. Finally, our study excludes expression of and thus a role for JAM-B on peripheral and CNS infiltrating myeloid cells. Taken together, although endothelial JAM-B is not required for immune cell trafficking across the BBB in EAE, in its absence accumulation of inflammatory cells mainly in CNS leptomeningeal spaces leads to amelioration of EAE.

The oncometabolite R-2-hydroxyglutarate activates NF-κB-dependent tumor-promoting stromal niche for acute myeloid leukemia cells.

Mutations of isocitrate dehydrogenase 1 (IDH1) and IDH2 in acute myeloid leukemia (AML) cells produce the oncometabolite R-2-hydroxyglutarate (R-2HG) to induce epigenetic alteration and block hematopoietic differentiation. However, the effect of R-2HG released by IDH-mutated AML cells on the bone marrow microenvironment is unclear. Here, we report that R-2HG induces IκB kinase-independent activation of NF-κB in bone marrow stromal cells. R-2HG acts via a reactive oxygen species/extracellular signal-regulated kinase (ERK)-dependent pathway to phosphorylate NF-κB on the Thr254 residue. This phosphorylation enhances the interaction of NF-κB and the peptidyl-prolyl cis-trans isomerase PIN1 and increases the protein stability and transcriptional activity of NF-κB. As a consequence, R-2HG enhances NF-κB-dependent expression of cytokines including IL-6, IL-8 and complement 5a to stimulate proliferation of AML cells. In addition, R-2HG also upregulates vascular endothelial adhesion molecule 1 and CXCR4 in stromal cells to enhance the contact between AML and stromal cells and attenuates chemotherapy-induced apoptosis. More importantly, we validated the R-2HG-activated gene signature in the primary bone marrow stromal cells isolated from IDH-mutated AML patients. Collectively, our results suggest that AML cell-derived R-2HG may be helpful for the establishment of a supportive bone marrow stromal niche to promote AML progression via paracrine stimulation.

Somatodendritic Expression of JAM2 Inhibits Oligodendrocyte Myelination.

Myelination occurs selectively around neuronal axons to increase the efficiency and velocity of action potentials. While oligodendrocytes are capable of myelinating permissive structures in the absence of molecular cues, structurally permissive neuronal somata and dendrites remain unmyelinated. Utilizing a purified spinal cord neuron-oligodendrocyte myelinating co-culture system, we demonstrate that disruption of dynamic neuron-oligodendrocyte signaling by chemical cross-linking results in aberrant myelination of the somatodendritic compartment of neurons. We hypothesize that an inhibitory somatodendritic cue is necessary to prevent non-axonal myelination. Using next-generation sequencing and candidate profiling, we identify neuronal junction adhesion molecule 2 (JAM2) as an inhibitory myelin-guidance molecule. Taken together, our results demonstrate that the somatodendritic compartment directly inhibits myelination and suggest a model in which broadly indiscriminate myelination is tailored by inhibitory signaling to meet local myelination requirements.

Influence of hypothermia and subsequent rewarming upon leukocyte-endothelial interactions and expression of Junctional-Adhesion-Molecules A and B.

Patients with risks of ischemic injury, e.g. during circulatory arrest in cardiac surgery, or after resuscitation are subjected to therapeutic hypothermia. For aortic surgery, the body is traditionally cooled down to 18 °C and then rewarmed to body temperature. The role of hypothermia and the subsequent rewarming process on leukocyte-endothelial interactions and expression of junctional-adhesion-molecules is not clarified yet. Thus, we investigated in an in-vitro model the influence of temperature modulation during activation and transendothelial migration of leukocytes through human endothelial cells. Additionally, we investigated the expression of JAMs in the rewarming phase. Exposure to low temperatures alone during transmigration scarcely affects leukocyte extravasation, whereas hypothermia during treatment and transendothelial migration improves leukocyte-endothelial interactions. Rewarming causes a significant up-regulation of transmigration with falling temperatures. JAM-A is significantly modulated during rewarming. Our data suggest that transendothelial migration of leukocytes is not only modulated by cell-activation itself. Activation temperatures and the rewarming process are essential. Continued hypothermia significantly inhibits transendothelial migration, whereas the rewarming process enhances transmigration strongly. The expression of JAMs, especially JAM-A, is strongly modulated during the rewarming process. Endothelial protection prior to warm reperfusion and mild hypothermic conditions reducing the difference between hypothermia and rewarming temperatures should be considered.

Zebrafish jam-b2 Gal4-enhancer trap line recapitulates endogenous jam-b2 expression in extraocular muscles.

BACKGROUND: Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS: We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS: The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

Transforming growth factor-ß3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-ß) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-ß3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-ß3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-ß3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-ß3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-ß3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-ß3-induced JAM-B protein degradation. In addition, TGF-ß3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-ß3 promotes the degradation of JAM-B transcript and TGF-ß3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-ß3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES.

Jam1a-Jam2a interactions regulate haematopoietic stem cell fate through Notch signalling.

Notch signalling plays a key role in the generation of haematopoietic stem cells (HSCs) during vertebrate development and requires intimate contact between signal-emitting and signal-receiving cells, although little is known regarding when, where and how these intercellular events occur. We previously reported that the somitic Notch ligands, Dlc and Dld, are essential for HSC specification. It has remained unclear, however, how these somitic requirements are connected to the later emergence of HSCs from the dorsal aorta. Here we show in zebrafish that Notch signalling establishes HSC fate as their shared vascular precursors migrate across the ventral face of the somite and that junctional adhesion molecules (JAMs) mediate this required Notch signal transduction. HSC precursors express jam1a (also known as f11r) and migrate axially across the ventral somite, where Jam2a and the Notch ligands Dlc and Dld are expressed. Despite no alteration in the expression of Notch ligand or receptor genes, loss of function of jam1a led to loss of Notch signalling and loss of HSCs. Enforced activation of Notch in shared vascular precursors rescued HSCs in jam1a or jam2a deficient embryos. Together, these results indicate that Jam1a-Jam2a interactions facilitate the transduction of requisite Notch signals from the somite to the precursors of HSCs, and that these events occur well before formation of the dorsal aorta.