Junctional Adhesion molecule-A Research Articles

Junctional adhesion molecule-A is down-regulated in anaplastic thyroid carcinomas and reduces cancer cell aggressiveness by modulating p53 and GSK3 α/β pathways.

Junctional adhesion molecule A (JAM-A) is a transmembrane protein that contributes to different biological process, including the epithelial to mesenchymal transition (EMT). Through an EMT profiler array, we explored the molecular players associated with human thyroid cancer progression and identified JAM-A as one of the genes mostly deregulated. The quantitative real-time polymerase chain reaction and immunohistochemistry analyses showed that downregulation of JAM-A occurred in anaplastic thyroid carcinoma (ATC) compared with normal thyroid (NT) and papillary thyroid carcinoma (PTC) tissues and correlated with extrathyroid infiltration, tumor size, and ATC histotype. In ATC cell lines, JAM-A restoration suppressed malignant hallmarks of transformation including cell proliferation, motility, and transendothelial migration. Accordingly, knockdown of JAM-A enhanced thyroid cancer cell proliferation and motility in PTC cells. Through the proteome profiler human phospho-kinase array, we demonstrated that higher expression of JAM-A was associated with a significant increased level of phosphorylation of p53 and GSK3 α/β proteins. In conclusion, our findings highlight a novel role of JAM-A in thyroid cancer progression and suggest that JAM-A restoration could have potential clinical relevance in thyroid cancer treatment.

Antibiotic Tetrocarcin-A Down-regulates JAM-A, IAPs and Induces Apoptosis in Triple-negative Breast Cancer Models.

Triple-negative breast cancers (TNBC) lack expression of three important receptors, and have limited treatment options. High expression of junctional adhesion molecule-A (JAM-A) has been linked with aggressive tumor phenotypes including TNBC. This study aimed to evaluate the bioactivity of a JAM-A-down-regulating compound, Tetrocarcin-A, in TNBC. TNBC cell viability, colony formation and xenograft growth were examined in Tetrocarcin-A-treated HCC38 human cells, 4T1 mouse cells or patient-derived primary cells. Protein expression of cell fate signaling effectors was examined by immunoblotting (versus transient JAM-A gene silencing). Apoptotic pathways were investigated in parallel. Tetrocarcin-A reduced TNBC cell viability in vitro and in an in ovo/semi-in vivo xenograft model. Tetrocarcin-A-induced JAM-A down-regulation and reduced ERK phosphorylation, followed by c-FOS phosphorylation on its transcription-regulating residue, which down-regulated several inhibitor of apoptosis (IAP) proteins and induced caspase-dependent intrinsic pathway of apoptosis. Tetrocarcin-A merits further investigation as a novel anti-tumor agent in TNBC.

Assembly of a portal-like structure in feline calicivirus following receptor engagement

The mechanism by which non-enveloped RNA viruses, such as the caliciviruses, escape the endosome, is poorly understood. The Caliciviridae are a family of viruses which include many important human and animal pathogens, most notably norovirus which causes winter vomiting disease. We used cryoEM and asymmetric three-dimensional reconstruction to investigate structural changes in the capsid of feline calicivirus (FCV) that occur upon virus binding to its cellular receptor; feline junctional adhesion molecule-A (fJAM-A). We discovered that following receptor engagement substantial conformational changes in the FCV capsid lead to the assembly of a portal-like structure at a unique three-fold symmetry axis. Atomic models of the major capsid protein, VP1, in the presence and absence of fJAM-A were calculated, revealing the conformational changes induced by the interaction. In the course of this analysis we discovered a large portal-like structure which assembles at a unique three-fold axis. The portal-like complex comprises 12 copies of the minor capsid protein VP2. We calculated an atomic model of VP2 and revealed structural changes in VP1 that lead to the formation of a pore in the capsid shell at the portal vertex. VP2 is encoded by all caliciviruses although despite being critical for the production of infectious virus, its function and structure were, until now, undetermined. We hypothesise that the VP2 portal-like complex is the method by which the virus escapes the endosome during virus entry, allowing delivery of the viral genome into the cytoplasm for replication to then ensue.

A peptide antagonist of F11R/JAM-A reduces plaque formation and prolongs survival in an animal model of atherosclerosis

Background and aimsThe F11 Receptor (F11R), AKA Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is an adhesion protein constitutively expressed on the membrane surface of circulating platelets and the luminal surface of inflamed endothelial cells (EC).Platelet adhesion to an inflamed endothelium is one of the early steps of atherosclerotic plaque formation. Our previous studies, conducted with cultured EC in vitro, have demonstrated the expression of F11R/JAM-A on the luminal surface of inflamed EC, platelet adhesion to inflamed EC through F11R/JAM-A interactions, and inhibition of this interaction by the presence of F11R/JAM-A antagonistic peptide (F11Rpeptide 4D). In the present study, we examined in vivo the overall health-benefits and cardiovascular effects of long-term treatment of animals prone to atherosclerosis, ApoE−/− mice, with F11R-peptide 4D. MethodsTwenty ApoE−/− mice were assigned to daily treatment with peptide 4D and compared to their counterparts control untreated mice. Mice were observed for wellness and survival. Plaque size in the aorta and heart was measured using histological analysis. Effects of peptide 4D (or scramble control) on platelet adhesion to inflamed endothelium were measured using intravital microscopy. ResultsSignificant reductions in atherosclerotic plaques number and size, an overall robust health with longer survival were found in the peptide 4D treated group of ApoE−/− mice. Intravital microscopic studies conducted in exposed vessels of ApoE−/− mice demonstrated significant inhibition by peptide 4D of platelet adhesion to the cytokine-inflamed endothelium. ConclusionsOur results demonstrate that peptide 4D significantly reduces atherosclerotic plaque formation in ApoE−/− mice and inhibits platelet adhesion to the inflamed arterial endothelium.

Macrophage-dependent neutrophil recruitment is impaired under conditions of increased intestinal permeability in JAM-A-deficient mice.

Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-afl/fl). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.

TGR5-dependent hepatoprotection through the regulation of biliary epithelium barrier function

ObjectiveWe explored the hypothesis that TGR5, the bile acid (BA) G-protein-coupled receptor highly expressed in biliary epithelial cells, protects the liver against BA overload through the regulation of biliary epithelium...

Naringenin promotes recovery from colonic damage through suppression of epithelial tumor necrosis factor–α production and induction of M2-type macrophages in colitic mice

Our previous study demonstrated that supplemental naringenin reduced the development of colitis induced by dextran sodium sulfate (DSS) in mice, however, the effect of naringenin on the recovery from colonic damage was totally unknown. The primary purpose was to investigate if naringenin promoted recovery from colonic damage in DSS-administered mice and colonic tissues. When mice were fed diets lacking or containing naringenin (0.3%, w/w) for 11 days after colitis induction through DSS administration, the supplemental naringenin was found to promote a reversal of body weight loss and suppress tumor necrosis factor (TNF)-α mRNA expression in the DSS-administered mice. Moreover, protein expression of two tight junction proteins, claudin-3 and junctional adhesion molecule-A, was higher in DSS-administered mice that were fed naringenin than in the mice that did not receive naringenin. To examine the early mechanisms underlying the naringenin-mediated reduction of colonic damage, the inflamed colonic tissues of DSS-administered mice were incubated with or without naringenin for 24 hours; in tissues incubated with naringenin, TNF-α production was lower and interleukin (IL)-10 and CD206 mRNA expression was higher than in tissues incubated without naringenin, but naringenin did not affect the expression of the tight junction proteins. Flow cytometry results further demonstrated that naringenin reduced TNF-α–positive epithelial cells, but not macrophages, and promoted the polarization of M2-type macrophages in the colonic tissues. Thus, supplemental naringenin promoted recovery from colonic damage in mice with colitis, and suppression of epithelial TNF-α production and induction of M2-type macrophages might represent the early mechanisms underlying this naringenin effect.

Role of JAM-A tyrosine phosphorylation in epithelial barrier dysfunction during intestinal inflammation.

Junctional adhesion molecule-A (JAM-A), an epithelial tight junction protein, plays an important role in regulating intestinal permeability through association with a scaffold signaling complex containing ZO-2, Afadin, and the small GTPase Rap2. Under inflammatory conditions, we report that the cytoplasmic tail of JAM-A is tyrosine phosphorylated (p-Y280) in association with loss of barrier function. While barely detectable Y280 phosphorylation was observed in confluent monolayers of human intestinal epithelial cells under basal conditions, exposure to cytokines TNFα, IFNγ, IL-22, or IL-17A, resulted in compromised barrier function in parallel with increased p-Y280. Phosphorylation was Src kinase dependent, and we identified Yes-1 and PTPN13 as a major kinase and phosphatase for p-JAM-A Y280, respectively. Moreover, cytokines IL-22 or IL-17A induced increased activity of Yes-1. Furthermore, the Src kinase inhibitor PP2 rescued cytokine-induced epithelial barrier defects and inhibited phosphorylation of JAM-A Y280 in vitro. Phosphorylation of JAM-A Y280 and increased permeability correlated with reduced JAM-A association with active Rap2. Finally, we observed increased phosphorylation of Y280 in colonic epithelium of individuals with ulcerative colitis and in mice with experimentally induced colitis. These findings support a novel mechanism by which tyrosine phosphorylation of JAM-A Y280 regulates epithelial barrier function during inflammation.

Highlight on Endothelial Activation and Beyond.

Highlight on Endothelial Activation and Beyond.

Junctional Adhesion Molecule-a Regulates Vascular Endothelial Growth Factor-a-Dependent Permeability

Introduction: Vascular barrier function is important for maintaining vascular integrity and is regulated by endothelial tight junctions. Breaches in the tight junction result in increased endothelial cell (EC) permeability, which leads to a variety of pathological conditions such as edema, vascular degeneration, diabetic retinopathy, and ischemia reperfusion injury. Junctional adhesion molecule A (JAM-A), is an integral part of the tight junctions of ECs. It has been shown that a lack of Jam-A affects neutrophil extravasation and FGF-2-induced angiogenesis. Role of JAM-A in regulation of vascular permeability is not known.Methods: Jam-A knockout (KO) and age-matched wild-type (WT) control mice of both sexes were used in this study. In vivo vascular permeability was assessed using Miles assay and quantified colorimetrically by extracting Evans blue in formamide at 56oC. The circulating plasma level of vascular endothelial growth factor A (VEGF-A) in WT and Jam-A KO mice was measured by ELISA assay. Surface expression of VEGF receptor 2 (VEGFR2) was analyzed by flow cytometry. Quantitative real- time PCR was used to determine gene transcription. Protein expression was determined by western blotting. VEGF-induced WT and Jam-A KO EC migration was analyzed by both scratch and trans-well assays.Results: Although Jam-A resides at the endothelial tight junctions, genetic ablation of Jam-A does not affect blood vessel structure. However, basal vascular permeability was significantly enhanced (P<0.05) in Jam-A KO mice compared to the WT controls, suggesting that a lack of Jam-A in EC compromises vascular integrity. This was further supported by the fact that there was increased edema in the lungs of the Jam-A KO mice compared to WT controls. Since VEGF is also known as vascular permeability factor, we evaluated the effect of VEGF-A on vascular permeability in Jam-A KO mice. Upon intravenous injection of VEGF-A (100ng/ml), EC permeability was significantly increased in both WT and Jam-A KO mice. In order to determine the cause of this enhanced basal permeability, we evaluated the levels of VEGF-A in WT and Jam-A KO mice. Jam-A deficient mice showed a three-fold (P<0.004) increased level of plasma VEGF-A compared to WT mice. Additionally, VEGF-A protein level was also increased in the ECs from Jam-A KO mice compared to WT controls. Furthermore, mRNA levels of VEGFR2, but not sFlt1, were increased in the Jam-A KO ECs compared to WT ECs. Protein levels as well as surface expression of VEGFR2 were significantly increased (P<0.001) in Jam-A KO ECs compared to WT ECs, suggesting that lack of Jam-A results in increased levels of VEGF-A/VEGFR2. We next evaluated the protein and mRNA levels of both hypoxia-inducible factor-1 α (HIF-1α), a transcription factor known to regulate expression of VEGF-A, and inhibitor of DNA binding 1 (Id1), a transcriptional factor known to regulate expression of VEGFR2. We found that bothHIF-1α and Id1 mRNA as well as protein levels were significantly (P<0.001 and P<0.05 respectively) augmented in Jam-A KO ECs compared to WT ECs. Additionally, overexpression of JAM-A in human umbilical vein endothelial cells (HUVECs) resulted in significantly reduced expression of bothHIF-1α and Id1 proteins. Furthermore, ECs isolated from Jam-A KO mouse showed significantly enhanced (P<0.05) cell migration in response to VEGF-A as compared to WT ECs, suggesting that Jam-A ECs respond favorably to VEGF-A due to increased VEGFR2 on their surface. To confirm that the observed augmented permeability in Jam-A KO mice was primarily due to enhanced VEGFR2 signaling, we inhibited VEGFR2 in mice using DC101 (200μg/kg), a function-blocking antibody. Isotype specific IgG (200μg/kg) was used as control. Inhibition of VEGFR2 completely abrogated basal as well as VEGF-A-induced vascular permeability in both Jam-A KO and WT mice, suggesting that the augmented EC permeability in Jam-A KO mice is not due to structural defect in tight junctions, but due to enhanced VEGF-A/VEGFR2 signaling.Conclusion: Our results suggest that JAM-A positively regulates EC tight junction integrity by suppressing levels of VEGF-A and VEGFR2 through downregulation ofHIF-1α and Id1 transcription factors. DisclosuresNo relevant conflicts of interest to declare.

Central Function for JAM-a in Multiple Myeloma Patients with Extramedullary Disease

During the last years, multiple myeloma (MM) has been recognized to present in some patients with extra-medullary disease (EMD) manifestation with different pathophysiological and clinical characteristics. Recently, we reported junctional adhesion molecule-A (JAM-A) as a novel target and a clinical biomarker in MM [Solimando et al. Leukemia 32(3):736-743, 2018]. Here, we hypothesized that alterations in the adhesion molecules, particularly JAM-A expression, on MM plasma cells (MM-PCs) may play a pivotal role in high-risk EMD patients.Therefore, we evaluated JAM-A expression in 60 MM patient bone marrow (BM) aspirates and biopsies at different disease stages with flow cytometry and immunohistochemistry. We compared these results with RNA-Seq data from 647 newly diagnosed MM (NDMM) patients collected in the MMRF (MM Research Foundation) CoMMpass study. Using bioinformatics, we investigated JAM-A related pathways with hierarchical cluster analysis. Subsequently, we functionally tested these JAM-A related pathways regarding epithelial mesenchymal transition, invasion and MM dissemination in vitro.The median OS differed significantly in subjects with elevated membrane JAM-A expression levels, dividing the patients in EMD JAM-Ahighgroup with a median OS of 84.1 months, whereas median OS in JAM-Alowpatients was not reached, irrespective from the EMD status (log-rank=4.19, P=0.04). Immunohistochemistry analysis of BM biopsies confirmed these findings. RNA-Seq analysis corroborated the prognostic impact of JAM-A (log-rank= 3.8, P= 0.051).High-risk myeloma patients with EMD and JAM-A expression displayed a unique gene-expression signature. Additionally, we constructed a novel expression cluster subgroup MM model, revealing complex molecular disease patterns and associations between clinical traits and expression markers. MM patients clustering according to JAM-A expression levels and EMD manifestation revealed a clear link to the epithelial-mesenchymal-transition and focal adhesion pathways. Notably, functional knockdown of JAM-A in vitro not only reduced cell viability, but also diminished MM adhesion molecules and CD138 surface expression. Furthermore, mTOR/PI3K in vitro inhibition with BEZ235 significantly down regulated surface expression in JAM-Ahigh MM cell lines. Interestingly, when we inhibited mTOR/PI3K in JAM-AlowMM cells, we observed JAM-A induction.In the present study we shed more light on the EMD pathophysiology. Conclusively, our results support that JAM-A can serve as promising prognostic marker for high risk EMD MM patients. Our data provide a biological rationale for a JAM-A guided decision-making process to better combine molecular and immunochemo-therapy modalities in the EMD MM patient subset. DisclosuresNo relevant conflicts of interest to declare.

Oncolytic Reovirus Is an Effective Treatment for Histone Deacetylase Inhibitor Resistant T-Cell Lymphoma

Aberrant gene expression plays a pivotal role during tumorigenesis and cancer progression. The acetylation status of histones is an important determinant of gene expression and is controlled by two opposing classes of enzymes: histones acetyl transferases (HATs) and histone deacetylases (HDACs). The deacetylation of histones is associated with repression of key tumor suppressor genes and has been linked to HDAC overexpression in multiple forms of cancer including lymphomas. Several HDAC inhibitors have been FDA approved for T-cell lymphoma (TCL) therapy including belinostat, vorinostat, and romidepsin. Despite the promising anti-lymphoma activity of HDAC inhibitors as a drug class, resistance is a significant clinical issue. Identification of new strategies that are more effective in the drug resistant patient population is a high priority, but the mechanisms underlying HDAC inhibitor-induced cell death and the development of drug resistance are not completely understood. Elucidating the molecular mechanisms driving HDAC inhibitor resistance and/or the specific targets that are altered in drug-resistant cells may facilitate the development of strategies that overcome drug resistance and are effective for refractory patients. To pursue this goal, we generated novel TCL cell line models of acquired HDAC inhibitor resistance through repeated exposure to belinostat. The sensitivity of parental and resistant TCL cells to belinostat and other clinically relevant HDAC inhibitors was initially characterized using cell viability and flow cytometric assays. Notably, belinostat-resistant cells displayed significant cross-resistance to other HDAC inhibitors including vorinostat, romidepsin, panobinostat, and ricolinostat. This indicates that TCL patients that fail one HDAC inhibitor regimen may not benefit significantly from subsequent treatment with other drugs of this class. Consistent with a lack of sensitivity to HDAC inhibitors, the resistant cells failed to induce increased acetylated histones, tubulin acetylation (an HDAC6 target), and exhibited reduced upregulation of the CDK inhibitor p21 following belinostat treatment. In agreement with the absence of these hallmark characteristics of HDAC inhibition, belinostat also failed to cleave caspase-3 in the belinostat-resistant cells. Comprehensive transcriptome analysis was conducted to further characterize these new drug-resistant models and identify potential mechanisms of resistance and targets for second-line treatment. Drug resistant cells featured significantly increased basal levels of the reovirus receptor junctional adhesion molecule-A (JAM-A) as well as decreased JAK/STAT activity, a key antiviral response pathway. Based on these findings, we investigated the efficacy of the proprietary clinical formulation of reovirus (Reolysin) in parental and drug-resistant models. Our investigation revealed that belinostat-resistant cells displayed enhanced sensitivity to oncolytic reovirus-induced cell death compared to their parental counterparts. The increased benefit of Reolysin in resistant models was linked to elevated viral loads and more efficient endoplasmic reticular stress-mediated apoptosis. The heightened sensitivity of HDAC-resistant TCL cells to Reolysin was further validated in parental and belinostat-resistant T-cell lymphoma xenograft models. Collectively, these data demonstrate that oncolytic reovirus may be a novel therapeutic approach to treat T-cell lymphoma patients that are relapsed/refractory to HDAC inhibitors. We are currently planning an early phase clinical trial to further test the safety and benefit of this new approach. DisclosuresPersky:Morphosys (IDMC): Consultancy; Merck: Research Funding; Spectrum: Research Funding; Genentech: Honoraria.

Cleavage of the extracellular domain of junctional adhesion molecule-A is associated with resistance to anti-HER2 therapies in breast cancer settings

BackgroundJunctional adhesion molecule-A (JAM-A) is an adhesion molecule whose overexpression on breast tumor tissue has been associated with aggressive cancer phenotypes, including human epidermal growth factor receptor-2 (HER2)-positive disease. Since JAM-A has been described to regulate HER2 expression in breast cancer cells, we hypothesized that JAM-dependent stabilization of HER2 could participate in resistance to HER2-targeted therapies.MethodsUsing breast cancer cell line models resistant to anti-HER2 drugs, we investigated JAM-A expression and the effect of JAM-A silencing on biochemical/functional parameters. We also tested whether altered JAM-A expression/processing underpinned differences between drug-sensitive and -resistant cells and acted as a biomarker of patients who developed resistance to HER2-targeted therapies.ResultsSilencing JAM-A enhanced the anti-proliferative effects of anti-HER2 treatments in trastuzumab- and lapatinib-resistant breast cancer cells and further reduced HER2 protein expression and Akt phosphorylation in drug-treated cells. Increased epidermal growth factor receptor expression observed in drug-resistant models was normalized upon JAM-A silencing. JAM-A was highly expressed in all of a small cohort of HER2-positive patients whose disease recurred following anti-HER2 therapy. High JAM-A expression also correlated with metastatic disease at the time of diagnosis in another patient cohort resistant to trastuzumab therapy. Importantly, cleavage of JAM-A was increased in drug-resistant cell lines in conjunction with increased expression of ADAM-10 and -17 metalloproteases. Pharmacological inhibition or genetic silencing studies suggested a particular role for ADAM-10 in reducing JAM-A cleavage and partially re-sensitizing drug-resistant cells to the anti-proliferative effects of HER2-targeted drugs. Functionally, recombinant cleaved JAM-A enhanced breast cancer cell invasion in vitro and both invasion and proliferation in a semi-in vivo model. Finally, cleaved JAM-A was detectable in the serum of a small cohort of HER2-positive patients and correlated significantly with resistance to HER2-targeted therapy.ConclusionsCollectively, our data suggest a novel model whereby increased expression and cleavage of JAM-A drive tumorigenic behavior and act as a biomarker and potential therapeutic target for resistance to HER2-targeted therapies.

Analysis of leukocyte transepithelial migration using an in vivo murine colonic loop model.

Molecular mechanisms that control leukocyte migration across the vascular endothelium (transendothelial migration; TEndoM) have been extensively characterized in vivo, but details of leukocyte transepithelial migration (TEpM) and its dysregulation (a pathologic feature of many mucosal diseases) are missing due to the lack of suitable animal models. Here, we describe a murine model that utilizes a vascularized proximal colonic segment (pcLoop) and enables quantitative studies of leukocyte trafficking across colonic epithelium. Consistent with previous in vitro studies, intraluminal injection of antibodies against integrin CD11b/CD18 reduced recruitment of polymorphonuclear neutrophils (PMN) into the lumen of pcLoops, and it increased subepithelial accumulation of PMN. We extended studies using the pcLoop to determine contributions of Junctional Adhesion Molecule-A (JAM-A, or F11R) in PMN TEpM and confirmed that mice with total loss of JAM-A or mice with intestinal epithelial selective loss of JAM-A had increased colonic permeability. Furthermore, there was reduced PMN migration into the colonic lumen that paralleled subepithelial accumulation of PMN in global-KO mice, as well as in intestinal epithelial-targeted JAM-A-deficient mice. These findings highlight a potentially novel role for JAM-A in regulating PMN TEpM in vivo and demonstrate utility of this model for identifying receptors that may be targeted in vivo to reduce pathologic intestinal inflammation.

Natural compound Tetrocarcin-A downregulates Junctional Adhesion Molecule-A in conjunction with HER2 and inhibitor of apoptosis proteins and inhibits tumor cell growth

Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the antibiotic Tetrocarcin-A as a novel downregulator of JAM-A and human epidermal growth factor receptor-2 (HER2) protein expression in breast cancer cells. Lysosomal inhibition partially rescued the downregulation of JAM-A and HER2 caused by Tetrocarcin-A, and attenuated its cytotoxic activity. Tetrocarcin-A treatment or JAM-A silencing reduced AKT and ERK phosphorylation, inhibited c-FOS phosphorylation at Threonine-232 (its transcriptional regulation site), inhibited nuclear localization of c-FOS, and downregulated expression of the inhibitor of apoptosis proteins (IAP). This was accompanied by Tetrocarcin-A-induced caspase-dependent apoptosis. To begin evaluating the potential clinical relevance of our findings, we extended our studies to other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cells and lung cancer stem cells, and inhibited the growth of xenografts in a semi-in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A warrants future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, reduce tumorigenic signaling and induce apoptosis.

Citrus kawachiensis Peel Powder Reduces Intestinal Barrier Defects and Inflammation in Colitic Mice

The anti-inflammatory effect of Citrus kawachiensis peel powder was examined in a murine model of dextran sodium sulfate (DSS)-induced colitic mice. In addition to the whole powder, its ethanol extract rich in polyphenolic compounds and ethanol extraction residue rich in dietary fibers were used. The whole powder ameliorated the DSS-induced body weight loss (body weight changes on day 9, Control 108 ± 2, DSS 91 ± 4, DSS+whole peel powder 106 ± 1%, p < 0.05), colon shortening (colon length, Control 5.0 ± 0.1, DSS 3.9 ± 0.1, DSS+whole peel powder 4.7 ± 0.1 cm, p < 0.05), increased expression of pro-inflammatory cytokines (e.g., TNF-α, Control 1.0 ± 0.1, DSS 22.2 ± 5.8, DSS+whole peel powder 4.3 ± 1.5 arbitrary unit, p < 0.05), and decreased expression of colonic tight junctions (TJs) (e.g., occludin, Control 1.00 ± 0.07, DSS 0.21 ± 0.07, DSS+whole peel powder 0.70 ± 0.06 arbitrary unit, p < 0.05). The resolution of abnormalities barring the decreased expression of zonula occludens-2, junctional adhesion molecule-A, and claudin-7 by the extraction residue was comparable to that achieved using the powder (body weight change 107 ± 1%; colon length 4.7 ± 0.1 cm; TNF-α 4.1 ± 0.7; occludin 0.58 ± 0.06 arbitrary unit, p < 0.05). The ethanol extract alone did not have any influence on these abnormalities (body weight change 94 ± 2%; colon length 4.1 ± 0.1 cm; TNF-α 40.5 ± 9.0 arbitrary unit; occludin 0.18 ± 0.02 arbitrary unit, p < 0.05). The powder and ethanol extraction residue, but not ethanol extract, increased fecal acetic acid concentration (Control 4.9 ± 0.6, DSS 5.0 ± 0.9, DSS+whole peel powder 8.8 ± 1.8, DSS+ethanol extract 5.3 ± 0.8, DSS+ethanol extraction residue 12.5 ± 1.1 mmol/L, p < 0.05). Taken together, DFs in the ethanol extraction residue largely contributed to the peel powder-mediated reduction of TJ barrier defect and inflammation in colitic mice.

Isolation and screening of microbial strains for potential antimicrobial activity

The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro-inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper-cholesterol diet to induce the loss of intestinal epithelial layer integrity.The components of the experimental mixture were the main oxysterols stemming from heat-induced cholesterol auto-oxidation, namely 7-ketocholesterol, 5α,6α-and 5β,6β-epoxycholesterol, 7α- and 7β-hydroxycholesterol. These compounds added to monolayers of differentiated CaCo-2 cells in combination or singularly, caused a time-dependent induction of matrix metalloproteinases (MMP)-2 and -9, also known as gelatinases. The hyperactivation of MMP-2 and -9 was found to be associated with decreased levels of the tight junctions zonula occludens-1 (ZO-1), occludin and Junction Adhesion Molecule-A (JAM-A). Together with such a protein loss, particularly evident for ZO-1, a net perturbation of spatial localization of the three tight junctions was observed.Cell monolayer pre-treatment with the selective inhibitor of MMPs ARP100 or polyphenol (-)-epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro-oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent - but incomplete - prevention of oxysterols-dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO-1, with possible significant recovery of intestinal monolayer integrity.

JAM-A knockdown accelerates the proliferation and migration of human keratinocytes, and improves wound healing in rats via FAK/Erk signaling

Junctional adhesion molecule-A (JAM-A) belongs to the immunoglobulin superfamily, it predominantly exists at the tight junctions of epithelial and endothelial cells. JAM-A is known to regulate leukocyte trans-endothelial migration, however, how it affects the proliferation and migration of keratinocytes, the two essential steps during wound healing, has less been explored. In this study, we showed that JAM-A was significantly expressed in normal skin epidermis. RNAi-mediated JAM-A knockdown remarkably promoted the proliferation and migration of keratinocytes. We also found that loss of JAM-A increased the protein levels of p-FAK, p-Erk1/2, and p-JNK; however, FAK inhibitor PF-562271 restrained the expression of p-FAK and p-Erk1/2 elevated by JAM-A RNAi, but not p-JNK, and also slowed down keratinocyte proliferation and migration. Finally, in a rat wound model we showed that absence of JAM-A significantly promoted the wound healing process, while the use of PF-562271 or Erk1/2 inhibitor PD98059 repressed those effects. These data collectively demonstrate that suppressing JAM-A expression could promote the proliferation and migration of keratinocytes and accelerate the healing process of rat skin wounds, potentially via FAK/Erk pathway, indicating that JAM-A might serve as a potential therapeutic target for the treatment of chronic refractory wounds.

The junctional adhesion molecule-А (JAM-A) and pathogenetic mechanisms of essential hypertension

Recent studies demonstrated an important role of the junctional adhesion molecule-A (JAM-A) in the pathogenesis of essential hypertension. Our studies showed higher content of JAM-A in women with essential hypertension. Also hypertensive women have higher levels of very low–density lipoprotein (VLDL), higher atherogenic index and lower concentrations of estrogen, progesterone and prolactin, indicating an increased risk of atherosclerosis and premature aging. By the laser speckle showed that the hemodynamic indices considerably change in essential hypertension. The levels of JAM-A correlate with blood pressure, lipids and hemodynamic indices, both in healthy subjects and in women with essential hypertension. The data demonstrate the importance of JAM-A in the pathogenesis of essential hypertension.

Characterization of a replicating expanded tropism oncolytic reovirus carrying the adenovirus E4orf4 gene.

While the mammalian orthoreovirus type 3 dearing (reovirus T3D) infects many different tumour cells, various cell lines resist the induction of reovirus-mediated cell death. In an effort to increase the oncolytic potency, we introduced transgenes into the S1 segment of reovirus T3D. The adenovirus E4orf4 gene was selected as transgene since the encoded E4orf4 protein induces cell death in transformed cells. The induction of cell death by E4orf4 depends in part on its binding to phosphatase 2A (PP2A). In addition to the S1-E4orf4 reovirus, two other reoviruses were employed in our studies. The reovirus rS1-RFA encodes an E4orf4 double-mutant protein that cannot interact with PP2A and the rS1-iLOV virus encoding the fluorescent marker iLOV as a reporter. The replacement of the codons for the junction adhesion molecule-A (JAM-A) binding head domain of the truncated spike protein blocks the entry of these recombinant viruses via the reovirus receptor JAM-A. Instead these viruses rely on internalization via binding to sialic acids on the cell surface. This expands their tropism and allows infection of JAM-A-deficient tumour cells. Here we not only demonstrate the feasibility of this approach but also established that the cytolytic activity of these recombinant viruses is largely transgene independent.