A 3D biomimetic model of lymphatics reveals cell-cell junction tightening and lymphedema via a cytokine-induced ROCK2/JAM-A complex.

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

Calicivirus VP2 forms a portal-like assembly following receptor engagement.

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.

Effect of F11 Receptor/Junctional Adhesion Molecule-A-derived Peptide on Neointimal Hyperplasia in a Murine Model.

PURPOSE: To determine whether inhibition of the F11 receptor/JAM-A (F11R) using F11R-specific antagonist peptide 4D results in inhibition of smooth muscle cell (SMC) proliferation and migration in vivo, known as neointimal hyperplasia (NIH), using a mouse focal carotid artery stenosis model (FCASM). MATERIALS AND METHODS: The mouse FCASM was chosen to test the hypothesis because the dominant cell type at the site of stenosis is SMC, similar to that in vascular access stenosis. Fourteen C57BL/6 mice underwent left carotid artery (LCA) partial ligation to induce stenosis, followed by daily injection of peptide 4D in 7 mice and saline in the remaining 7 mice, and these mice were observed for 21 days and then euthanized. Bilateral carotid arteries were excised for histologic analysis of the intima and media areas. RESULTS: The mean intimal area was significantly larger in control mice compared with peptide 4D-treated mice (0.031 mm2 [SD ± 0.024] vs 0.0082 mm2 [SD ± 0.0103]; P = .011). The mean intima-to-intima + media area ratio was significantly larger in control mice compared with peptide 4D-treated mice (0.27 [SD ± 0.13] vs 0.089 [SD ± 0.081]; P = .0079). NIH was not observed in the right carotid arteries in both groups. CONCLUSIONS: Peptide 4D, an F11R antagonist, significantly inhibited NIH in C57BL/6 mice in a FCASM.

The Murine Neuronal Receptor NgR1 Is Dispensable for Reovirus Pathogenesis.

Engagement of host receptors is essential for viruses to enter target cells and initiate infection. Expression patterns of receptors in turn dictate host range, tissue tropism, and disease pathogenesis during infection. Mammalian orthoreovirus (reovirus) displays serotype-dependent patterns of tropism in the murine central nervous system (CNS) that are dictated by the viral attachment protein σ1. However, the receptor that mediates reovirus CNS tropism is unknown. Two proteinaceous receptors have been identified for reovirus, junctional adhesion molecule A (JAM-A) and Nogo-66 receptor 1 (NgR1). Engagement of JAM-A is required for reovirus hematogenous dissemination but is dispensable for neural spread and infection of the CNS. To determine whether NgR1 functions in reovirus neuropathogenesis, we compared virus replication and disease in wild-type (WT) and NgR1-/- mice. Genetic ablation of NgR1 did not alter reovirus replication in the intestine or transmission to the brain following peroral inoculation. Viral titers in neural tissues following intramuscular inoculation, which provides access to neural dissemination routes, also were comparable in WT and NgR1-/- mice, suggesting that NgR1 is dispensable for reovirus neural spread to the CNS. The absence of NgR1 also did not alter reovirus replication, neural tropism, and virulence following direct intracranial inoculation. In agreement with these findings, we found that the human but not the murine homolog of NgR1 functions as a receptor and confers efficient reovirus binding and infection of nonsusceptible cells in vitro. Thus, neither JAM-A nor NgR1 is required for reovirus CNS tropism in mice, suggesting that other unidentified receptors support this function. IMPORTANCE Viruses engage diverse molecules on host cell surfaces to navigate barriers, gain cell entry, and establish infection. Despite discovery of several reovirus receptors, host factors responsible for reovirus neurotropism are unknown. Human NgR1 functions as a reovirus receptor in vitro and is expressed in CNS neurons in a pattern overlapping reovirus tropism. We used mice lacking NgR1 to test whether NgR1 functions as a reovirus neural receptor. Following different routes of inoculation, we found that murine NgR1 is dispensable for reovirus dissemination to the CNS, tropism and replication in the brain, and resultant disease. Concordantly, expression of human but not murine NgR1 confers reovirus binding and infection of nonsusceptible cells in vitro. These results highlight species-specific use of alternate receptors by reovirus. A detailed understanding of species- and tissue-specific factors that dictate viral tropism will inform development of antiviral interventions and targeted gene delivery and therapeutic viral vectors.

F11R/JAM-A: why do platelets express a molecule which is also present in tight junctions?

F11 receptor (F11R)/Junctional Adhesion Molecule -A (JAM-A) is a transmembrane protein which belongs to the immunoglobulin superfamily of cell adhesion molecules. F11R/JAM-A is present in epithelial cells, endothelial cells, leukocytes, and blood platelets. In epithelial and endothelial cells, it takes part in the formation of tight junctions. In these structures, molecules of F11R/JAM-A located on adjacent cells form homodimers and thus take part in stabilization of cellular layer integrity. In leukocytes, F11R/JAM-A was shown to play role in their transmigration through the vascular wall. Paradoxically, the function of F11R/JAM-A in blood platelets, where it was primarily discovered, is much less understood. It has been proven to regulate downstream signaling of αIIbß3 integrin and to mediate platelet adhesion under static conditions. It was also shown to contribute to transient interactions of platelets with inflamed vascular wall. The review is aimed at summarizing the current state of knowledge of the platelet pool of F11R/JAM-A. The article also presents perspectives of the future research to better understand the role of this protein in hemostasis, thrombosis, and other processes where blood platelets are involved.

The molecule of a complex name F11R/JAM-A is a protein which was primarily discovered on blood platelets. Later, the presence of the same molecule was confirmed on endothelial cells and epithelial cells. From the moment of the discovery, most of the research was focused on the role of this protein in the latter types of cells. It was found to be an important element of so-called tight junctions. These structures are crucial for maintaining of integrity and selective permeability of cellular layers composed of these types of cells. In the following years, the presence of F11R/JAM-A has also been reported on leukocytes. An important role of specific type of leukocytes is their penetration to the sites of inflammation. Interplay of F11R/JAM-A present on endothelium and that on leukocyte is involved in this process. But what about the role of this protein in blood platelets where it was originally discovered? There is limited knowledge regarding this issue. It was found to play a role in the ability of platelets to adhere to a surface under static conditions, but it is not known if the same is true under flow. Is the protein necessary for platelets to aggregate and form thrombus? Genetically engineered mice were created which lack this protein in blood platelets to answer this question. These platelets were abnormally reactive, as it transpired that the protein plays a role of a negative regulator to one of the most important mechanisms, which triggers platelet aggregation. But is this inhibitory function the only task F11R/JAM-A has to fulfil in platelets? Presented review collects all the knowledge regarding this protein in blood platelets and tries to show interesting routes which need exploration.

JAM-A is a multifaceted regulator in hepatic fibrogenesis, supporting LSEC integrity and stellate cell quiescence.

BACKGROUND AND AIMS: Leukocyte infiltration is a hallmark of hepatic inflammation. The Junctional Adhesion Molecule A (JAM-A) is a crucial regulator of leukocyte extravasation and is upregulated in human viral fibrosis. Reduced shear stress within hepatic sinusoids and the specific phenotype of liver sinusoidal endothelial cells (LSEC) cumulate in differing adhesion characteristics during liver fibrosis. The aim of this study was to define the functional role of cell-specific adhesion molecule JAM-A during hepatic fibrogenesis. METHODS: Complete, conditional (intestinal epithelial; endothelial) and bone marrow chimeric Jam-a knockout animals and corresponding C57Bl/6 wild-type animals were treated with carbon tetrachloride (CCl4 , 6 weeks). For functional analyses of JAM-A, comprehensive in vivo studies, co-culture models and flow-based adhesion assays were performed. RESULTS: Complete and bone marrow-derived Jam-a-/- animals showed aggravated fibrosis with increased non-sinusoidal, perivascular accumulation of CD11b+ F4/80+ monocyte-derived macrophages in contrast to wild-type mice. Despite being associated with disturbed epithelial barrier function, an intestinal epithelial Jam-a knockout did not affect fibrogenesis. In endothelial-specific Jam-a-/- animals, liver fibrosis was aggravated alongside sinusoid capillarization and hepatic stellate cell (HSC) activation. HSC activation is induced via Jam-a-/- LSEC-derived secretion of soluble factors. Sinusoid CD31 expression and hedgehog gene signalling were increased, but leukocyte infiltration and adhesion to LSECs remained unaffected. CONCLUSIONS: Our models decipher cell-specific JAM-A to exert crucial functions during hepatic fibrogenesis. JAM-A on bone marrow-derived cells regulates non-sinusoidal vascular immune cell recruitment, while endothelial JAM-A controls liver sinusoid capillarization and HSC quiescence.

Lentivirus-mediated subcutaneous JAM-A modification promotes skin wound healing in a mouse model by strengthening the secretory function and proliferation of fibroblasts.

A better understanding of the molecular regulation of wound healing may provide novel therapeutic targets. A previous study revealed that junctional adhesion molecule A (JAM-A)-modified mesenchymal stem cells promoted wound healing. However, whether direct JAM-A modification in the skin wound edge area accelerates the wound repair process is not clear. We determined whether JAM-A modification at the skin wound edge accelerated the wound healing process. We established JAM-A modification mouse wound models and mouse primary fibroblast cell models. Wound pictures were taken to compare the wound size. H&E staining was performed to monitor the morphology of the wound and quality of the newborn skin. CCK-8 assays and immunofluorescence (IF) for Ki67 were used to measure the cell proliferation of mouse primary fibroblasts. Quantitative real-time PCR, immunohistochemistry, IF, and Western blot analysis were used to detect bFGF and EGF expression in vivo and in vitro. The JAM-A-overexpressing group exhibited a smaller residual wound size than the control group at Day 7. Thicker epidermal layers and more hair follicle-like structures were found in the JAM-A-overexpressing group at Day 21. Cell proliferation capacity was higher in JAM-A-modified mouse fibroblasts. Elevated levels of bFGF and EGF were found in the JAM-A-modified group in vivo and in vitro. JAM-A modification significantly promoted fibroblast proliferation and wound healing. Increased levels of bFGF and EGF growth factors may be part of the mechanism.

Generation of Genetically RGD σ1-Modified Oncolytic Reovirus That Enhances JAM-A-Independent Infection of Tumor Cells.

Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy.IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus, family Reoviridae, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.

Low junctional adhesion molecule-A expression is associated with an epithelial to mesenchymal transition and poorer outcomes in high-grade serous carcinoma of uterine adnexa.

High-grade serous carcinoma of uterine adnexa (HGSC) is the most frequent histotype of epithelial ovarian cancer and has a poor 5-year survival rate due to late-stage diagnosis and the poor efficacy of standard treatments. Novel biomarkers of cancer outcome are needed to identify new targetable pathways and improve personalized treatments. Cell-surface screening of 26 HGSC cell lines by high-throughput flow cytometry identified junctional adhesion molecule 1 (JAM-A, also known as F11R) as a potential biomarker. Using a multi-labeled immunofluorescent staining coupled with digital image analysis, protein levels of JAM-A were quantified in tissue microarrays from three HGSC patient cohorts: a discovery cohort (n = 101), the Canadian Ovarian Experimental Unified Resource cohort (COEUR, n = 1158), and the Canadian Cancer Trials Group OV16 cohort (n = 267). Low JAM-A level was associated with poorer outcome in the three cohorts by Kaplan-Meier (p = 0.023, p < 0.001, and p = 0.036, respectively) and was an independent marker of shorter survival in the COEUR cohort (HR = 0.517 (0.381-703), p < 0.001). When analyses were restricted to patients treated by taxane-platinum-based chemotherapy, low JAM-A protein expression was associated with poorer responses in the COEUR (p < 0.001) and OV16 cohorts (p = 0.006) by Kaplan-Meier. Decreased JAM-A gene expression was an indicator of poor outcome in gene expression datasets including The Cancer Genome Atlas (n = 606, p = 0.002) and Kaplan-Meier plotter (n = 1816, p = 0.024). Finally, we observed that tumors with decreased JAM-A expression exhibited an enhanced epithelial to mesenchymal transition (EMT) signature. Our results demonstrate that JAM-A expression is a robust prognostic biomarker of HGSC and may be used to discriminate tumors responsive to therapies targeting EMT.

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.

Junctional adhesion molecule-A: functional diversity through molecular promiscuity.

Cell adhesion molecules (CAMs) of the immunoglobulin superfamily (IgSF) regulate important processes such as cell proliferation, differentiation and morphogenesis. This activity is primarily due to their ability to initiate intracellular signaling cascades at cell-cell contact sites. Junctional adhesion molecule-A (JAM-A) is an IgSF-CAM with a short cytoplasmic tail that has no catalytic activity. Nevertheless, JAM-A is involved in a variety of biological processes. The functional diversity of JAM-A resides to a large part in a C-terminal PDZ domain binding motif which directly interacts with nine different PDZ domain-containing proteins. The molecular promiscuity of its PDZ domain motif allows JAM-A to recruit protein scaffolds to specific sites of cell-cell adhesion and to assemble signaling complexes at those sites. Here, we review the molecular characteristics of JAM-A, including its dimerization, its interaction with scaffolding proteins, and the phosphorylation of its cytoplasmic domain, and we describe how these characteristics translate into diverse biological activities.

Junctional adhesion molecule A: expression in the murine epididymal tract and accessory organs and acquisition by maturing sperm.

STUDY QUESTION: Is junctional adhesion molecule A (JAM-A), a sperm protein essential for normal motility, expressed in the murine post-testicular pathway and involved in sperm maturation? SUMMARY ANSWER: JAM-A is present in the prostate and seminal vesicles and in all three regions of the epididymis where it is secreted in epididymosomes in the luminal fluid and can be delivered to sperm in vitro. WHAT IS KNOWN ALREADY: JAM-A shares with the plasma membrane Ca2+ATPase 4 (PMCA4, the major Ca2+ efflux pump in murine sperm) a common interacting partner, CASK (Ca2+/CaM-dependent serine kinase). JAM-A, like PMCA4, plays a role in Ca2+ regulation, since deletion of Jam-A results in significantly elevated intracellular Ca2+ levels and reduced sperm motility. Recently, PMCA4 was reported to be expressed in the epididymis and along with CASK was shown to be in a complex on epididymosomes where it was transferred to sperm. Because of the association of JAM-A with CASK in sperm and because of the presence of PMCA4 and CASK in the epididymis, the present study was performed to determine whether JAM-A is expressed in the epididymis and delivered to sperm during their maturation. STUDY DESIGN, SIZE, DURATION: The epididymides, prostate and seminal vesicles were collected from sexually mature C57BL/6J and Institute for Cancer Research mice and antibodies specific for JAM-A and Ser285 -phosphorylated JAM-A (pJAM-A) were used for the analysis. Tissues, sperm and epididymal luminal fluid (ELF) were studied. Epididymosomes were also isolated for study. Caput and caudal sperm were co-incubated with ELF individually to determine their abilities to acquire JAM-A in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sections of all three regions of the epididymis were subjected to indirect immunofluorescence analysis. Epididymal tissues, fluid, sperm, prostate and seminal vesicle tissues were analyzed for JAM-A and/or pJAM-A via western blotting analysis. The relative amounts of JAM-A and pJAM-A among epididymal tissues, ELF and sperm were detected by western blot via quantification of band intensities. Epididymosomes were isolated by ultracentrifugation of the ELF after it was clarified to remove cells and tissue fragments, and the proteins western blotted for JAM-A and pJAM-A, and exosomal biochemical markers. FACS analysis was used to quantify the amount of JAM-A present on caput and caudal sperm, as well as the amount of JAM-A acquired in vitro after their co-incubation with ELF. MAIN RESULTS AND THE ROLE OF CHANCE: Western blots revealed that JAM-A is expressed in all three regions of the epididymis, the prostate and seminal vesicles. As confirmed by indirect immunofluorescence, a western blot showed that JAM-A has a higher expression in the corpus and caudal regions, where it is significantly (P < 0.01) more abundant than in the caput. Both JAM-A and Ser285-phosphorylated JAM-A (pJAM-A) are secreted into the ELF where it is highest in the distal regions. In the ELF, both JAM-A and pJAM-A were detected in epididymosomes. Western blotting of sperm proteins showed a significant (P < 0.01) increase of JAM-A and pJAM-A in caudal, compared with caput, sperm. Flow-cytometric analysis confirmed the increase in JAM-A in caudal sperm where it was 1.4-fold higher than in caput ones. Co-incubation of caput and caudal sperm with ELF demonstrated ~2.3- and ~1.3-fold increases, respectively, in JAM-A levels indicating that epididymosomes transfer more JAM-A to caput sperm that are less saturated with the protein than caudal ones. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: First, although the ELF was clarified prior to ultracentrifugation for epididymosome isolation, we cannot rule out contamination of the epididymosomal proteins by those from epididymal epithelial cells. Second, the JAM-A detected in the prostate and seminal vesicles might not necessarily be secreted from those organs and may only be present within the tissues, where it would be unable to impact sperm in the ejaculate. WIDER IMPLICATIONS OF THE FINDINGS: Although performed in the mouse the study has implications for humans, as the highly conserved JAM-A is a signaling protein in human sperm. There is physiological significance to the finding that JAM-A, which regulates sperm motility and intracellular Ca2+, exists in elevated levels in the cauda where sperm gain motility and fertilizing ability. The study suggests that the acquisition of JAM-A in the epididymal tract is involved in the mechanism by which sperm gain their motility during epididymal maturation. This increased understanding of sperm physiology is important for aspects of ART. STUDY FUNDING AND COMPETING INTEREST(S): The work was supported by NIH-RO3HD073523 and NIH-5P20RR015588 grants to P.A.M.-D. The authors declare there are no conflicts of interests.

Junctional Adhesion Molecule-A Is Highly Expressed on Human Hematopoietic Repopulating Cells and Associates with the Key Hematopoietic Chemokine Receptor CXCR4.

Hematopoietic stem/progenitor cells (HSPCs) reside in specialized bone marrow microenvironmental niches, with vascular elements (endothelial/mesenchymal stromal cells) and CXCR4-CXCL12 interactions playing particularly important roles for HSPC entry, retention, and maintenance. The functional effects of CXCL12 are dependent on its local concentration and rely on complex HSPC-niche interactions. Two Junctional Adhesion Molecule family proteins, Junctional Adhesion Molecule-B (JAM)-B and JAM-C, are reported to mediate HSPC-stromal cell interactions, which in turn regulate CXCL12 production by mesenchymal stromal cells (MSCs). Here, we demonstrate that another JAM family member, JAM-A, is most highly expressed on human hematopoietic stem cells with in vivo repopulating activity (p < .01 for JAM-A(high) compared to JAM-A(Int or Low) cord blood CD34(+) cells). JAM-A blockade, silencing, and overexpression show that JAM-A contributes significantly (p < .05) to the adhesion of human HSPCs to IL-1ß activated human bone marrow sinusoidal endothelium. Further studies highlight a novel association of JAM-A with CXCR4, with these molecules moving to the leading edge of the cell upon presentation with CXCL12 (p < .05 compared to no CXCL12). Therefore, we hypothesize that JAM family members differentially regulate CXCR4 function and CXCL12 secretion in the bone marrow niche. Stem Cells 2016;34:1664-1678.

Short communication: Glucagon-like peptide-2 and coccidiosis alter tight junction gene expression in the gastrointestinal tract of dairy calves.

Tight junction (TJ) proteins are integral factors involved in gut barrier function, and therapy with glucagon-like peptide-2 (GLP-2) enhances gut integrity. Our aim was to assess effects of GLP-2 treatment on mRNA expression of 8 TJ complex proteins in the intestine of dairy calves not infected or infected with Eimeria bovis at 11±3d of age. Mucosal epithelium from jejunum, ileum, and cecum was collected at slaughter from Holstein bull calves assigned to 4 groups: noninfected, buffer-treated (n=5); noninfected, GLP-2 treated (n=4); E. bovis-infected, buffer-treated (n=5); and E. bovis-infected, GLP-2-treated (n=4). Infected calves were orally dosed with 100,000 to 200,000 sporulated E. bovis oocysts on d 0; GLP-2-treated calves received 50 µg of GLP-2/kg of body weight subcutaneously twice daily for 10d beginning on d 18; and buffer-treated calves received an equal injection volume of 0.01 M Na bicarbonate buffer. All calves were killed on d 28. The mRNA expression of coxsackie and adenovirus receptor (CXADR), claudins 1, 2, and 4 (CLDN1, CLDN2, and CLDN4), F11 receptor (F11R), junction adhesion molecule 2 (JAM2), occludin (OCLN), and tight junction protein ZO-1 (TJP1) was determined by real-time quantitative PCR. In jejunum and ileum, an interaction of E. bovis infection and GLP-2 treatment on gene expression was noted. In jejunum of noninfected calves, GLP-2 increased CXADR, CLDN2, OCLN, and TJP1 mRNA expression but had no effect on mRNA expression in infected calves. Treatment with GLP-2 also increased tight junction protein ZO-1 protein expression in jejunum of noninfected calves as determined by immunohistochemistry. In ileum, E. bovis decreased expression of JAM2, OCLN, and TJP1 in buffer-treated calves, and GLP-2 increased TJP1 expression in infected calves. In cecum, E. bovis infection reduced expression of CXADR, CLDN4, F11R, and OCLN, and GLP-2 therapy increased expression of CLDN4, F11R, OCLN, and TJP1. Results are consistent with studies in nonruminants showing decreased expression of TJ complex proteins in the intestinal tract during pathogen-induced diarrhea and increased TJ protein expression in intestinal tissues in response to GLP-2 treatment. In conclusion, E. bovis reduces gene expression of TJ proteins primarily in cecum of calves 28d postinfection, and GLP-2 increases expression of selected TJ genes in intestinal tissues. Use of GLP-2 to improve gut barrier function in ruminants during pathogen-induced diarrhea warrants additional study.

Generation and characterization of mAb 61H9 against junctional adhesion molecule-a with potent antitumor activity.

Junctional adhesion molecule-A (JAM-A) is an adhesion molecule that exists on the surface of certain types of cells, including white blood cells, endothelial cells, and dendritic cells. In this study, the cDNA sequences of JAM-A-Fc were chemically synthesized with optimization for mammalian expression. Afterward, we analyzed JAM-A protein expression through transient transfection in HEK293 cell lines. Mice were immunized with JAM-A-Fc protein, and hybridoma was prepared by fusing myeloma cells and mouse spleen cells. Antibodies were purified from the hybridoma supernatant and four monoclonal strains were obtained and numbered 61H9, 70E5, 71A8, and 74H3 via enzyme-linked immunosorbent assay screening. Immunofluorescence staining assay showed 61H9 was the most suitable cell line for mAb production due to its fluorescence signal being the strongest. Flow cytometric analysis proved that 61H9 possessed high affinity. Moreover, antagonism of JAM-A mAb could attenuate the proliferative, migrative, and invasive abilities of ESCC cells and significantly inhibit tumor growth in mice. By examining hematoxylin-eosin staining mice tumor tissues, we found inflammatory cells infiltrated lightly in the anti-JAM-A group. The expression of BCL-2 and IκBα in the anti-JAM-A group were decreased in mice tumor tissues compared to the control group. Ultimately, a method for preparing high-yield JAM-A-Fc protein was created and a high affinity mAb against JAM-A with an antitumor effect was prepared.

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus).

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORF) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV.

Research Note: Junctional adhesion molecule A is expressed in epithelial cells of the crypt and villi whereas junctional adhesion molecule 2 is expressed in vascular cells.

A functional intestinal barrier is essential for a healthy intestine. This barrier includes an apical tight junctional complex between adjacent intestinal epithelial cells. The tight junctions (TJ) are multiprotein junctional complexes that consist of a number of members of the occludin, claudin, zona occludens, and junctional adhesion molecule families. The mRNA expression of junctional adhesin molecule A (JAMA) and junctional adhesion molecule 2 (JAM2) are 2 TJ mRNAs that are often used to assess intestinal barrier integrity. The objective of this study was to use in situ hybridization to identify cells that express JAMA and JAM2 mRNA in the small intestine of chickens. In the jejunum of a 21 d old broiler, JAMA mRNA was highly expressed in the epithelial cells of the villi and crypt. By contrast, JAM2 mRNA was located in the vascular system in the center of the villi and in the lamina propria. These results demonstrate that JAMA and not JAM2 is the appropriate gene to use when assessing TJ between intestinal epithelial cells.

LINC01146/F11R facilitates growth and metastasis of prostate cancer under the regulation of TGF-ß.

The effect of long intergenic non-protein coding RNAs (lncRNAs) was verified in prostate cancer (PCa), but the mechanism of LINC01146 in PCa is unclear. Bioinformatics was applied to analyze LINC01146 expression in PCa and predict target genes of LINC01146, followed by the verification of qRT-PCR, RNA pull-down and co-immunoprecipitation (Co-IP). The correlation between LINC01146 expression and clinicopathological characteristics was investigated. The location of LINC01146 in PCa cells was detected by fluorescence in situ hybridization (FISH). After interference with LINC01146 or/and F11 receptor (F11R) or treated with transforming growth factor beta 1 (TGF-ß1), the function of LINC01146 in PCa in vitro or in vivo was determined by CCK-8, colony formation, flow cytometry, scratch test, transwell assay, xenograft experiment and western blot. LINC01146 and F11R were over-expressed in PCa and positively correlated with poor prognosis. LINC01146 located in the cytoplasm and combined with F11R. LINC01146 overexpression impeded apoptosis, facilitated viability, proliferation, migration and invasion in PCa cells in vitro, promoted tumor growth in vivo, downregulated E-cadherin, Bax and Cleaved caspase-3, and upregulated N-cadherin, Vimentin and PCNA, but LINC01146 silencing did the opposite. F11R was positively regulated by LINC01146 and F11R depletion negated the effect of LINC01146 overexpression on malignant phenotypes of PCa cells. The expression of LINC01146 and F11R was regulated by TGF-ß1. The promoting role of TGF-ß1 in migration, invasion and F11R in PCa cells was reversed by LINC01146 silencing. LINC01146 upregulated F11R to facilitate malignant phenotypes of PCa cells, which was regulated by TGF-ß.

Aging-induced impaired endothelial wall shear stress mechanosensing causes arterial remodeling via JAM-A/F11R shedding by ADAM17.

Physiological and pathological vascular remodeling is uniquely driven by mechanical forces from blood flow in which wall shear stress (WSS) mechanosensing by the vascular endothelium plays a pivotal role. This study aimed to determine the novel role for a disintegrin and metalloproteinase 17 (ADAM17) in impaired WSS mechanosensing, which was hypothesized to contribute to aging-associated abnormal vascular remodeling. Without changes in arterial blood pressure and blood flow rate, skeletal muscle resistance arteries of aged mice (30-month-old vs. 12-week-old) exhibited impaired WSS mechanosensing and displayed inward hypertrophic arterial remodeling. These vascular changes were recapitulated by in vivo confined, AAV9-mediated overexpression of ADAM17 in the resistance arteries of young mice. An aging-related increase in ADAM17 expression reduced the endothelial junction level of its cleavage substrate, junctional adhesion molecule-A/F11 receptor (JAM-A/F11R). In cultured endothelial cells subjected to steady WSS ADAM17 activation or JAM-A/F11R knockdown inhibited WSS mechanosensing. The ADAM17-activation induced, impaired WSS mechanosensing was normalized by overexpression of ADAM17 cleavage resistant, mutated JAM-AV232Y both in cultured endothelial cells and in resistance arteries of aged mice, in vivo. These data demonstrate a novel role for ADAM17 in JAM-A/F11R cleavage-mediated impaired endothelial WSS mechanosensing and subsequently developed abnormal arterial remodeling in aging. ADAM17 could prove to be a key regulator of WSS mechanosensing, whereby it can also play a role in pathological vascular remodeling in diseases.