RESUMEN
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.
Asunto(s)
Molécula A de Adhesión de Unión , Molécula B de Adhesión de Unión , Animales , Molécula A de Adhesión de Unión/genética , Molécula A de Adhesión de Unión/metabolismo , Molécula B de Adhesión de Unión/metabolismo , Pollos/genética , Células Epiteliales/metabolismo , Uniones Estrechas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ocludina/genéticaRESUMEN
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.
Asunto(s)
Molécula A de Adhesión de Unión , MicroARNs , Neoplasias de la Próstata , ARN Largo no Codificante , Masculino , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/genética , Molécula A de Adhesión de Unión/genética , Molécula A de Adhesión de Unión/metabolismo , Regulación Neoplásica de la Expresión Génica , Movimiento Celular/genética , Hibridación Fluorescente in Situ , Línea Celular Tumoral , Neoplasias de la Próstata/metabolismo , ARN Largo no Codificante/genética , Proliferación Celular/genética , MicroARNs/genética , Receptores de Superficie Celular/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismoRESUMEN
Overexpression of the human epidermal growth factor receptor-2 (HER2) is associated with aggressive disease in breast and certain other cancers. At a cellular level, the adhesion protein Junctional Adhesion Molecule-A (JAM-A) has been reported to regulate the expression of HER3 via a transcriptional pathway involving FOXA1. Since FOXA1 is also a suggested transcription factor for HER2, this study set out to determine if JAM-A regulates HER2 expression via a similar mechanism. An integrated tripartite approach was taken, involving cellular expression studies after targeted disruption of individual players in the putative pathway, in silico identification of relevant HER2 promoter regions and, finally, interrogation of cancer patient survival databases to deconstruct functionally important links between HER2, JAM-A and FOXA1 gene expression. The outcome of these investigations revealed a unidirectional pathway in which JAM-A expression transcriptionally regulates that of HER2 by influencing the binding of FOXA1 to a specific site in the HER2 gene promoter. Moreover, a correlation between JAM-A and HER2 gene expression was identified in 75% of a sample of 40 cancer types from The Cancer Genome Atlas, and coincident high mean mRNA expression of JAM-A, HER2 and FOXA1 was associated with poorer survival outcomes in HER2-positive (but not HER2-negative) patients with either breast or gastric tumors. These investigations provide the first evidence of a transcriptional pathway linking JAM-A, HER2 and FOXA1 in cancer settings, and support potential future pharmacological targeting of JAM-A as an upstream regulator of HER2.
Asunto(s)
Neoplasias de la Mama , Factor Nuclear 3-alfa del Hepatocito , Molécula A de Adhesión de Unión , Receptor ErbB-2 , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Femenino , Factor Nuclear 3-alfa del Hepatocito/genética , Humanos , Molécula A de Adhesión de Unión/genética , Receptor ErbB-2/genética , Receptores de Superficie Celular/genéticaRESUMEN
Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cell-cell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here, we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen-I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen-I-binding α3ß1 integrin. We also find that JAM-A interacts with tetraspanins CD151 and CD9, which both interact with α3ß1 integrin and regulate α3ß1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with α3ß1 integrin and tetraspanins CD151 and CD9 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3ß1 integrin or tetraspanins CD151 and CD9 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A exists with α3ß1 integrin and tetraspanins CD151 and CD9 in a functional complex to regulate collective cell migration of polarized epithelial cells.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Integrina alfa3beta1/metabolismo , Tetraspanina 24/metabolismo , Tetraspanina 29/metabolismo , Animales , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Perros , Doxorrubicina/farmacología , Humanos , Molécula A de Adhesión de Unión/antagonistas & inhibidores , Molécula A de Adhesión de Unión/genética , Células de Riñón Canino Madin Darby , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
Helicobacter pylori infects approximately half of the world's population and is the strongest risk factor for peptic ulcer disease and gastric cancer, representing a major global health concern. H. pylori persistently colonizes the gastric epithelium, where it subverts the highly organized structures that maintain epithelial integrity. Here, a unique strategy used by H. pylori to disrupt the gastric epithelial junctional adhesion molecule-A (JAM-A) is disclosed, using various experimental models that include gastric cell lines, primary human gastric cells, and biopsy specimens of infected and non-infected individuals. H. pylori preferentially cleaves the cytoplasmic domain of JAM-A at Alanine 285. Cells stably transfected with full-length JAM-A or JAM-A lacking the cleaved sequence are used in a range of functional assays, which demonstrate that the H. pylori cleaved region is critical to the maintenance of the epithelial barrier and of cell-cell adhesion. Notably, by combining chromatography techniques and mass spectrometry, PqqE (HP1012) is purified and identified as the H. pylori virulence factor that cleaves JAM-A, uncovering a previously unreported function for this bacterial protease. These findings propose a novel mechanism for H. pylori to disrupt epithelial integrity and functions, breaking new ground in the understanding of the pathogenesis of this highly prevalent and clinically relevant infection.
Asunto(s)
Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/enzimología , Molécula A de Adhesión de Unión/metabolismo , Factores de Virulencia/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Humanos , Molécula A de Adhesión de Unión/química , Molécula A de Adhesión de Unión/genética , Dominios Proteicos , Factores de Virulencia/genéticaRESUMEN
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.
Asunto(s)
Molécula A de Adhesión de Unión/genética , Molécula A de Adhesión de Unión/metabolismo , Oligopéptidos/metabolismo , Reoviridae/genética , Reoviridae/metabolismo , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas , Moléculas de Adhesión Celular , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Humanos , Orthoreovirus Mamífero 3/genética , Orthoreovirus Mamífero 3/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Desnudos , Viroterapia Oncolítica , Virus Oncolíticos/genética , Orthoreovirus/genética , Orthoreovirus/metabolismo , Receptores de Superficie Celular , Replicación ViralRESUMEN
Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA-124 (miR-124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR-124 in NPC CSCs. qRT-PCR was performed to measure miR-124 expression in NPC tissues and cell lines and the effects of miR-124 on stem-like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR-124 with the 3'UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR-124 in an animal model and clinical samples. Down-regulation of miR-124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR-124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up-regulation of miR-124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR-124 can inhibit stem-like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.
Asunto(s)
Molécula A de Adhesión de Unión/genética , MicroARNs/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Tolerancia a Radiación/genética , Regiones no Traducidas 3'/genética , Animales , Línea Celular Tumoral , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Metástasis Linfática/genética , Ratones , Ratones Endogámicos BALB C , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND/AIM: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: Tetrocarcin-A merits further investigation as a novel anti-tumor agent in TNBC.
Asunto(s)
Aminoglicósidos/farmacología , Antineoplásicos/farmacología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Embrión de Pollo , Membrana Corioalantoides , Regulación hacia Abajo , Silenciador del Gen , Humanos , Molécula A de Adhesión de Unión/genética , Ratones , ARN Interferente Pequeño/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Cell adhesion in the multiple myeloma (MM) microenvironment has been recognized as a major mechanism of MM cell survival and the development of drug resistance. Here we addressed the hypothesis that the protein junctional adhesion molecule-A (JAM-A) may represent a novel target and a clinical biomarker in MM. We evaluated JAM-A expression in MM cell lines and in 147 MM patient bone marrow aspirates and biopsies at different disease stages. Elevated JAM-A levels in patient-derived plasma cells were correlated with poor prognosis. Moreover, circulating soluble JAM-A (sJAM-A) levels were significantly increased in MM patients as compared with controls. Notably, in vitro JAM-A inhibition impaired MM migration, colony formation, chemotaxis, proliferation and viability. In vivo treatment with an anti-JAM-A monoclonal antibody (αJAM-A moAb) impaired tumor progression in a murine xenograft MM model. These results demonstrate that therapeutic targeting of JAM-A has the potential to prevent MM progression, and lead us to propose JAM-A as a biomarker in MM, and sJAM-A as a serum-based marker for clinical stratification.
Asunto(s)
Biomarcadores de Tumor , Molécula A de Adhesión de Unión/sangre , Mieloma Múltiple/sangre , Mieloma Múltiple/mortalidad , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Médula Ósea/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica , Humanos , Molécula A de Adhesión de Unión/genética , Masculino , Ratones , Terapia Molecular Dirigida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , PronósticoRESUMEN
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.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Eucariotas/metabolismo , Inmunoglobulinas/metabolismo , Molécula A de Adhesión de Unión/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Animales , Proteínas Portadoras/genética , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Células Eucariotas/ultraestructura , Regulación de la Expresión Génica , Humanos , Inmunoglobulinas/genética , Molécula A de Adhesión de Unión/genética , Proteínas de Microfilamentos/genética , Morfogénesis/genética , Proteínas Nucleares/genética , Dominios PDZ , Fosforilación , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructuraRESUMEN
BACKGROUND: Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. METHODS: Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. RESULTS: Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. CONCLUSIONS: Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour.
Asunto(s)
Toxina del Cólera/sangre , Disbiosis/inmunología , Endotelio/metabolismo , Ileítis/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Espondilitis Anquilosante/inmunología , Enfermedad Aguda , Proteínas de Fase Aguda , Uniones Adherentes/genética , Animales , Antibacterianos/farmacología , Antígenos CD/genética , Bacterias/aislamiento & purificación , Células CACO-2 , Cadherinas/genética , Proteínas Portadoras/sangre , Proteínas Portadoras/genética , Estudios de Casos y Controles , Toxina del Cólera/genética , Enfermedad Crónica , Disbiosis/microbiología , Proteínas de Unión a Ácidos Grasos/sangre , Expresión Génica , Antígeno HLA-B27/genética , Haptoglobinas , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ileítis/sangre , Íleon/inmunología , Íleon/microbiología , Interleucina-8 , Mucosa Intestinal/microbiología , Molécula A de Adhesión de Unión/genética , Lipopolisacáridos/sangre , Glicoproteínas de Membrana/sangre , Proteínas de la Membrana/genética , Monocitos/inmunología , Permeabilidad , Precursores de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Transgénicas , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/genética , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Epidídimo/metabolismo , Molécula A de Adhesión de Unión/genética , Maduración del Esperma/genética , Motilidad Espermática/genética , Espermatozoides/metabolismo , Animales , Señalización del Calcio , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Epidídimo/citología , Epidídimo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Humanos , Molécula A de Adhesión de Unión/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Próstata/citología , Próstata/crecimiento & desarrollo , Próstata/metabolismo , Transporte de Proteínas , Vesículas Seminales/citología , Vesículas Seminales/crecimiento & desarrollo , Vesículas Seminales/metabolismo , Espermatozoides/citología , Espermatozoides/crecimiento & desarrolloRESUMEN
Tight junctions are the outermost structures of intercellular junctions and are classified as transmembrane proteins. These factors form selective permeability barriers between cells, act as paracellular transporters and regulate structural and functional polarity of cells. Although tight junctions have been previously studied, comparison of the transcriptionaltranslational levels of these molecules in canine organs remains to be investigated. In the present study, organspecific expression of the tight junction proteins, claudin, occludin, junction adhesion molecule A and zona occludens 1 was examined in the canine duodenum, lung, liver and kidney. Results of immunohistochemistry analysis demonstrated that the tight junctions were localized in intestinal villi and glands of the duodenum, bronchiolar epithelia and alveolar walls of the lung, endometrium and myometrium of the hepatocytes, and the distal tubules and glomeruli of the kidney. These results suggest that tight junctions are differently expressed in organs, and therefore may be involved in organspecific functions to maintain physiological homeostasis.
Asunto(s)
Claudinas/análisis , Perros/genética , Molécula A de Adhesión de Unión/análisis , Ocludina/análisis , Proteína de la Zonula Occludens-1/genética , Animales , Claudinas/genética , Duodeno/metabolismo , Duodeno/ultraestructura , Femenino , Expresión Génica , Molécula A de Adhesión de Unión/genética , Riñón/metabolismo , Riñón/ultraestructura , Hígado/metabolismo , Hígado/ultraestructura , Pulmón/metabolismo , Pulmón/ultraestructura , Ocludina/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Proteína de la Zonula Occludens-1/análisisRESUMEN
MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial-mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3' UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Molécula A de Adhesión de Unión/genética , MicroARNs/genética , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patología , Interferencia de ARN , Regiones no Traducidas 3' , Sitios de Unión , Brassica/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Molécula A de Adhesión de Unión/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN de PlantaRESUMEN
Junctional adhesion molecule A (JAM-A) is a broadly expressed adhesion molecule that regulates cell-cell contacts and facilitates leukocyte transendothelial migration. The latter occurs through interactions with the integrin LFA-1. Although we understand much about JAM-A, little is known regarding the protein's role in mechanotransduction or as a modulator of RhoA signaling. We found that tension imposed on JAM-A activates RhoA, which leads to increased cell stiffness. Activation of RhoA in this system depends on PI3K-mediated activation of GEF-H1 and p115 RhoGEF. These two GEFs are further regulated by FAK/ERK and Src family kinases, respectively. Finally, we show that phosphorylation of JAM-A at Ser-284 is required for RhoA activation in response to tension. These data demonstrate a direct role of JAM-A in mechanosignaling and control of RhoA and implicate Src family kinases in the regulation of p115 RhoGEF.
Asunto(s)
Molécula A de Adhesión de Unión/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Moléculas de Adhesión Celular , Técnicas de Cultivo de Célula , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Molécula A de Adhesión de Unión/genética , Molécula A de Adhesión de Unión/fisiología , Mecanotransducción Celular/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Familia-src QuinasasRESUMEN
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.
Asunto(s)
Comunicación Celular , Células Endoteliales/fisiología , Hipotermia , Molécula A de Adhesión de Unión/metabolismo , Molécula B de Adhesión de Unión/metabolismo , Leucocitos/fisiología , Recalentamiento , Membrana Celular/metabolismo , Expresión Génica , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Molécula A de Adhesión de Unión/genética , Molécula B de Adhesión de Unión/genética , Migración Transendotelial y TransepitelialRESUMEN
Planar spindle orientation in polarized epithelial cells depends on the precise localization of the dynein-dynactin motor protein complex at the lateral cortex. The contribution of cell adhesion molecules to the cortical localization of the dynein-dynactin complex is poorly understood. Here we find that junctional adhesion molecule-A (JAM-A) regulates the planar orientation of the mitotic spindle during epithelial morphogenesis. During mitosis, JAM-A triggers a transient activation of Cdc42 and PI(3)K, generates a gradient of PtdIns(3,4,5)P3 at the cortex and regulates the formation of the cortical actin cytoskeleton. In the absence of functional JAM-A, dynactin localization at the cortex is reduced, the mitotic spindle apparatus is misaligned and epithelial morphogenesis in three-dimensional culture is compromised. Our findings indicate that a PI(3)K- and cortical F-actin-dependent pathway of planar spindle orientation operates in polarized epithelial cells to regulate epithelial morphogenesis, and we identify JAM-A as a junctional regulator of this pathway.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Dineínas/metabolismo , Molécula A de Adhesión de Unión/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Huso Acromático/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Actinas/metabolismo , Animales , Polaridad Celular , Perros , Complejo Dinactina , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Molécula A de Adhesión de Unión/metabolismo , Células de Riñón Canino Madin Darby , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/genéticaRESUMEN
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.
Asunto(s)
Coccidiosis/tratamiento farmacológico , Tracto Gastrointestinal/parasitología , Expresión Génica , Péptido 2 Similar al Glucagón/farmacología , Proteína de la Zonula Occludens-1/genética , Animales , Animales Recién Nacidos , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Enfermedades de los Bovinos/parasitología , Claudina-1/genética , Claudina-1/metabolismo , Claudina-2/genética , Claudina-2/metabolismo , Claudina-4/genética , Claudina-4/metabolismo , Coccidiosis/veterinaria , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Eimeria/efectos de los fármacos , Eimeria/aislamiento & purificación , Tracto Gastrointestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Molécula A de Adhesión de Unión/genética , Molécula A de Adhesión de Unión/metabolismo , Ocludina/genética , Ocludina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
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.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Molécula A de Adhesión de Unión/metabolismo , Molécula B de Adhesión de Unión/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Aorta/citología , Aorta/crecimiento & desarrollo , Aorta/metabolismo , Diferenciación Celular , Movimiento Celular , Molécula A de Adhesión de Unión/genética , Molécula B de Adhesión de Unión/genética , Fenotipo , Receptores de Superficie Celular/genética , Somitos/citología , Somitos/embriología , Somitos/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
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.