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Moskal J, Michalak S. Tight junction proteins in glial tumors development and progression. Front Cell Neurosci 2025; 19:1541885. [PMID: 39963115 PMCID: PMC11830821 DOI: 10.3389/fncel.2025.1541885] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2024] [Accepted: 01/09/2025] [Indexed: 02/20/2025] Open
Abstract
Tight junctions form a paracellular barrier in epithelial and endothelial cells, and they regulate the diffusion of fluids, molecules, and the penetration of cells across tissue compartments. Tight junctions are composed of a group of integral membrane proteins, which include the claudin family, tight junction-associated Marvel protein family, junctional adhesion molecule family, and proteins that anchor the cytoskeleton, such as zonula occludens proteins and the cingulin family. Several factors, such as neurotransmitters or cytokines, and processes like ischemia/hypoxia, inflammation, tumorigenesis, phosphorylation/dephosphorylation, ubiquitination, and palmitoylation, regulate tight junction proteins. Claudins are involved in tumorigenesis processes that lead to glioma formation. In gliomas, there is a noticeable dysregulation of claudins, occludin, and zonula occludens-1 abundance, and their dislocation has been observed. The weakening of intercellular adhesion and cell detachment is responsible for glioma infiltration into surrounding tissues. Furthermore, the paracellular permeability of the blood-brain barrier, formed with the involvement of tight junction proteins, influences the development of peritumoral edema - and, simultaneously, the rate of drug delivery to the glial tumor. Understanding the junctional and paracellular environments in brain tumors is crucial to predicting glial tumor progression and the feasibility of chemotherapeutic drug delivery. This knowledge may also illuminate differences between high and low-grade gliomas.
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Affiliation(s)
- Jakub Moskal
- Department of Neurosurgery and Neurotraumatology, Poznan University of Medical Sciences, Poznan, Poland
| | - Slawomir Michalak
- Department of Neurosurgery and Neurotraumatology, Poznan University of Medical Sciences, Poznan, Poland
- Department of Neurochemistry and Neuropathology, Poznan University of Medical Sciences, Poznan, Poland
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Bendinelli P, De Noni I, Cattaneo S, Silvetti T, Brasca M, Piazzalunga F, Donetti E, Ferraretto A. Surface layer proteins from Lactobacillus helveticus ATCC® 15009™ affect the gut barrier morphology and function. Tissue Barriers 2024; 12:2289838. [PMID: 38059583 PMCID: PMC11583618 DOI: 10.1080/21688370.2023.2289838] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Revised: 10/30/2023] [Accepted: 11/28/2023] [Indexed: 12/08/2023] Open
Abstract
Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.
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Affiliation(s)
- Paola Bendinelli
- Department of Biomedical Sciences for Health, University of Milan, Milan, Italy
| | - Ivano De Noni
- Department of Food, Environmental and Nutritional Sciences, University of Milan, Milan, Italy
| | - Stefano Cattaneo
- Department of Food, Environmental and Nutritional Sciences, University of Milan, Milan, Italy
| | - Tiziana Silvetti
- Institute of Sciences of Food Production, National Research Council (CNR-ISPA), Milan, Italy
| | - Milena Brasca
- Institute of Sciences of Food Production, National Research Council (CNR-ISPA), Milan, Italy
| | | | - Elena Donetti
- Department of Biomedical Sciences for Health, University of Milan, Milan, Italy
| | - Anita Ferraretto
- Department of Biomedical Sciences for Health, University of Milan, Milan, Italy
- Laboratory of Experimental Biochemistry & Molecular Biology, IRCCS Istituto Galeazzi-Sant’Ambrogio, Milan, Italy
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Fernández-Lainez C, Aan de Stegge M, Silva-Lagos LA, López-Velázquez G, de Vos P. β(2 → 1)-β(2 → 6) and β(2 → 1) fructans protect from impairment of intestinal tight junction's gene expression and attenuate human dendritic cell responses in a fructan-dependent fashion. Carbohydr Polym 2023; 320:121259. [PMID: 37659831 DOI: 10.1016/j.carbpol.2023.121259] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 05/12/2023] [Accepted: 08/02/2023] [Indexed: 09/04/2023]
Abstract
β(2 → 1)-β(2 → 6) branched graminan-type fructans (GTFs) and β(2 → 1) linear fructans (ITFs) possess immunomodulatory properties and protect human intestinal barrier function, however the mechanisms underlying these effects are not well studied. Herein, GTFs and ITFs effects with different degree of polymerization (DP) values on tight junctions (TJs) genes CLDN-1, -2 and -3, CDH1, OCLN and TJP1 were studied in Caco-2 gut epithelial cells, under homeostatic and inflammatory conditions. Also, cytokine production in dendritic cells (DCs) was studied. Higher DP fructans decreased the expression of the pore forming CLDN-2. Higher DP GTFs enhanced CLDN-3, OCLN, and TJP-1. Fructans prevented mRNA dysregulation of CLDN-1, -2 and -3 induced by the barrier disruptors A23187 and deoxynivalenol in a fructan-type dependent fashion. The production of pro-inflammatory cytokines MCP-1/CCL2, MIP-1α/CCL3 and TNFα by DCs was also attenuated in a fructan-type dependent manner and was strongly attenuated by DCs cultured with medium of Caco-2 cells which were pre-exposed to fructans. Our data show that specific fructans have TJs and DCs modulating effects and contribute to gut homeostasis. This might serve to design effective dietary means to prevent intestinal inflammation.
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Affiliation(s)
- Cynthia Fernández-Lainez
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands; Laboratorio de Errores innatos del Metabolismo y Tamiz, Instituto Nacional de Pediatría, Ciudad de México, Mexico; Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Cuidad de México, Mexico.
| | - Myrthe Aan de Stegge
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
| | - Luis Alfredo Silva-Lagos
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
| | - Gabriel López-Velázquez
- Laboratorio de Biomoléculas y Salud Infantil, Instituto Nacional de Pediatría, Cuidad de México, Mexico.
| | - Paul de Vos
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
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Enniatin B and beauvericin affect intestinal cell function and hematological processes in Atlantic salmon (Salmo salar) after acute exposure. Food Chem Toxicol 2023; 172:113557. [PMID: 36526092 DOI: 10.1016/j.fct.2022.113557] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2022] [Revised: 12/05/2022] [Accepted: 12/11/2022] [Indexed: 12/15/2022]
Abstract
Unintentional use of mold-infested plant-based feed ingredients are sources of mycotoxins in fish feeds. The presence of the emerging mycotoxins ENNB and BEA in Norwegian commercial fish feeds and plant-based feed ingredients has raised concerns regarding the health effects on farmed Atlantic salmon (Salmon salar). Atlantic salmon pre-smolts were exposed to non-lethal doses of BEA and ENNB (ctrl, 50 and 500 μg/kg feed for 12 h), after which total RNA sequencing of the intestine and liver was carried out to evaluate gut health and identify possible hepatological changes after acute dietary exposure. ENNB and BEA did not trigger acute toxicity, however ENNB caused the onset of pathways linked to acute intestinal inflammation and BEA exposures caused the onset of hepatic hematological disruption. The prevalence and concentration of ENNB found in today's commercial feed could affect the fish health if consumed over a longer time-period.
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Fernández-Lainez C, Logtenberg MJ, Tang X, Schols HA, López-Velázquez G, de Vos P. β(2→1) chicory and β(2→1)-β(2→6) agave fructans protect the human intestinal barrier function in vitro in a stressor-dependent fashion. Food Funct 2022; 13:6737-6748. [PMID: 35665791 DOI: 10.1039/d2fo00534d] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Dietary fibers such as fructans can protect the intestinal epithelial barrier integrity, but the mechanisms underlying this protection are not completely understood. We aimed to study the protective effect of β(2→1)-β(2→6) branched graminan-type fructans (GTFs) on gut epithelial barrier function that was disrupted by three different agents which impact the barrier function via different cellular mechanisms. The effects of GTFs were compared with those of linear β(2→1) inulin-type fructans (ITFs). T84 intestinal epithelial monolayers were incubated with GTFs and ITFs. Afterwards, the monolayers were challenged with the barrier disruptors calcium ionophore A23187, 12-myristate 13-acetate (PMA) and deoxynivalenol (DON). Transepithelial resistance was measured with an electric cell-substrate impedance sensing system. All fructans studied prevented the barrier disruption induced by A23187. ITF II protected from the disruptive effects of PMA. However, none of the studied fructans influenced the disruption induced by DON. As a measure of disruption-induced inflammation, interleukin-8 (IL-8) production by the intestinal epithelium was determined by ELISA. The production of IL-8 induced by A23187 was decreased by all fructans, whereas IL-8 production induced by DON decreased only upon pre-treatment with ITF II. None of the studied fructans prevented PMA induced IL-8 production. GTFs just like ITFs can influence the barrier function and inflammatory processes in gut epithelial cells in a structure-dependent fashion. These distinct protective effects are dependent on the different signaling pathways that lead to gut barrier disruption.
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Affiliation(s)
- Cynthia Fernández-Lainez
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.
- Laboratorio de Errores innatos del Metabolismo y Tamiz, Instituto Nacional de Pediatría, Av. Iman 1, 04530, Ciudad de México, Mexico
- Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México. Edificio D, 1° Piso. Circuito de Posgrados, Ciudad Universitaria, 04510, Ciudad de México, Mexico
| | - Madelon J Logtenberg
- Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands.
| | - Xin Tang
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.
| | - Henk A Schols
- Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708 WG, Wageningen, The Netherlands.
| | - Gabriel López-Velázquez
- Laboratorio de Biomoléculas y Salud Infantil, Instituto Nacional de Pediatría, Av. Iman 1, 04530, Cuidad de México, Mexico.
| | - Paul de Vos
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University Medical Centre Groningen, Hanzeplein 1, 9713 GZ, Groningen, The Netherlands.
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Ren C, Zhang Q, de Haan BJ, Faas MM, Zhang H, de Vos P. Protective effects of lactic acid bacteria on gut epithelial barrier dysfunction are Toll like receptor 2 and protein kinase C dependent. Food Funct 2020; 11:1230-1234. [PMID: 32043507 DOI: 10.1039/c9fo02933h] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Lactic acid bacteria (LAB) are recognized for support of host gut homeostasis but the precise mechanisms remain to be identified. LABs interact with Toll-like receptors (TLRs) which might stimulate barrier function of gut epithelial cells. We previously identified six TLR2-signalling LAB strains. As TLR2 is involved in barrier-function enhancement in gut-epithelium, the epithelial barrier-protective effect of these TLR2-signalling strains was studied by using T84 human colorectal cancer cell monolayer as an in vitro gut epithelial barrier model. The protein kinase C (PKC) dependent barrier disruptor A23187 and mitogen-activated protein kinase dependent barrier stressor deoxynivalenol were tested to determine which pathways LAB influenced. We found that exclusively the PKC dependent disruption was prevented by the selected TLR2-signalling LAB strains. This study suggests that TLR2 is a pivotal epithelial barrier modulator, and provides novel insight in the molecular mechanisms by which LAB contribute to intestinal health.
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Affiliation(s)
- Chengcheng Ren
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen and University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. and School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
| | - Qiuxiang Zhang
- School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
| | - Bart J de Haan
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen and University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
| | - Marijke M Faas
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen and University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
| | - Hao Zhang
- School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
| | - Paul de Vos
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen and University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
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Clostridium perfringens Epsilon-Toxin Impairs the Barrier Function in MDCK Cell Monolayers in a Ca 2+-Dependent Manner. Toxins (Basel) 2020; 12:toxins12050286. [PMID: 32365779 PMCID: PMC7291203 DOI: 10.3390/toxins12050286] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Revised: 04/21/2020] [Accepted: 04/28/2020] [Indexed: 12/27/2022] Open
Abstract
Epsilon-toxin produced by Clostridium perfringens significantly contributes to the pathogeneses of enterotoxemia in ruminants and multiple sclerosis in humans. Epsilon-toxin forms a heptameric oligomer in the host cell membrane, promoting cell disruption. Here, we investigate the effect of epsilon-toxin on epithelial barrier functions. Epsilon-toxin impairs the barrier integrity of Madin-Darby Canine Kidney (MDCK) cells, as demonstrated by decreased transepithelial electrical resistance (TEER), increased paracellular flux marker permeability, and the decreased cellular localization of junctional proteins, such as occludin, ZO-1, and claudin-1. U73122, an endogenous phospholipase C (PLC) inhibitor, inhibited the decrease in TEER and the increase in the permeability of flux marker induced by epsilon-toxin. The application of epsilon-toxin to MDCK cells resulted in the biphasic formation of 1,2-diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). U73122 blocked the formation of DAG and IP3 induced by the toxin. Epsilon-toxin also specifically activated endogenous PLC-γ1. Epsilon-toxin dose-dependently increased the cytosolic calcium ion concentration ([Ca2+]i). The toxin-induced elevation of [Ca2+]i was inhibited by U73122. Cofilin is a key regulator of actin cytoskeleton turnover and tight-junction (TJ) permeability regulation. Epsilon-toxin caused cofilin dephosphorylation. These results demonstrate that epsilon-toxin induces Ca2+ influx through activating the phosphorylation of PLC-γ1 and then causes TJ opening accompanied by cofilin dephosphorylation.
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Fusarium mycotoxins and in vitro species-specific approach with porcine intestinal and brain in vitro barriers: A review. Food Chem Toxicol 2018; 121:666-675. [DOI: 10.1016/j.fct.2018.09.050] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2018] [Revised: 09/19/2018] [Accepted: 09/21/2018] [Indexed: 01/03/2023]
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Kiewiet MBG, González Rodríguez MI, Dekkers R, Gros M, Ulfman LH, Groeneveld A, de Vos P, Faas MM. The epithelial barrier-protecting properties of a soy hydrolysate. Food Funct 2018; 9:4164-4172. [PMID: 30066013 DOI: 10.1039/c8fo00913a] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Enhancing the epithelial barrier function could be a possible strategy to prevent food allergy or reduce its symptoms. Soy hydrolysates containing bioactive peptides could be instrumental in this. In this study, the protective effects of pretreatment with 6 soy hydrolysates on calcium ionophore A23187-induced TEER reduction were studied in T84 cells. The effects of the most potent soy hydrolysate on tight junction gene expression were studied. In order to identify the underlying pathways involved, the barrier disruptor specificity of the effect was studied by comparing the protective effects on TEER and Lucifer Yellow flux after the exposure to barrier disruptors that work via different intracellular pathways, i.e. the disruptors A23187, mellitin, and deoxynivalenol (DON). Preincubation with one of the six hydrolysates protected the epithelial cells from a decrease in TEER induced by A23187 (restored to 105% of the starting point, while A23187 alone decreased to 53% of the starting value) and mellitin (restored to 11% of the starting point, while mellitin alone decreased to 3.8% of the starting value). This soy hydrolysate was found to increase claudin-1 and decrease claudin-2 expression. The protective effect of the hydrolysate on TEER was specific for the barrier disruptors A23187 and mellitin, but was not observed for DON. This observation suggests that the soy hydrolysate may act via PKC isoforms, which are known to lead to changes in the expression of claudin-1 and 2. Our data suggest that specific soy hydrolysates may be designed to strengthen the epithelial barrier which might be instrumental in the management of the barrier function in individuals at risk of developing food allergy.
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Affiliation(s)
- Mensiena B G Kiewiet
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands.
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Hasan S, Kulkarni NN, Asbjarnarson A, Linhartova I, Osicka R, Sebo P, Gudmundsson GH. Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers. Infect Immun 2018; 86:e00445-17. [PMID: 29203545 PMCID: PMC5820963 DOI: 10.1128/iai.00445-17] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2017] [Accepted: 11/28/2017] [Indexed: 12/14/2022] Open
Abstract
The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.
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Affiliation(s)
- Shakir Hasan
- Institute of Microbiology of the CAS, v.v.i., Prague, Czech Republic
| | | | | | - Irena Linhartova
- Institute of Microbiology of the CAS, v.v.i., Prague, Czech Republic
| | - Radim Osicka
- Institute of Microbiology of the CAS, v.v.i., Prague, Czech Republic
| | - Peter Sebo
- Institute of Microbiology of the CAS, v.v.i., Prague, Czech Republic
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Phospholipase Cγ1 links inflammation and tumorigenesis in colitis-associated cancer. Oncotarget 2017; 9:5752-5763. [PMID: 29464031 PMCID: PMC5814171 DOI: 10.18632/oncotarget.23430] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2017] [Accepted: 11/29/2017] [Indexed: 12/19/2022] Open
Abstract
Colorectal cancer (CRC) is the third diagnosed cancer and the second leading cause of cancer-related deaths in the United States. Colorectal cancer is linked to inflammation and phospholipase Cγ1 (PLCγ1) is associated with tumorigenesis and the development of colorectal cancer; however, evidence of mechanisms connecting them remains unclear. The tight junctions (TJ), as intercellular junctional complexes, have an important role for integrity of the epithelial barrier to regulate the cellular permeability. Here we found that PLCγ1 regulated colitis and tumorigenesis in intestinal epithelial cells (IEC). To induce the colitis-associated cancer (CAC), we used the AOM/DSS model. Mice were sacrificed at 100 days (DSS three cycles) and 120 days (DSS one cycle). In a CAC model, we showed that the deletion of PLCγ1 in IEC decreased the incidence of tumors by enhancing apoptosis and inhibiting proliferation during tumor development. Accordingly, the deletion of PLCγ1 in IEC reduced colitis-induced epithelial inflammation via inhibition of pro-inflammatory cytokines and mediators. The PLCγ1 pathway in IEC accelerated colitis-induced epithelial damage via regulation of TJ proteins. Conclusions: Our findings suggest that PLCγ1 is a critical regulator of colitis and colorectal cancer and could further help in the development of therapy for colitis-associated cancer.
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de Almeida RMC, Clendenon SG, Richards WG, Boedigheimer M, Damore M, Rossetti S, Harris PC, Herbert BS, Xu WM, Wandinger-Ness A, Ward HH, Glazier JA, Bacallao RL. Transcriptome analysis reveals manifold mechanisms of cyst development in ADPKD. Hum Genomics 2016; 10:37. [PMID: 27871310 PMCID: PMC5117508 DOI: 10.1186/s40246-016-0095-x] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2016] [Accepted: 11/04/2016] [Indexed: 12/18/2022] Open
Abstract
Background Autosomal dominant polycystic kidney disease (ADPKD) causes progressive loss of renal function in adults as a consequence of the accumulation of cysts. ADPKD is the most common genetic cause of end-stage renal disease. Mutations in polycystin-1 occur in 87% of cases of ADPKD and mutations in polycystin-2 are found in 12% of ADPKD patients. The complexity of ADPKD has hampered efforts to identify the mechanisms underlying its pathogenesis. No current FDA (Federal Drug Administration)-approved therapies ameliorate ADPKD progression. Results We used the de Almeida laboratory’s sensitive new transcriptogram method for whole-genome gene expression data analysis to analyze microarray data from cell lines developed from cell isolates of normal kidney and of both non-cystic nephrons and cysts from the kidney of a patient with ADPKD. We compared results obtained using standard Ingenuity Volcano plot analysis, Gene Set Enrichment Analysis (GSEA) and transcriptogram analysis. Transcriptogram analysis confirmed the findings of Ingenuity, GSEA, and published analysis of ADPKD kidney data and also identified multiple new expression changes in KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways related to cell growth, cell death, genetic information processing, nucleotide metabolism, signal transduction, immune response, response to stimulus, cellular processes, ion homeostasis and transport and cofactors, vitamins, amino acids, energy, carbohydrates, drugs, lipids, and glycans. Transcriptogram analysis also provides significance metrics which allow us to prioritize further study of these pathways. Conclusions Transcriptogram analysis identifies novel pathways altered in ADPKD, providing new avenues to identify both ADPKD’s mechanisms of pathogenesis and pharmaceutical targets to ameliorate the progression of the disease. Electronic supplementary material The online version of this article (doi:10.1186/s40246-016-0095-x) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Rita M C de Almeida
- Biocomplexity Institute and Department of Physics, Indiana University, Bloomington, IN, 47405, USA.,Instituto de Física and Instituto Nacional de Ciência e Tecnologia, Universidade Federal do Rio Grande do Sul, 91501-970, Porto Alegre, RS, Brazil
| | - Sherry G Clendenon
- Biocomplexity Institute and Department of Intelligent Systems Engineering, Indiana University, Bloomington, IN, 47405, USA
| | | | | | - Michael Damore
- AMGEN Inc., One Amgen Center Drive, Thousand Oaks, CA, 91320-1799, USA
| | - Sandro Rossetti
- Division of Nephrology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA
| | - Peter C Harris
- Division of Nephrology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA
| | - Britney-Shea Herbert
- Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Wei Min Xu
- Division of Nephrology, Department of Medicine, Richard Roudebush VAMC and Indiana University School of Medicine, Indianapolis, IN, 46202, USA
| | - Angela Wandinger-Ness
- Department of Pathology MSC08-4640 and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA
| | - Heather H Ward
- Division of Nephrology, Department of Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA
| | - James A Glazier
- Biocomplexity Institute and Department of Intelligent Systems Engineering, Indiana University, Bloomington, IN, 47405, USA
| | - Robert L Bacallao
- Division of Nephrology, Department of Medicine, Richard Roudebush VAMC and Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
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Effect of Fusarium-Derived Metabolites on the Barrier Integrity of Differentiated Intestinal Porcine Epithelial Cells (IPEC-J2). Toxins (Basel) 2016; 8:toxins8110345. [PMID: 27869761 PMCID: PMC5127141 DOI: 10.3390/toxins8110345] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2016] [Revised: 10/20/2016] [Accepted: 11/15/2016] [Indexed: 12/20/2022] Open
Abstract
The human, animal and plant pathogen Fusarium, which contaminates agricultural commodities worldwide, produces numerous secondary metabolites. An example is the thoroughly-investigated deoxynivalenol (DON), which severely impairs gastrointestinal barrier integrity. However, to date, the toxicological profile of other Fusarium-derived metabolites, such as enniatins, beauvericin, moniliformin, apicidin, aurofusarin, rubrofusarin, equisetin and bikaverin, are poorly characterized. Thus we examined their effects—as metabolites alone and as metabolites in combination with DON—on the intestinal barrier function of differentiated intestinal porcine epithelial cells (IPEC-J2) over 72 h. Transepithelial electrical resistance (TEER) was measured at 24-h intervals, followed by evaluation of cell viability using neutral red (NR) assay. Enniatins A, A1, B and B1, apicidin, aurofusarin and beauvericin significantly reduced TEER. Moniliformin, equisetin, bikaverin and rubrofusarin had no effect on TEER. In the case of apicidin, aurofusarin and beauvericin, TEER reductions were further substantiated by the addition of otherwise no-effect DON concentrations. In all cases, viability was unaffected, confirming that TEER reductions were not due to compromised viability. Considering the prevalence of mycotoxin contamination and the diseases associated with intestinal barrier disruption, consumption of contaminated food or feed may have substantial health implications.
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14
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The impact of lemon pectin characteristics on TLR activation and T84 intestinal epithelial cell barrier function. J Funct Foods 2016. [DOI: 10.1016/j.jff.2016.02.002] [Citation(s) in RCA: 58] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
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15
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He B, Yuan L, Dai W, Gao W, Zhang H, Wang X, Fang W, Zhang Q. Dynamic bio-adhesion of polymer nanoparticles on MDCK epithelial cells and its impact on bio-membranes, endocytosis and paracytosis. NANOSCALE 2016; 8:6129-45. [PMID: 26932537 DOI: 10.1039/c5nr08858e] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/20/2023]
Abstract
Nowadays, concern about the use of nanotechnology for biomedical application is unprecedentedly increasing. In fact, nanosystems applied for various potential clinical uses always have to cross the primary biological barrier consisting of epithelial cells. However, little is really known currently in terms of the influence of the dynamic bio-adhesion of nanosystems on bio-membranes as well as on endocytosis and transcytosis. This was investigated here using polymer nanoparticles (PNs) and MDCK epithelial cells as the models. Firstly, the adhesion of PNs on cell membranes was found to be time-dependent with a shift of both location and dispersion pattern, from the lateral adhesion of mainly mono-dispersed PNs initially to the apical coverage of the PN aggregate later. Then, it was interesting to observe in this study that the dynamic bio-adhesion of PNs only affected their endocytosis but not their transcytosis. It was important to find that the endocytosis of PNs was not a constant process. A GM1 dependent CDE (caveolae dependent endocytosis) pathway was dominant in the preliminary stage, followed by the co-existence of a CME (clathrin-mediated endocytosis) pathway for the PN aggregate at a later stage, in accordance with the adhesion features of PNs, suggesting the modification of PN adhesion patterns on the endocytosis pathways. Next, the PN adhesion was noticed to affect the structure of cell junctions, via altering the extra- and intra-cellular calcium levels, leading to the enhanced paracellular transport of small molecules, but not favorably enough for the obviously increased passing of PNs themselves. Finally, FRAP and other techniques all demonstrated the obvious impact of PN adhesion on the membrane confirmation, independent of the adhesion location and time, which might lower the threshold for the internalization of PNs, even their aggregates. Generally, these findings confirm that the transport pathway mechanism of PNs through epithelial cells is rather dynamic, and is remarkably affected by the adhesion patterns of PNs on the cell membrane.
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Affiliation(s)
- Bing He
- Department of Pathology, School of Basic Medical Sciences, Peking University, Beijing 100191, China. and Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.
| | - Lan Yuan
- Medical and Healthy Analytical Center, Peking University, Beijing 100191, China
| | - Wenbing Dai
- Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.
| | - Wei Gao
- Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
| | - Hua Zhang
- Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.
| | - Xueqing Wang
- Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.
| | - Weigang Fang
- Department of Pathology, School of Basic Medical Sciences, Peking University, Beijing 100191, China.
| | - Qiang Zhang
- Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China and State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.
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16
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Cellulose alters the expression of nuclear factor kappa B-related genes and Toll-like receptor-related genes in human peripheral blood mononuclear cells. J Funct Foods 2015. [DOI: 10.1016/j.jff.2015.08.011] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
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17
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Vogt LM, Meyer D, Pullens G, Faas MM, Venema K, Ramasamy U, Schols HA, de Vos P. Toll-like receptor 2 activation by β2→1-fructans protects barrier function of T84 human intestinal epithelial cells in a chain length-dependent manner. J Nutr 2014; 144:1002-8. [PMID: 24790027 DOI: 10.3945/jn.114.191643] [Citation(s) in RCA: 83] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells, confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan-mediated health effects.
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Affiliation(s)
- Leonie M Vogt
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;
| | | | - Gerdie Pullens
- Cosun Food Technology Centre, Roosendaal, The Netherlands
| | - Marijke M Faas
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
| | - Koen Venema
- TNO Quality of Life, Department of Biosciences, Zeist, The Netherlands; and
| | - Uttara Ramasamy
- Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands
| | - Henk A Schols
- Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands
| | - Paul de Vos
- Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
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18
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Clark CA, Thomas LK, Azghani AO. Inhibition of protein kinase C attenuates Pseudomonas aeruginosa elastase-induced epithelial barrier disruption. Am J Respir Cell Mol Biol 2011; 45:1263-71. [PMID: 21757681 DOI: 10.1165/rcmb.2010-0459oc] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Pseudomonas aeruginosa pulmonary infection compromises the human airway epithelium, and can be especially devastating to immunocompromised or debilitated individuals. We have reported earlier that P. aeruginosa elastase (PE) increases paracellular permeability in epithelial cell monolayers by mechanisms involving tight junction (TJ) disruption and cytoskeletal reorganization, leading to destruction of epithelial barrier function. The aim of this study was to investigate putative TJ targets and potential mechanisms by which PE induces barrier disruption. We found that PE decreased localization of TJ proteins, occludin and zonula occludens (ZO)-1, in membrane fractions, and induced reorganization of F-actin within 1 hour. Although inhibition of protein kinase (PK) C α/β signaling modestly altered the extent of cytoskeletal disruption and ZO-1 translocation, we found PKC signaling to play a significant role in decreased occludin functionality during PE exposure. Furthermore, elevated PKC levels correlated with decreased levels of TJ proteins in membrane fractions, and increased paracellular permeability in a time-dependent manner. Therefore, we conclude that PKC signaling is involved during PE-induced epithelial barrier disruption via TJ translocation and cytoskeletal reorganization. Specifically, occludin, as well as associated ZO-1 and F-actin, may be early targets of PE pathogenesis occurring via a PKC-dependent pathway.
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Affiliation(s)
- Curtis A Clark
- Department of Biology, The University of Texas at Tyler, 75799, USA
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19
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Hicks MA, Hosgood GL, Morgan TW, Briere CA, McConnico RS. In vitro effect of carprofen and meloxicam on the conductance and permeability to mannitol and the histologic appearance of the gastric mucosa of dogs. Am J Vet Res 2011; 72:570-7. [DOI: 10.2460/ajvr.72.4.570] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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20
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Roig-Pérez S, Cortadellas N, Moretó M, Ferrer R. Intracellular mechanisms involved in docosahexaenoic acid-induced increases in tight junction permeability in Caco-2 cell monolayers. J Nutr 2010; 140:1557-63. [PMID: 20660281 DOI: 10.3945/jn.109.120469] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
We recently showed that enrichment of Caco-2 cells with docosahexaenoic acid (DHA) increases lipid peroxidation and the formation of hydrogen peroxide and peroxynitrite, which disrupt the epithelial barrier function. Studies were designed to test whether the participation of phospholipase C (PLC)/Ca(2+)/protein kinase C (PKC), cyclooxygenase (COX), and 5-lipooxygenase pathways are involved in mediating the effects of DHA. Paracellular permeability was assessed from D-mannitol flux and transepithelial electrical resistance (TER) in differentiated Caco-2 cell monolayers incubated in control or DHA-enriched conditions (100 micromol/L). The effect of DHA was prevented by U73122 (PLC inhibitor), chelerytrine (PKC inhibitor), and 1-[5-iodonaphtalene-1-sulfonyl]-1H-hexahydro-1,4-diazepine hydrochloride (myosin light chain kinase inhibitor). In contrast, the effect of DHA was enhanced by A23187 (Ca(2+) ionophore) and BAPTA-AM (Ca(2+) chelator). Indomethacin (COX inhibitor) and AA961 (5-lipooxygenase inhibitor) also prevented the changes in D-mannitol flux induced by DHA, but no effect was detected for TER. Moreover, occludin and ZO-1 immunogold staining microscopy showed that the increase in paracellular permeability was accompanied by the redistribution of both tight junction proteins. We conclude that the disruption of epithelial barrier function by DHA is partly mediated by the PLC/Ca(2+)/PKC pathway and by the formation of eicosanoids.
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Affiliation(s)
- Sònia Roig-Pérez
- Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Spain
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21
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Bourdeau RW, Malito E, Chenal A, Bishop BL, Musch MW, Villereal ML, Chang EB, Mosser EM, Rest RF, Tang WJ. Cellular functions and X-ray structure of anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis. J Biol Chem 2009; 284:14645-56. [PMID: 19307185 DOI: 10.1074/jbc.m807631200] [Citation(s) in RCA: 75] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.
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Affiliation(s)
- Raymond W Bourdeau
- Ben-May Department for Cancer Research, University of Chicago, Chicago, Illinois 60637, USA
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22
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Briere CA, Hosgood G, Morgan TW, Hedlund CS, Hicks M, McConnico RS. Effects of carprofen on the integrity and barrier function of canine colonic mucosa. Am J Vet Res 2008; 69:174-81. [PMID: 18241012 DOI: 10.2460/ajvr.69.2.174] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
OBJECTIVE To measure effects of carprofen on conductance and permeability to mannitol and histologic appearance in canine colonic mucosa. SAMPLE POPULATION Colonic mucosa from 13 mature mixed-breed dogs. Procedures-Sections of mucosa from the transverse colon and proximal and distal portions of the descending colon were obtained immediately after dogs were euthanized. Sections were mounted in Ussing chambers. Carprofen (400 microg/mL) was added to the bathing solution for treated sections. Conductance was calculated at 15-minute intervals for 240 minutes. Flux of mannitol was calculated for three 1-hour periods. Histologic examination of sections was performed after experiments concluded. Conductance was graphed against time for each chamber, and area under each curve was calculated. Conductance X time, flux of mannitol, and frequency distribution of histologic findings were analyzed for an effect of region and carprofen. RESULTS Carprofen significantly increased mean conductance X time, compared with values for control (untreated) sections for all regions of colon. Carprofen significantly increased mean flux of mannitol from period 1 to period 2 and from period 2 to period 3 for all regions of colon. Carprofen caused a significant proportion of sections to have severe sloughing of cells and erosions involving >or= 10% of the epithelium, compared with control sections. CONCLUSIONS AND CLINICAL RELEVANCE Carprofen increased in vitro conductance and permeability to mannitol in canine colonic mucosa. Carprofen resulted in sloughing of cells and erosion of the colonic mucosa. These findings suggested that carprofen can compromise the integrity and barrier function of the colonic mucosa of dogs.
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Affiliation(s)
- Catherine A Briere
- Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
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23
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Crosstalk of tight junction components with signaling pathways. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2007; 1778:729-56. [PMID: 17950242 DOI: 10.1016/j.bbamem.2007.08.018] [Citation(s) in RCA: 584] [Impact Index Per Article: 32.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/30/2007] [Revised: 08/03/2007] [Accepted: 08/16/2007] [Indexed: 12/28/2022]
Abstract
Tight junctions (TJs) regulate the passage of ions and molecules through the paracellular pathway in epithelial and endothelial cells. TJs are highly dynamic structures whose degree of sealing varies according to external stimuli, physiological and pathological conditions. In this review we analyze how the crosstalk of protein kinase C, protein kinase A, myosin light chain kinase, mitogen-activated protein kinases, phosphoinositide 3-kinase and Rho signaling pathways is involved in TJ regulation triggered by diverse stimuli. We also report how the phosphorylation of the main TJ components, claudins, occludin and ZO proteins, impacts epithelial and endothelial cell function.
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24
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Fiandra L, Casartelli M, Giordana B. The paracellular pathway in the lepidopteran larval midgut: modulation by intracellular mediators. Comp Biochem Physiol A Mol Integr Physiol 2006; 144:464-73. [PMID: 16765075 DOI: 10.1016/j.cbpa.2006.04.011] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2005] [Revised: 03/23/2006] [Accepted: 04/11/2006] [Indexed: 11/18/2022]
Abstract
The features of the paracellular pathway, an important route for the transfer of ions and molecules in epithelia, are in insects still poorly investigated and it has not yet been elucidated how the septate junction (SJ) acts as a transepithelial barrier. In this study, some properties of the paracellular pathway of Bombyx mori larval midgut, isolated in Ussing chambers, were determined and the modulation of SJ permeability by intracellular events disclosed. Diffusion potentials evoked by transepithelial gradients of different salts indicated that the junction bore weak negative charges and that the paracellular pathway was selective with respect to ion charge and size. In standard conditions, the transepithelial resistance was 28.2+/-2.1 Omega cm(2), a value indicating that the midgut is a low resistance epithelium. The modulation of midgut SJ by typical enhancers of mammalian tight junction permeability known to act on the cytoskeleton was studied by measuring the shunt resistance and the lumen-to-haemolymph flux of sucrose. An increase of the intracellular level of cAMP and Ca(2+) caused a significant decrease of the shunt resistance and an increase of SJ permeability. The attenuation of Ca(2+) effect in the presence of the calcium channel blocker nifedipine indicated that the influx of external Ca(2+) into the cytoplasm was important for the opening of the SJ, as well as the release of Ca(2+) from the intracellular stores.
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Affiliation(s)
- L Fiandra
- Department of Biology, University of Milan, Via Celoria 26, 20133 Milano, Italy.
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25
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Wu SK, Mathias NR, Kim KJ, Lee VHL. Functional and pharmacological mechanisms of nucleoside transport across the basolateral membrane of rabbit tracheal epithelial cells. Life Sci 2005; 78:310-20. [PMID: 16111717 DOI: 10.1016/j.lfs.2005.04.066] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2005] [Accepted: 04/22/2005] [Indexed: 10/25/2022]
Abstract
The role of basolateral membrane nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) was studied. Primary cultured RTEC were grown on permeable support at an air-interface. Transport studies were conducted in the uptake, efflux, and transepithelial transport configurations using (3)H-uridine as a model substrate. Time, temperature and concentration dependency of (3)H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of (3)H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of (3)H-uridine uptake were studied after treatment of RTEC with pharmacological levels of A23187, forskolin, tamoxifen, H89 and colchicine. The basolateral aspect has a low-affinity and high-capacity transport system that exhibits characteristics of bi-directionality, temperature/concentration dependency, and broad specificity towards purines and pyrimidines without requiring Na(+). Basolateral equilibrative-sensitive/insensitive (es/ei) type transport machinery manifested as a biphasic dose response to nitro-benzyl-mercapto-purine-ribose (NBMPR) inhibition. In addition, a number of therapeutically relevant nucleoside analogs appeared to compete with the uptake of uridine from basolateral fluid. Short-term pre-incubation of primary cultured RTEC with the calcium ionophore A23187 inhibited basolateral uridine uptake without affecting the J(max) and K(m). The inhibitory effect was not reversible with a protein kinase C (PKC) antagonist, tamoxifen. In contrast, basolateral uridine uptake was increased by adenylyl cyclase activator forskolin (reversible with protein kinase A (PKA) inhibitor H89), resulting in a decreased K(m), but a lower J(max). Uridine exit across the basolateral membrane of primary cultured RTEC occurs via a facilitative diffusion carrier, which can be modulated by intracellular Ca(2+) levels and PKA. Information about these carriers will help improve the transportability of antitumor and antiviral nucleoside analogs in the pulmonary setting.
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Affiliation(s)
- Sharon K Wu
- Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089-9121, USA.
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26
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Narai A, Watanabe H, Iwanaga T, Tomita T, Shimizu M. Effect of a pore-forming protein derived from Flammulina velutipes on the Caco-2 intestinal epithelial cell monolayer. Biosci Biotechnol Biochem 2005; 68:2230-8. [PMID: 15564659 DOI: 10.1271/bbb.68.2230] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We have previously found a transepithelial electrical resistance (TEER)-decreasing protein derived from Flammulina velutipes, which was revealed to be identical to flammutoxin (FTX) that is known as a hemolytic pore-forming protein. This protein induced a rapid decrease in TEER and parallel increase in paracellular permeability in the intestinal epithelial Caco-2 cell monolayer without any cytotoxicity. An immunoblotting analysis revealed that the FTX-induced decrease in TEER was accompanied by the formation of a high-molecular-weight complex on the surface of Caco-2 cells. Intracellular Ca(2+) imaging showed that exposure to FTX caused a rapid Ca(2+) influx. It was observed by electron microscopy that FTX induced swelling of microvilli and expansion of the cellular surface. Staining with fluorescent phalloidin showed a marked change to filamentous actin in the FTX-treated cells. These results suggest that TEER reduction could sensitively detect small membrane pore formation by FTX in the intestinal epithelium which causes a morphological alteration and disruption of the paracellular barrier function.
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Affiliation(s)
- Asako Narai
- Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
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27
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Brown RC, Davis TP. Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells. Biochem Biophys Res Commun 2005; 327:1114-23. [PMID: 15652512 DOI: 10.1016/j.bbrc.2004.12.123] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2004] [Indexed: 01/12/2023]
Abstract
The blood-brain barrier (BBB) serves as a critical organ in the maintenance of central nervous system homeostasis and is disrupted in a number of neurological disorders, including stroke. We examined the effects of hypoxia/aglycemia on the expression and localization of tight junction proteins, and on the function of the BBB in an in vitro model system. A receptor-operated/store-operated calcium channel blocker, SKF 96365, was used to determine if calcium flux was important in mediating hypoxia/aglycemia effects on the BBB. Expression of the tight junction protein occludin increased after hypoxic/aglycemic stress when cells were exposed to SKF 96365; this was correlated with partial protection of membrane localization of occludin and inhibition of the hypoxia-induced increase in permeability. Actin expression was dramatically reduced by hypoxia/aglycemia. Treatment with SKF 96365 during hypoxic stress protected monolayer permeability of sucrose, but transendothelial electrical resistances decreased with exposure to hypoxic stress regardless of treatment. Therefore, the presence of occludin at the membrane is dependent in part on calcium-sensitive signaling cascades; this provides a target for therapeutic intervention to minimize BBB disruption after stroke.
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Affiliation(s)
- Rachel C Brown
- Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, TX, USA
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28
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Zhu JX, Yang N, Zhang GH, Tsang LL, Gou YL, Wong HYC, Chung YW, Chan HC. Improvement of barrier function and stimulation of colonic epithelial anion secretion by Menoease Pills. World J Gastroenterol 2004; 10:2514-8. [PMID: 15300895 PMCID: PMC4572152 DOI: 10.3748/wjg.v10.i17.2514] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: Menoease Pills (MP), a Chinese medicine-based new formula for postmenopausal women, has been shown to modulate the endocrine and immune systems[1]. The present study investigated the effects of MP and one of its active ingredients, ligustrazine, on epithelial barrier and ion transport function in a human colonic cell line, T84.
METHODS: Colonic transepithelial electrophysiological characteristics and colonic anion secretion were studied using the short circuit current (ISC) technique. RT-PCR was used to examine the expression of cytoplasmic proteins associated with the tight junctions, ZO-1 (zonula occludens-1) and ZO-2 (zonula occludens-2).
RESULTS: Pretreatment of T84 cells with MP (15 μg/mL) for 72 h significantly increased basal potential difference, transepithelial resistance and basal ISC. RT-PCR results showed that the expressions of ZO-1 and ZO-2 were significantly increased after MP treatment, consistent with improved epithelial barrier function. Results of acute stimulation showed that apical addition of MP produced a concentration-dependent (10-5000 μg/mL, EC50 = 293.9 μg/mL) increase in ISC. MP-induced ISC was inhibited by basolateral treatment with bumetanide (100 μmol/L), an inhibitor of the Na + -K + -2Cl- cotransporter, apical addition of Cl- channel blockers, diphenylamine-2, 2’-dicarboxylic acid (1 mmol/L) or glibenclamide (1 mmol/L), but not 4, 4’-diisothiocyanostilbene-2, 2’-disulfonic acid or epithelial Na + channel blocker, amiloride. The effect of MP on ZO-1 and ZO-2 was mimicked by Ligustrazine and the ligustrazine-induced ISC was also blocked by basolateral application of bumetanide and apical addition of diphenylamine-2, 2'-dicarboxylic acid or glibenclamide, and reduced by a removal of extracellular Cl-.
CONCLUSION: The results of the present study suggest that MP and ligustrazine may improve epithelial barrier function and exert a stimulatory effect on colonic anion secretion, indicating the potential use of MP and its active ingredients for improvement of GI tract host defense and alleviation of constipation often seen in the elderly.
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Affiliation(s)
- Jin-Xia Zhu
- Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
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Suzuki T, Hara H. Various nondigestible saccharides open a paracellular calcium transport pathway with the induction of intracellular calcium signaling in human intestinal Caco-2 cells. J Nutr 2004; 134:1935-41. [PMID: 15284379 DOI: 10.1093/jn/134.8.1935] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Ingestion of soluble nondigestible saccharides increases calcium absorption, and it is suggested that paracellular calcium transport contributes to this effect. However, cellular mechanisms and the contribution of active transport have not been clarified. This study examined the effects of 4 nondigestible saccharides, difructose anhydride (DFA) III, DFAIV, fructooligosaccharides, and raffinose, on active and passive calcium transport, permeability of paracellular pathways, and intracellular calcium signaling in a human intestinal Caco-2 cell monolayer. Net, active, and passive calcium transport were evaluated using (45)Ca. Transepithelial electrical resistance (TEER) and transport of lucifer yellow were measured as indicators of paracellular passage in differentiated Caco-2 cell monolayers incubated with 0-100 mmol/L of the various saccharides. The changes in intracellular calcium ion concentrations ([Ca(2+)](i)) were measured by fura-2 loading before and after the addition of each saccharide (50 or 100 mmol/L). The addition of 100 mmol/L of each saccharide to the apical medium of the Caco-2 cells enhanced net calcium transport without any changes in active calcium transport. Relative TEER was dose dependently and reversibly decreased by the addition of saccharides, and the decreases in TEER were highly correlated with net calcium transport (P < 0.001). Basolateral application of the saccharides had a slight or no effect on indicators of the paracellular pathway. Each saccharide caused an immediate and dose-dependent rise in [Ca(2+)](i) in the cells. The 4 nondigestible saccharides increased net calcium transport in the cells via the paracellular route through tight junctions. The rise in [Ca(2+)](i) induced by these saccharides may be involved in the opening of tight junctions.
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Affiliation(s)
- Takuya Suzuki
- Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan
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Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterology 2004; 127:224-38. [PMID: 15236188 DOI: 10.1053/j.gastro.2004.04.015] [Citation(s) in RCA: 379] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND & AIMS Protein kinase C (PKC) has been implicated in regulation of intestinal epithelial integrity in response to lumenal bacteria. Intestinal epithelial cells (IECs) constitutively express Toll-like receptor (TLR)2, which contains multiple potential PKC binding sites. The aim of this study was to determine whether TLR2 may activate PKC in response to specific ligands, thus potentially modulating barrier function in IECs. METHODS TLR2 agonist (synthetic bacterial lipopeptide Pam(3)CysSK4, peptidoglycan)-induced activation of PKC-related signaling cascades were assessed by immunoprecipitation, Western blotting, immunofluorescence, and kinase assays-combined with functional transfection studies in the human model IEC lines HT-29 and Caco-2. Transepithelial electrical resistance characterized intestinal epithelial barrier function. RESULTS Stimulation with TLR2 ligands led to activation (phosphorylation, enzymatic activity, translocation) of specific PKC isoforms (PKCalpha and PKCdelta). Phosphorylation of PKC by TLR2 ligands was blocked specifically by transfection with a TLR2 deletion mutant. Ligand-induced activation of TLR2 greatly enhanced transepithelial resistance in IECs, which was prevented by pretreatment with PKC-selective antagonists. This effect correlated with apical tightening and sealing of tight junction (TJ)-associated ZO-1, which was mediated via PKC in response to TLR2 ligands, whereas morphologic changes of occludin, claudin-1, or actin cytoskeleton were not evident. Downstream the endogenous PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS), but not transcriptional factor activator protein-1 (AP-1), was activated significantly on stimulation. CONCLUSIONS The present study provides evidence that PKC is an essential component of the TLR2 signaling pathway with the physiologic consequence of directly enhancing intestinal epithelial integrity through translocation of ZO-1 on activation.
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Affiliation(s)
- Elke Cario
- Division of Gastroenterology and Hepatology, University Hospital of Essen, Germany.
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31
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Kiely B, Feldman G, Ryan MP. Modulation of renal epithelial barrier function by mitogen-activated protein kinases (MAPKs): mechanism of cyclosporine A-induced increase in transepithelial resistance. Kidney Int 2003; 63:908-16. [PMID: 12631071 DOI: 10.1046/j.1523-1755.2003.00804.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND Cyclosporine A (CsA) has been shown to increase transepithelial resistance in Madin-Darby canine kidney (MDCK) cells, and the mechanism may involve altered phosphorylation of junctional proteins. In this study, we examine the effect of the extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways on the basal transepithelial resistance (TER) and on the CsA-induced increase in TER across MDCK monolayers. Here we present evidence that CsA may be mediating some of its effects through activation of the ERK 1/2 MAPK pathway. METHODS MDCK cells were treated with CsA (4.2 micromol/L) and paracellular permeability was assessed by measuring TER. The role of the ERK 1/2 and the p38 MAPK pathways in modulating TER was investigated using the inhibitors PD98059 and U0126 for ERK 1/2 and SB203580 for p38. ERK 1/2 and p38 phosphorylation/activation was also examined by Western blot analysis. RESULTS CsA (4.2 micromol/L) increased the TER of MDCK monolayers. The ERK 1/2 inhibitor PD98059 decreased basal TER and also ameliorated the CsA-induced increase in TER. Similar results were found with the U0126 inhibitor of ERK 1/2. The p38 inhibitor SB203580 had no effect on the basal TER of the monolayers, however, SB203580 significantly augmented the CsA-induced increase in TER. CsA was shown to significantly activate ERK 1/2 and this activation by CsA was prevented by PD98059. Inhibition of the p38 pathway by SB203580 also resulted in activation of ERK 1/2 and this activation of ERK 1/2 was further enhanced by CsA. No effect of CsA or the inhibitors PD98059 or SB203580 on p38 phosphorylation was detected. CONCLUSION The results presented here suggest that activation of the ERK 1/2 MAPK cascade is important in the regulation of the paracellular permeability in MDCK cells. Activation of this pathway appears to be pivotal to the CsA-induced increase in TER.
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Affiliation(s)
- Breda Kiely
- Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland
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Ward PD, Ouyang H, Thakker DR. Role of phospholipase C-beta in the modulation of epithelial tight junction permeability. J Pharmacol Exp Ther 2003; 304:689-98. [PMID: 12538823 DOI: 10.1124/jpet.102.043638] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
The results presented in this study establish an association between phospholipase C-beta (PLC-beta) and tight junction permeability across Madin-Darby canine kidney (MDCK) cell monolayers, an in vitro model for epithelial tissue. These results further show that PLC-beta modulates tight junction permeability by affecting actin filament organization. Hexadecylphosphocholine (HPC) inhibited PLC-beta and increased tight junction permeability in MDCK cells. Interestingly, the analogs of HPC, a series of alkylphosphocholines containing various lengths of linear alkyl chains, inhibited PLC-beta and increased tight junction permeability with a wide range of potency. The potency of alkylphosphocholines as enhancers of tight junction permeability significantly correlated (p < 0.05) with their potency as PLC-beta inhibitors. U73122, a steroid derivative that is structurally unrelated to alkylphosphocholines, inhibited PLC-beta and increased tight junction permeability with potencies that fit into the correlation observed for the alkylphosphocholine series. U73122 and HPC induced disorganization of actin filaments in MDCK cell monolayers. The potencies to cause disorganization of actin filaments were consistent with the potencies of these agents as inhibitors of PLC-beta and enhancers of tight junction permeability. Furthermore, ATP, an activator of PLC-beta, attenuated U73122-induced increase in tight junction permeability as well as disorganization of actin filaments. These results provide strong evidence that PLC-beta inhibition leads to increased tight junction permeability across MDCK cell monolayers through disorganization of actin filaments.
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Affiliation(s)
- Peter D Ward
- Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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Ward PD, Klein RR, Troutman MD, Desai S, Thakker DR. Phospholipase C-gamma modulates epithelial tight junction permeability through hyperphosphorylation of tight junction proteins. J Biol Chem 2002; 277:35760-5. [PMID: 12101180 DOI: 10.1074/jbc.m203134200] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Phospholipase C-gamma (PLC-gamma) is stimulated by epidermal growth factor via activation of the epidermal growth factor receptors. The PLC inhibitor, 3-nitrocoumarin (3-NC), selectively inhibited PLC-gamma in Madin-Darby canine kidney cells without affecting the activity of PLC-beta. In contrast, inhibitors of PLC-beta, hexadecylphosphocholine and, had no effect on the activity of PLC-gamma. Inhibition of PLC-gamma by 3-NC was associated with an increase in tight junction permeability across Madin-Darby canine kidney cell monolayers, as evidenced by 3-NC-induced decrease in transepithelial electrical resistance and increase in mannitol flux over a concentration range that was inhibitory to PLC-gamma. An analog of 3-NC, 7-hydroxy-3-NC (7-OH-3-NC), which was inactive as an inhibitor of PLC-gamma, also had no effect on tight junction permeability. Treatment with 3-NC caused punctate disruption in the cortical actin filaments. The PLC-gamma inhibitor, 3-NC, but not the inactive analog, 7-OH-3-NC, caused hyperphosphorylation of the tight junction proteins, occludin, ZO-1, and ZO-2. The serine/threonine kinase inhibitor, staurosporine (50-200 nm), significantly attenuated 3-NC-induced hyperphosphorylation of ZO-2. This corresponded with attenuation by staurosporine of 3-NC-induced increase in tight junction permeability, suggesting a relationship between ZO-2 phosphorylation and tight junction permeability.
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Affiliation(s)
- Peter D Ward
- Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA
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Toriano R, Kierbel A, Ramirez MA, Malnic G, Parisi M. Spontaneous water secretion in T84 cells: effects of STa enterotoxin, bumetanide, VIP, forskolin, and A-23187. Am J Physiol Gastrointest Liver Physiol 2001; 281:G816-22. [PMID: 11518694 DOI: 10.1152/ajpgi.2001.281.3.g816] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The regulated Cl(-) secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca(2+), cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux (J(w) = -0.16 +/- 0.02 microl.min(-1).cm(-2)) in parallel with moderate short-circuit current values (I(sc) = 1.55 +/- 0.23 microA/cm(2)). The secretory J(w) reversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I(sc), but, unexpectedly, J(w) was not affected. Bumetanide, an inhibitor of basolateral Na(+)-K(+)-2Cl(-) cotransporter, completely blocked secretory J(w) with no change in I(sc). Conversely, serosal forskolin increased I(sc), but J(w) switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J(w) and I(sc). No difference between the absorptive and secretory unidirectional Cl(-) fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl(-) flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J(w) values cannot be shown by I(sc) and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.
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Affiliation(s)
- R Toriano
- Laboratorio de Biomembranas, Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, 1453 Buenos Aires, Argentina
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Song JC, Hanson CM, Tsai V, Farokhzad OC, Lotz M, Matthews JB. Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms. Am J Physiol Cell Physiol 2001; 281:C649-61. [PMID: 11443064 DOI: 10.1152/ajpcell.2001.281.2.c649] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl(-) secretion (short-circuit current, I(sc)) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I(sc) and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKC epsilon followed by the conventional PKC alpha and activated both isozymes by in vitro kinase assay. PKC delta was activated by PMA but did not translocate. By immunofluorescence, PKC epsilon redistributed to the basolateral domain in response to PMA, whereas PKC alpha moved apically. Inhibition of I(sc) by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 microM) but not the conventional isoform inhibitor Gö-6976 (5 microM) or the PKC delta inhibitor rottlerin (10 microM), implicating PKC epsilon in inhibition of Cl(-) secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKC alpha. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibited cAMP-elicited I(sc). However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 microM) translocated only PKC epsilon and inhibited I(sc) with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I(sc). We conclude that basolateral translocation of PKC epsilon inhibits Cl(-) secretion, while apical translocation of PKC alpha decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.
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Affiliation(s)
- J C Song
- Division of General and Gastrointestinal Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA
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Kimura Y, Hosoda Y, Yamaguchi M, Nagano H, Shima M, Adachi S, Matsuno R. Effects of medium-chain fatty acids on intracellular calcium levels and the cytoskeleton in human intestinal (Caco-2) cell monolayers. Biosci Biotechnol Biochem 2001; 65:743-51. [PMID: 11388448 DOI: 10.1271/bbb.65.743] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The effects of medium-chain fatty acids (MCFA) on intracellular calcium (Ca2+) levels and actin filaments in the Caco-2 monolayer were investigated. A site-dependent increase in intracellular Ca2+ levels caused by decanoic acid (C10) at 13 mM was observed by confocal laser scanning microscopy. The area in which the intracellular Ca2+ levels was increased was measured by image analysis, and increased to 11% of the total area of the monolayer within 1 minute. This was maintained for 5 minutes, and decreased thereafter. The other MCFAs did not significantly increase the intracellular Ca2+ levels. Obvious morphological changes of actin filaments were induced by only C10 among C8-C14. The area in which actin filaments were depleted was also quantified, and the increase in area became significant after 40 minutes. The area of the actin-depleted spot corresponded to the area occupied by 5 to 10 cells as well as that in which the intracellular Ca2+ level was increased. The effectiveness of only C10 suggested that the mechanism of the absorption enhancement by C10 would be different from that by the other MCFAs, or that C10 has some additional physiological functions although the mechanism of the enhancement is the same as for the other MCFAs.
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Affiliation(s)
- Y Kimura
- Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
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Karczewski J, Groot J. Molecular physiology and pathophysiology of tight junctions III. Tight junction regulation by intracellular messengers: differences in response within and between epithelia. Am J Physiol Gastrointest Liver Physiol 2000; 279:G660-5. [PMID: 11005751 DOI: 10.1152/ajpgi.2000.279.4.g660] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Tight junction permeability differs with the type of permeants, their size, and their charge. Selective changes in permeability do occur, and they illustrate the diversity in functional reactions of tight junctions. This suggests that special structures in the tight junctions are involved. More and more structural components of the tight junctions are becoming known. The divergence in behavior of native tissue and filter-grown epithelial monolayers with respect to the effects of intracellular messengers offers the possibility to relate structure and function. In addition to the tools for conventional permeability studies, probes have become available to detect changes in activation of intracellular effector proteins such as the protein kinase C isotypes, and with in situ imaging techniques the way is open for a functional approach in the study of tight junctions.
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Affiliation(s)
- J Karczewski
- Section Neurobiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, The Netherlands.
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38
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Roche JK, Martins CA, Cosme R, Fayer R, Guerrant RL. Transforming growth factor beta1 ameliorates intestinal epithelial barrier disruption by Cryptosporidium parvum in vitro in the absence of mucosal T lymphocytes. Infect Immun 2000; 68:5635-44. [PMID: 10992464 PMCID: PMC101516 DOI: 10.1128/iai.68.10.5635-5644.2000] [Citation(s) in RCA: 47] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Exposure to oocysts of the protozoan Cryptosporidium parvum causes intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor beta1 (TGF-beta1)-a cytokine synthesized and released by cells in the intestine-to preserve the barrier function of human colonic epithelia when challenged with C. parvum oocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-beta1 were localized by confocal microscopy, and TGF-beta1-induced protein kinase C activation was detected intracellularly by translocation of its alpha isozyme. TGF-beta1 alone enhanced intestinal epithelial barrier function, while exposure to C. parvum oocysts (> or =10(5)/monolayer) markedly reduced barrier function to < or =40% of that of the control. When epithelial monolayers were pretreated with TGF-beta1 at 5.0 ng/ml, the barrier-disrupting effect of C. parvum oocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-beta1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-beta1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-beta1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-beta1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa alpha isozyme within 30 s of epithelial exposure to TGF-beta1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-beta1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-beta1 when human colonic epithelium is exposed to an important human pathogen, C. parvum.
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Affiliation(s)
- J K Roche
- Divisions of Gastroenterology and of Geographic and International Medicine, Department of Medicine, University of Virginia Health System, Charlottesville, Virginia 22908, USA.
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Ward PD, Tippin TK, Thakker DR. Enhancing paracellular permeability by modulating epithelial tight junctions. PHARMACEUTICAL SCIENCE & TECHNOLOGY TODAY 2000; 3:346-358. [PMID: 11050459 DOI: 10.1016/s1461-5347(00)00302-3] [Citation(s) in RCA: 158] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The intestinal epithelium is a major barrier to the absorption of hydrophilic drugs. The presence of intercellular junctional complexes, particularly the tight junctions (zona occludens), renders the epithelium impervious to hydrophilic drugs, which cannot diffuse across the cells through the lipid bilayer of the cell membranes. There have been significant advances in understanding the structure and cellular regulation of tight junctions over the past decade. This article reviews current knowledge regarding the physiological regulation of tight junctions and paracellular permeability, and recent progress towards the rational design of agents that can effectively and safely increase paracellular permeability via modulation of tight junctions.
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Affiliation(s)
- PD Ward
- Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, 27599, Chapel Hill NC, USA
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40
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Rickard KL, Gibson PR, Wilson NJ, Mariadason JM, Phillips WA. Short-chain fatty acids reduce expression of specific protein kinase C isoforms in human colonic epithelial cells. J Cell Physiol 2000; 182:222-31. [PMID: 10623886 DOI: 10.1002/(sici)1097-4652(200002)182:2<222::aid-jcp11>3.0.co;2-b] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
LIM1215 colon cancer cells were used as a model of human colonic epithelium to examine the effects of butyrate on protein kinase C (PKC) activity and isoform expression. On Western blot analysis, LIM1215 cells express the PKC isoforms alpha, beta, epsilon, zeta, and lambda, but not gamma, straight theta, or micro. Treatment with 2 mM butyrate for 48 h reduced cellular PKC activity up to 50% and specifically reduced the expression of PKC alpha and PKC epsilon. Similar results were obtained using Caco-2 colon cancer cells. These effects were neither a consequence of the induction of differentiation itself nor the result of direct or indirect activation of PKC. Although dependent on gene transcription and protein synthesis, the effect was not due to a reduction in the synthesis of PKC protein. Butyrate's effect was independent of its beta-oxidation but was mimicked, at least in part, by trichostatin A, an inhibitor of histone deacetylase.
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Affiliation(s)
- K L Rickard
- Department of Medicine, The Royal Melbourne Hospital, University of Melbourne, Victoria, Australia
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Keely S, Barrett K. Chapter 7 Integrated signaling mechanisms that regulate intestinal chloride secretion. CURRENT TOPICS IN MEMBRANES 2000. [DOI: 10.1016/s1063-5823(00)50009-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
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Turner JR, Angle JM, Black ED, Joyal JL, Sacks DB, Madara JL. PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase. THE AMERICAN JOURNAL OF PHYSIOLOGY 1999; 277:C554-62. [PMID: 10484342 DOI: 10.1152/ajpcell.1999.277.3.c554] [Citation(s) in RCA: 99] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G214-G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 (Cell Physiol. 42): C1378-C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.
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Affiliation(s)
- J R Turner
- Department of Pathology,Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
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Liu L, Zuurbier AEM, Mul FPJ, Verhoeven AJ, Lutter R, Knol EF, Roos D. Triple Role of Platelet-Activating Factor in Eosinophil Migration Across Monolayers of Lung Epithelial Cells: Eosinophil Chemoattractant and Priming Agent and Epithelial Cell Activator. THE JOURNAL OF IMMUNOLOGY 1998. [DOI: 10.4049/jimmunol.161.6.3064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Abstract
Infiltration of eosinophils into the lung lumen is a hallmark of allergic asthmatic inflammation. To reach the lung lumen, eosinophils must migrate across the vascular endothelium, through the interstitial matrix, and across the lung epithelium. The regulation of this process is obscure. In this study, we investigated the migration of human eosinophils across confluent monolayers of either human lung H292 epithelial cells or primary human bronchial epithelial cells. Established eosinophil chemoattractants (IL-8, RANTES, platelet-activating factor (PAF), leukotriene B4, and complement fragment 5a (C5a)) or activation of the epithelial cells with IL-1β induced little eosinophil transmigration (<7% in 2 h). In contrast, addition of PAF in combination with C5a induced extensive (>20%) transepithelial migration of unprimed and IL-5-primed eosinophils. Eosinophil migration assessed in a Boyden chamber assay, i.e., without an epithelial monolayer, was only slightly increased upon addition of PAF and C5a. Preincubation of eosinophils with the PAF receptor antagonist WEB 2086 only inhibited migration of unprimed eosinophils toward PAF and C5a, whereas preincubation of epithelial cells with WEB 2086 abolished migration of both IL-5-primed and unprimed eosinophils. This latter result indicated the presence of PAF receptors on epithelial cells. Indeed, addition of PAF to epithelial cells induced an increase in cytosolic free Ca2+, which was blocked by the PAF receptor antagonists WEB 2086 and TCV-309. Our results show that PAF induces permissive changes in epithelial cells, and that PAF acts as a chemoattractant and priming agent for the eosinophils.
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Affiliation(s)
- Lixin Liu
- *Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, and
| | - Astrid E. M. Zuurbier
- *Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, and
| | - Frederik P. J. Mul
- *Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, and
| | - Arthur J. Verhoeven
- *Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, and
| | - René Lutter
- †Department of Pulmonology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Edward F. Knol
- *Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, and
| | - Dirk Roos
- *Central Laboratory of The Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, and
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Hofman P, Flatau G, Selva E, Gauthier M, Le Negrate G, Fiorentini C, Rossi B, Boquet P. Escherichia coli cytotoxic necrotizing factor 1 effaces microvilli and decreases transmigration of polymorphonuclear leukocytes in intestinal T84 epithelial cell monolayers. Infect Immun 1998; 66:2494-500. [PMID: 9596707 PMCID: PMC108229 DOI: 10.1128/iai.66.6.2494-2500.1998] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/1997] [Accepted: 03/05/1998] [Indexed: 02/07/2023] Open
Abstract
Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic Escherichia coli strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho. Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A. Nusrat, M. Giry, J. R. Turner, S. P. Colgan, C. A. Parkos, E. Lemichez, P. Boquet, and J. L. Madara, Proc. Natl. Acad. Sci. USA 92:10629-10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration. Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin. However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction. CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs. Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria.
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Affiliation(s)
- P Hofman
- INSERM Unité 364, Faculté de Médecine, 06107 Nice Cedex 02, France.
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Groot J. Correlation between electrophysiological phenomena and transport of macromolecules in intestinal epithelium. Vet Q 1998. [DOI: 10.1080/01652176.1998.9694968] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/15/2022] Open
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