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In JoVE (1)
- Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis
Other Publications (26)
- Journal of Cell Science
- Advanced Drug Delivery Reviews
- Molecular Biology of the Cell
- The Journal of Experimental Medicine
- Journal of Immunology (Baltimore, Md. : 1950)
- World Journal of Gastroenterology : WJG
- Journal of Immunology (Baltimore, Md. : 1950)
- American Journal of Rhinology
- Toxicology and Applied Pharmacology
- Molecular Biology of the Cell
- Molecular Biology of the Cell
- EMBO Reports
- Cell Adhesion & Migration
- Infection and Immunity
- Tissue Barriers
- The Journal of Biological Chemistry
- Molecular Biology of the Cell
- Molecular Biology of the Cell
- Experimental Cell Research
- Oxidative Medicine and Cellular Longevity
- Scientific Reports
- The Biochemical Journal
Articles by Porfirio Nava in JoVE
Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis
Hilda Vargas Robles1, Karla Fabiola Castro Ochoa1, Porfirio Nava2, Angélica Silva Olivares3, Mineko Shibayama3, Michael Schnoor1
1Department of Molecular Biomedicine, Center for Research and Advanced Studies of the National Polytechnic Institute, 2Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies of the National Polytechnic Institute, 3Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute
Other articles by Porfirio Nava on PubMed
The Rotavirus Surface Protein VP8 Modulates the Gate and Fence Function of Tight Junctions in Epithelial Cells
Journal of Cell Science. Nov, 2004 | Pubmed ID: 15494377
Rotaviruses constitute a major cause of diarrhea in young mammals. Rotaviruses utilize different integrins as cell receptors, therefore upon their arrival to the intestinal lumen their integrin receptors will be hidden below the tight junction (TJ), on the basolateral membrane. Here we have studied whether the rotavirus outer capsid proteins are capable of opening the paracellular space sealed by the TJ. From the outermost layer of proteins of the rotavirus, 60 spikes formed of protein VP4 are projected. VP4 is essential for virus-cell interactions and is cleaved by trypsin into peptides VP5 and VP8. Here we found that when these peptides are added to confluent epithelial monolayers (Madin-Darby canine kidney cells), VP8 is capable of diminishing in a dose dependent and reversible manner the transepithelial electrical resistance. VP5 exerted no effect. VP8 can also inhibit the development of newly formed TJs in a Ca-switch assay. Treatment with VP8 augments the paracellular passage of non-ionic tracers, allows the diffusion of a fluorescent lipid probe and the apical surface protein GP135, from the luminal to the lateral membrane, and triggers the movement of the basolateral proteins Na+-K+-ATPase, alphanubeta3 integrin and beta1 integrin subunit, to the apical surface. VP8 generates a freeze-fracture pattern of TJs characterized by the appearance of loose end filaments, that correlates with an altered distribution of several TJ proteins. VP8 given orally to diabetic rats allows the enteral administration of insulin, thus indicating that it can be employed to modulate epithelial permeability.
Tight Junctions, from Tight Intercellular Seals to Sophisticated Protein Complexes Involved in Drug Delivery, Pathogens Interaction and Cell Proliferation
Advanced Drug Delivery Reviews. Apr, 2005 | Pubmed ID: 15820554
Tight junctions (TJ) are intercellular seals that regulate the passage of ions and molecules through the paracellular pathway. This property allows epithelial and endothelial sheets to function as barriers between different body compartments. Although TJ were identified by electron microscopy since the 1950's, the analysis in recent years of their molecular composition, has depicted TJ as signaling complexes involved in a wide spectrum of physiological and pathological processes, such as cell growth and differentiation, cancer and metastasis, and pathogen interaction. This new knowledge has in turn given rise to novel strategies for enhancing the paracellular flux of poorly absorbed therapeutics.
Molecular Biology of the Cell. Nov, 2007 | Pubmed ID: 17804817
Intestinal epithelial intercellular junctions regulate barrier properties, and they have been linked to epithelial differentiation and programmed cell death (apoptosis). However, mechanisms regulating these processes are poorly defined. Desmosomes are critical elements of intercellular junctions; they are punctate structures made up of transmembrane desmosomal cadherins termed desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) that affiliate with the underlying intermediate filaments via linker proteins to provide mechanical strength to epithelia. In the present study, we generated an antibody, AH12.2, that recognizes Dsg2. We show that Dsg2 but not another desmosomal cadherin, Dsc2, is cleaved by cysteine proteases during the onset of intestinal epithelial cell (IEC) apoptosis. Small interfering RNA-mediated down-regulation of Dsg2 protected epithelial cells from apoptosis. Moreover, we report that a C-terminal fragment of Dsg2 regulates apoptosis and Dsg2 protein levels. Our studies highlight a novel mechanism by which Dsg2 regulates IEC apoptosis driven by cysteine proteases during physiological differentiation and inflammation.
The Journal of Experimental Medicine. Dec, 2007 | Pubmed ID: 18039951
Recent evidence has linked intestinal permeability to mucosal inflammation, but molecular studies are lacking. Candidate regulatory molecules localized within the tight junction (TJ) include Junctional Adhesion Molecule (JAM-A), which has been implicated in the regulation of barrier function and leukocyte migration. Thus, we analyzed the intestinal mucosa of JAM-A-deficient (JAM-A(-/-)) mice for evidence of enhanced permeability and inflammation. Colonic mucosa from JAM-A(-/-) mice had normal epithelial architecture but increased polymorphonuclear leukocyte infiltration and large lymphoid aggregates not seen in wild-type controls. Barrier function experiments revealed increased mucosal permeability, as indicated by enhanced dextran flux, and decreased transepithelial electrical resistance in JAM-A(-/-) mice. The in vivo observations were epithelial specific, because monolayers of JAM-A(-/-) epithelial cells also demonstrated increased permeability. Analyses of other TJ components revealed increased expression of claudin-10 and -15 in the colonic mucosa of JAM-A(-/-) mice and in JAM-A small interfering RNA-treated epithelial cells. Given the observed increase in colonic inflammation and permeability, we assessed the susceptibility of JAM-A(-/-) mice to the induction of colitis with dextran sulfate sodium (DSS). Although DSS-treated JAM-A(-/-) animals had increased clinical disease compared with controls, colonic mucosa showed less injury and increased epithelial proliferation. These findings demonstrate a complex role of JAM-A in intestinal homeostasis by regulating epithelial permeability, inflammation, and proliferation.
Formyl Peptide Receptor-1 Activation Enhances Intestinal Epithelial Cell Restitution Through Phosphatidylinositol 3-kinase-dependent Activation of Rac1 and Cdc42
Journal of Immunology (Baltimore, Md. : 1950). Dec, 2007 | Pubmed ID: 18056353
Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.
World Journal of Gastroenterology : WJG. Jan, 2008 | Pubmed ID: 18200662
A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the interstitium. The single layer of intestinal epithelial cells (IECs) serves as a dynamic interface between the host and its environment. Cell polarity and structural properties of the epithelium is complex and is important in the development of epithelial barrier function. Epithelial cells associate with each other via a series of intercellular junctions. The apical most intercellular junctional complex referred to as the Apical Junction Complex (AJC) is important in not only cell-cell recognition, but also in the regulation of paracellular movement of fluid and solutes. Defects in the intestinal epithelial barrier function have been observed in a number of intestinal disorders such as inflammatory bowel disease (IBD). It is now becoming evident that an aberrant epithelial barrier function plays a central role in the pathophysiology of IBD. Thus, a better understanding of the intestinal epithelial barrier structure and function in healthy and disease states such as IBD will foster new ideas for the development of therapies for such chronic disorders.
Journal of Immunology (Baltimore, Md. : 1950). Oct, 2008 | Pubmed ID: 18802107
During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (-/-) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (-/-) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.
American Journal of Rhinology. Nov-Dec, 2008 | Pubmed ID: 19178795
Many mucosal inflammatory conditions are associated with alterations in epithelial intercellular junctions and barrier function; however, little is known about the role of intercellular junctions in inflammatory diseases of the upper airways. In this study, we examined nasal polyps for altered intercellular junctions and protein expression.
Effects of Phenol on Barrier Function of a Human Intestinal Epithelial Cell Line Correlate with Altered Tight Junction Protein Localization
Toxicology and Applied Pharmacology. Nov, 2009 | Pubmed ID: 19679145
Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.
Dkk-1 Inhibits Intestinal Epithelial Cell Migration by Attenuating Directional Polarization of Leading Edge Cells
Molecular Biology of the Cell. Nov, 2009 | Pubmed ID: 19776352
Wnt signaling pathways regulate proliferation, motility, and survival in a variety of human cell types. Dickkopf-1 (Dkk-1) is a secreted Wnt antagonist that has been proposed to regulate tissue homeostasis in the intestine. In this report, we show that Dkk-1 is secreted by intestinal epithelial cells after wounding and that it inhibits cell migration by attenuating the directional orientation of migrating epithelial cells. Dkk-1 exposure induced mislocalized activation of Cdc42 in migrating cells, which coincided with a displacement of the polarity protein Par6 from the leading edge. Consequently, the relocation of the microtubule organizing center and the Golgi apparatus in the direction of migration was significantly and persistently inhibited in the presence of Dkk-1. Small interfering RNA-induced down-regulation of Dkk-1 confirmed that extracellular exposure to Dkk-1 was required for this effect. Together, these data demonstrate a novel role of Dkk-1 in the regulation of directional polarization of migrating intestinal epithelial cells, which contributes to the effect of Dkk-1 on wound closure in vivo.
Interferon-gamma Regulates Intestinal Epithelial Homeostasis Through Converging Beta-catenin Signaling Pathways
Immunity. Mar, 2010 | Pubmed ID: 20303298
Inflammatory cytokines have been proposed to regulate epithelial homeostasis during intestinal inflammation. We report here that interferon-gamma (IFN-gamma) regulates the crucial homeostatic functions of cell proliferation and apoptosis through serine-threonine protein kinase AKT-beta-catenin and Wingless-Int (Wnt)-beta-catenin signaling pathways. Short-term exposure of intestinal epithelial cells to IFN-gamma resulted in activation of beta-catenin through AKT, followed by induction of the secreted Wnt inhibitor Dkk1. Consequently, we observed an increase in Dkk1-mediated apoptosis upon extended IFN-gamma treatment and reduced proliferation through depletion of the Wnt coreceptor LRP6. These effects were enhanced by tumor necrosis factor-alpha (TNF-alpha), suggesting synergism between the two cytokines. Consistent with these results, colitis in vivo was associated with decreased beta-catenin-T cell factor (TCF) signaling, loss of plasma membrane-associated LRP6, and reduced epithelial cell proliferation. Proliferation was partially restored in IFN-gamma-deficient mice. Thus, we propose that IFN-gamma regulates intestinal epithelial homeostasis by sequential regulation of converging beta-catenin signaling pathways.
Loss of Desmocollin-2 Confers a Tumorigenic Phenotype to Colonic Epithelial Cells Through Activation of Akt/Î²-catenin Signaling
Molecular Biology of the Cell. Apr, 2011 | Pubmed ID: 21325624
Desmocollin-2 (Dsc2) and desmoglein-2 (Dsg2) are transmembrane cell adhesion proteins of desmosomes. Reduced expression of Dsc2 has been reported in colorectal carcinomas, suggesting that Dsc2 may play a role in the development and/or progression of colorectal cancer. However, no studies have examined the mechanistic contribution of Dsc2 deficiency to tumorigenesis. Here we report that loss of Dsc2 promotes cell proliferation and enables tumor growth in vivo through the activation of Akt/Î²-catenin signaling. Inhibition of Akt prevented the increase in Î²-catenin-dependent transcription and proliferation following Dsc2 knockdown and attenuated the in vivo growth of Dsc2-deficient cells. Taken together, our results provide evidence that loss of Dsc2 contributes to the growth of colorectal cancer cells and highlight a novel mechanism by which the desmosomal cadherins regulate Î²-catenin signaling.
EMBO Reports. Apr, 2011 | Pubmed ID: 21372850
Expression of the tight junction protein junctional adhesion molecule-A (JAM-A) has been linked to proliferation and tumour progression. However, a direct role for JAM-A in regulating proliferative processes has not been shown. By using complementary in vivo and in vitro approaches, we demonstrate that JAM-A restricts intestinal epithelial cell (IEC) proliferation in a dimerization-dependent manner, by inhibiting Akt-dependent β-catenin activation. Furthermore, IECs from transgenic JAM-A(-/-)/β-catenin/T-cell factor reporter mice showed enhanced β-catenin-dependent transcription. Finally, inhibition of Akt reversed colonic crypt hyperproliferation in JAM-A-deficient mice. These data establish a new link between JAM-A and IEC homeostasis.
Gastroenterology. Jul, 2011 | Pubmed ID: 21440550
Dkk1 is a secreted antagonist of the Wnt/β-catenin signaling pathway. It is induced by inflammatory cytokines during colitis and exacerbates tissue damage by promoting apoptosis of epithelial cells. However, little is known about the physiologic role of Dkk1 in normal intestinal homeostasis and during wound repair following mucosal injury. We investigated whether inhibition of Dkk1 affects the morphology and function of the adult intestine.
Characterization of Full-length and Proteolytic Cleavage Fragments of Desmoglein-2 in Native Human Colon and Colonic Epithelial Cell Lines
Cell Adhesion & Migration. Jul-Aug, 2011 | Pubmed ID: 21715983
The desmosomal cadherin desmoglein-2 (Dsg2) is a transmembrane cell adhesion protein that is widely expressed in epithelial and non-epithelial tissues, such as the intestine, epidermis, testis, and heart. Dsg2 has been shown to regulate numerous cellular processes, including proliferation and apoptosis, and we have previously reported that intracellular fragments of Dsg2 promote apoptosis in colonic epithelial cells. While several studies have shown that both the extracellular and intracellular domains of Dsg2 can be targeted by proteases, identification of these putative Dsg2 fragments in colonic epithelial cells has not been performed. Here, we report that the mouse monoclonal antibody (mAb) AH12.2 binds to the first extracellular domain of Dsg2. Using this antibody along with previously described mAb against the extracellular (6D8) and intracellular (DG3.10) domains of Dsg2, we characterize the expression and identify the cleavage fragments of Dsg2 in colonic epithelial cells. This study provides a detailed description of the extracellular and intracellular Dsg2 cleavage fragments that are generated in the simple epithelium of the colon and will guide future studies examining the relationship of these fragments to cellular fate and disease states.
Compromised Intestinal Epithelial Barrier Induces Adaptive Immune Compensation That Protects from Colitis
Immunity. Sep, 2012 | Pubmed ID: 22981539
Mice lacking junctional adhesion molecule A (JAM-A, encoded by F11r) exhibit enhanced intestinal epithelial permeability, bacterial translocation, and elevated colonic lymphocyte numbers, yet do not develop colitis. To investigate the contribution of adaptive immune compensation in response to increased intestinal epithelial permeability, we examined the susceptibility of F11r(-/-)Rag1(-/-) mice to acute colitis. Although negligible contributions of adaptive immunity in F11r(+/+)Rag1(-/-) mice were observed, F11r(-/-)Rag1(-/-) mice exhibited increased microflora-dependent colitis. Elimination of T cell subsets and cytokine analyses revealed a protective role for TGF-β-producing CD4(+) T cells in F11r(-/-) mice. Additionally, loss of JAM-A resulted in elevated mucosal and serum IgA that was dependent upon CD4(+) T cells and TGF-β. Absence of IgA in F11r(+/+)Igha(-/-) mice did not affect disease, whereas F11r(-/-)Igha(-/-) mice displayed markedly increased susceptibility to acute injury-induced colitis. These data establish a role for adaptive immune-mediated protection from acute colitis under conditions of intestinal epithelial barrier compromise.
Quorum-sensing Systems LuxS/autoinducer 2 and Com Regulate Streptococcus Pneumoniae Biofilms in a Bioreactor with Living Cultures of Human Respiratory Cells
Infection and Immunity. Apr, 2013 | Pubmed ID: 23403556
Streptococcus pneumoniae forms organized biofilms in the human upper respiratory tract that may play an essential role in both persistence and acute respiratory infection. However, the production and regulation of biofilms on human cells is not yet fully understood. In this work, we developed a bioreactor with living cultures of human respiratory epithelial cells (HREC) and a continuous flow of nutrients, mimicking the microenvironment of the human respiratory epithelium, to study the production and regulation of S. pneumoniae biofilms (SPB). SPB were also produced under static conditions on immobilized HREC. Our experiments demonstrated that the biomass of SPB increased significantly when grown on HREC compared to the amount on abiotic surfaces. Additionally, pneumococcal strains produced more early biofilms on lung cells than on pharyngeal cells. Utilizing the bioreactor or immobilized human cells, the production of early SPB was found to be regulated by two quorum-sensing systems, Com and LuxS/AI-2, since a mutation in either comC or luxS rendered the pneumococcus unable to produce early biofilms on HREC. Interestingly, while LuxS/autoinducer 2 (AI-2) regulated biofilms on both HREC and abiotic surfaces, Com control was specific for those structures produced on HREC. The biofilm phenotypes of strain D39-derivative ΔcomC and ΔluxS QS mutants were reversed by genetic complementation. Of note, SPB formed on immobilized HREC and incubated under static conditions were completely lysed 24 h postinoculation. Biofilm lysis was also regulated by the Com and LuxS/AI-2 quorum-sensing systems.
Tissue Barriers. Apr, 2013 | Pubmed ID: 24665393
Epithelial tissues form a selective barrier that separates the external environment from the internal tissue milieu. Single epithelial cells are densely packed and associate via distinct intercellular junctions. Intercellular junction proteins not only control barrier properties of the epithelium but also play an important role in regulating epithelial homeostasis that encompasses cell proliferation, migration, differentiation and regulated shedding. Recent studies have revealed that several proteases target epithelial junction proteins during physiological maturation as well as in pathologic states such as inflammation and cancer. This review discusses mechanisms and biological consequences of transmembrane junction protein cleavage. The influence of junction protein cleavage products on pathogenesis of inflammation and cancer is discussed.
The Journal of Biological Chemistry. Apr, 2014 | Pubmed ID: 24567334
The desmosomal cadherins, desmogleins, and desmocollins mediate strong intercellular adhesion. Human intestinal epithelial cells express the desmoglein-2 isoform. A proteomic screen for Dsg2-associated proteins in intestinal epithelial cells identified a lectin referred to as galectin-3 (Gal3). Gal3 bound to N-linked β-galactosides in Dsg2 extracellular domain and co-sedimented with caveolin-1 in lipid rafts. Down-regulation of Gal3 protein or incubation with lactose, a galactose-containing disaccharide that competitively inhibits galectin binding to Dsg2, decreased intercellular adhesion in intestinal epithelial cells. In the absence of functional Gal3, Dsg2 protein was internalized from the plasma membrane and degraded in the proteasome. These results report a novel role of Gal3 in stabilizing a desmosomal cadherin and intercellular adhesion in intestinal epithelial cells.
Molecular Biology of the Cell. Oct, 2014 | Pubmed ID: 25079689
The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin-mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation.
Molecular Biology of the Cell. Sep, 2015 | Pubmed ID: 26224314
Desmosomal cadherins mediate intercellular adhesion and control epithelial homeostasis. Recent studies show that proteinases play an important role in the pathobiology of cancer by targeting epithelial intercellular junction proteins such as cadherins. Here we describe the proinflammatory cytokine-induced activation of matrix metalloproteinase 9 and a disintegrin and metalloproteinase domain-containing protein 10, which promote the shedding of desmosomal cadherin desmoglein-2 (Dsg2) ectodomains in intestinal epithelial cells. Epithelial exposure to Dsg2 ectodomains compromises intercellular adhesion by promoting the relocalization of endogenous Dsg2 and E-cadherin from the plasma membrane while also promoting proliferation by activation of human epidermal growth factor receptor 2/3 signaling. Cadherin ectodomains were detected in the inflamed intestinal mucosa of mice with colitis and patients with ulcerative colitis. Taken together, our findings reveal a novel response pathway in which inflammation-induced modification of columnar epithelial cell cadherins decreases intercellular adhesion while enhancing cellular proliferation, which may serve as a compensatory mechanism to promote repair.
Heterogeneity Between Triple Negative Breast Cancer Cells Due to Differential Activation of Wnt and PI3K/AKT Pathways
Experimental Cell Research. Nov, 2015 | Pubmed ID: 26453937
The lack of a successful treatment for triple-negative breast cancer demands the study of the heterogeneity of cells that constitute these tumors. With this aim, two clones from triple negative breast MDA-MB-231 cancer cells were isolated: One with fibroblast-like appearance (F) and another with semi-epithelial (SE) morphology. Cells of the F clone have a higher migration and tumorigenesis capacity than SE cells, suggesting that these cells are in a more advanced stage of epithelial to mesenchymal transformation. In agreement, F cells have a diminished expression of the tight junction proteins claudins 1 and 4, and an increased content of β-catenin. The latter is due to an augmented activity of the canonical Wnt route and of the EGFR/PI3K/mTORC2/AKT pathway favoring the cytoplasmic accumulation of β-catenin and its transcriptional activity. In addition, F cells display increased phosphorylation of β-catenin at Tyr654 by Src. These changes favor in F cells, the over-expression of Snail that promotes EMT. Finally, we observe that both F and SE cells display markers of cancer stem cells, which are more abundant in the F clone.
Experimental Colitis Is Attenuated by Cardioprotective Diet Supplementation That Reduces Oxidative Stress, Inflammation, and Mucosal Damage
Oxidative Medicine and Cellular Longevity. 2016 | Pubmed ID: 26881044
Inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD) are multifactorial, relapsing disorders of the gastrointestinal tract. However, the etiology is still poorly understood but involves altered immune responses, epithelial dysfunction, environmental factors, and nutrition. Recently, we have shown that the diet supplement corabion has cardioprotective effects due to reduction of oxidative stress and inflammation. Since oxidative stress and inflammation are also prominent risk factors in IBD, we speculated that corabion also has beneficial effects on experimental colitis. Colitis was induced in male mice by administration of 3.5% (w/v) dextran sulfate sodium (DSS) in drinking water for a period of 3 or 7 days with or without daily gavage feeding of corabion consisting of vitamin C, vitamin E, L-arginine, and eicosapentaenoic and docosahexaenoic acid. We found that corabion administration attenuated DSS-induced colon shortening, tissue damage, and disease activity index during the onset of colitis. Mechanistically, these effects could be explained by reduced neutrophil recruitment, oxidative stress, production of proinflammatory cytokines, and internalization of the junctional proteins ZO-1 and E-cadherin leading to less edema formation. Thus, corabion may be a useful diet supplement for the management of chronic inflammatory intestinal disorders such as IBD.
The CpAL System Regulates Changes of the Trans-epithelial Resistance of Human Enterocytes During Clostridium Perfringens Type C Infection
Anaerobe. Jun, 2016 | Pubmed ID: 27063897
Clostridium perfringens type C strains produce severe disease in humans and animals including enterotoxaemia and hemorrhagic diarrhea. Type C disease is mediated by production of toxins that damage the site of infection inducing loss of bloody fluids. Production of type C toxins, such as CPA, PFO, and, CPB is regulated by the C. perfringens Agr-like (CpAL) quorum sensing (QS) system. The CpAL system is also required to recapitulate, in vivo, intestinal signs of C. perfringens type C-induced disease, including hemorrhagic diarrhea and accumulation of fluids. The intestinal epithelium forms a physical barrier, made up of a series of intercellular junctions including tight junctions (TJs), adherens junctions (AJs) and desmosomes (DMs). This selective barrier regulates important physiological processes, including paracellular movement of ions and solutes, which, if altered, results in loss of fluids into the intestinal lumen. In this work, the effects of C. perfringens infection on the barrier function of intestinal epithelial cells was evaluated by measuring trans-epithelial resistance (TEER). Our studies demonstrate that infection of human enterocytes with C. perfringens type C strain CN3685 induced a significant drop on TEER. Changes in TEER were mediated by the CpAL system as a CN3685ΔagrB mutant did not induce such a drop. Physical contact between bacteria and enterocytes produced more pronounced changes in TEER and this phenomenon appeared also to be mediated by the CpAL system. Finally, immunofluorescence studies demonstrate that C. perfringens type C infection redistribute TJs protein occludin, and Claudin-3, and DMs protein desmoglein-2, but did not affect the AJs protein E-cadherin.
Loss of Cortactin Causes Endothelial Barrier Dysfunction Via Disturbed Adrenomedullin Secretion and Actomyosin Contractility
Scientific Reports. Jun, 2016 | Pubmed ID: 27357373
Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity.
The Pro-inflammatory Cytokines IFNγ/TNFα Increase Chromogranin A-positive Neuroendocrine Cells in the Colonic Epithelium
The Biochemical Journal. Nov, 2016 | Pubmed ID: 27538402
The gastrointestinal tract is the largest hormone-producing organ in the body due to a specialized cell population called enteroendocrine cells (EECs). The number of EECs increases in the mucosa of inflammatory bowel disease patients; however, the mechanisms responsible for these changes remain unknown. Here, we show that the pro-inflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα) or dextran sulfate sodium (DSS)-induced colitis increase the number of EECs producing chromogranin A (CgA) in the colonic mucosa of C57BL/6J mice. CgA-positive cells were non-proliferating cells enriched with inactive phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and autophagy markers. Moreover, inhibition of Akt and autophagy prevented the increase in CgA-positive cells after IFNγ/TNFα treatment. Similarly, we observed that CgA-positive cells in the colonic mucosa of patients with colitis expressed Akt and autophagy markers. These findings suggest that Akt signaling and autophagy control differentiation of the intestinal EEC lineage during inflammation.