Biomedical subjects

A Nusrat

Publications and source records attributed to A Nusrat.

At least 19 recordsLinked to original sources

PubMed2006

Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug.

Although dysregulation of tight junction (TJ) proteins is observed in epithelial malignancy, their participation in epithelial transformation is poorly understood. Recently we demonstrated that expression of oncogenic Raf 1 in Pa4 epithelial cells disrupts TJs and induces an oncogenic phenotype by downregulating expression of the TJ protein, occludin. Here we report the mechanism by which Raf 1 regulates occludin expression. Raf 1 inhibited occludin transcription by repressing a minimal segment of the occludin promoter in concert with upregulation of the transcriptional repressor, Slug without influencing the well-documented transcriptional repressor, Snail. Overexpression of Slug in Pa4 cells recapitulated the effect of Raf 1 on occludin expression, and depletion of Slug by small interfering RNA abrogated the effect of Raf 1 on occludin. Finally, chromatin immunoprecipitation assays and site-directed mutagenesis demonstrated a direct interaction between Slug and an E-box within the minimal Raf 1-responsive segment of the occludin promoter. These findings support a role of Slug in mediating Raf 1-induced transcriptional repression of occludin and subsequent epithelial to mesenchymal transition.

Animals↗

PubMed2001

Junction adhesion molecule is a receptor for reovirus.

Virus attachment to cells plays an essential role in viral tropism and disease. Reovirus serotypes 1 and 3 differ in the capacity to target distinct cell types in the murine nervous system and in the efficiency to induce apoptosis. The binding of viral attachment protein sigma1 to unidentified receptors controls these phenotypes. We used expression cloning to identify junction adhesion molecule (JAM), an integral tight junction protein, as a reovirus receptor. JAM binds directly to sigma1 and permits reovirus infection of nonpermissive cells. Ligation of JAM is required for reovirus-induced activation of NF-kappaB and apoptosis. Thus, reovirus interaction with cell-surface receptors is a critical determinant of both cell-type specific tropism and virus-induced intracellular signaling events that culminate in cell death.

Animals↗

PubMed2001

Interaction of bacteria and bacterial toxins with intestinal epithelial cells.

The epithelium of the intestinal tract is a key barrier between the external environment and the internal body environment. Intestinal epithelial cells are targets for luminal bacteria and viruses and must discriminate between pathogenic and nonpathogenic commensal organisms. Pathogenic bacteria and their secreted products influence epithelial cell function and induce diarrhea by numerous mechanisms that range from an effect on epithelial cell-cell associations to intracellular signal transduction pathways. These effects lead to an inflammatory response and an influx of neutrophils into the epithelium. Infiltrating neutrophils, in turn, signal to epithelial cells, induce a secretory response, and perpetuate the diarrhea. Conversely, commensal bacteria have the ability to suppress inflammatory responses by inhibiting specific intracellular signal transduction pathways. Some of these diverse host pathogenic responses are addressed in this review.

Animals↗

PubMed2001

Neutrophil transmigration in inflammatory bowel disease is associated with differential expression of epithelial intercellular junction proteins.

Inflammatory bowel disease (IBD) consisting of ulcerative colitis (UC) and Crohn's (CD) typically displays a waxing and waning course punctuated by disease flares that are characterized by transepithelial migration of neutrophils (PMN) and altered barrier function. Since epithelial barrier function is primarily regulated by the apical most intercellular junction referred to as the tight junction (TJ), our aim was to examine expression of TJ and adherens junction (AJ) proteins in relation to PMN infiltration in mucosal tissue samples from patients with active IBD. Expression of epithelial intercellular TJ proteins (occludin, ZO-1, claudin-1, and JAM) and subjacent AJ (beta-catenin and E-cadherin) proteins were examined by immunoflourescence/confocal microscopy, immunohistochemistry, and Western blotting. Colonic mucosa from patients with UC revealed dramatic, global down-regulation of the key TJ transmembrane protein occludin in regions of actively transmigrating PMN and in quiescent areas in the biopsy samples. Significant decreases in occludin expression were observed at the protein and mRNA levels by Western and Northern blotting. In contrast, expression of other TJ and AJ proteins such as ZO-1, claudin-1, JAM, beta-catenin, and E-cadherin were down-regulated only in epithelial cells immediately adjacent to transmigrating PMN. Analysis of inflamed mucosa from Crohn's disease patients mirrored the results obtained with UC patients. No change in TJ and AJ protein expression was observed in colonic epithelium from patients with collagenous colitis or lymphocytic colitis that are respectively characterized by a thickened subepithelial collagen plate and increased intraepithelial lymphocytes. These results suggest that occludin expression is diminished in IBD by mechanisms distinct from those regulating expression of other intercellular junction proteins. We speculate that down-regulation of epithelial occludin may play a role in enhanced paracellular permeability and PMN transmigration that is observed in active inflammatory bowel disease.

Adherens Junctions↗

PubMed2001

Reduced expression of junctional adhesion molecule and platelet/endothelial cell adhesion molecule-1 (CD31) at human vascular endothelial junctions by cytokines tumor necrosis factor-alpha plus interferon-gamma Does not reduce leukocyte transmigration under flow.

The combination of tumor necrosis factor (TNF)-alpha plus interferon (IFN)-gamma has been shown previously to promote redistribution of platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31), junctional adhesion molecule (JAM), and VE-cadherin away from lateral junctions of human umbilical vein endothelial cell monolayers. In parallel, neutrophil transmigration was significantly reduced. Because PECAM-1 and JAM have been implicated in leukocyte transmigration, the observed redistribution by cytokine activation was presumed to represent the mechanism causing decreased transmigration under static conditions. The current results confirm that culture of human umbilical vein endothelial cells with TNF-alpha plus IFN-gamma caused a decrease in surface-expressed and junctional-localized JAM and PECAM-1, but did not cause decreased leukocyte transmigration in an in vitro flow assay. Furthermore, blocking monoclonal antibody to PECAM-1 still significantly reduced monocyte transmigration, demonstrating that it retains a functional role even though its levels were reduced and redistributed away from junctions, whereas a panel of monoclonal antibodies to JAM failed to reduce leukocyte transmigration. Given the alterations in junction protein location, permeability function was assessed. IFN-gamma alone or TNF-alpha plus IFN-gamma significantly increased permeability, but TNF-alpha alone did not, suggesting lack of correlation between transmigration and loss of permeability. In conclusion, cytokine activation induced loss and redistribution of PECAM-1 and JAM away from lateral junctions, but per se does not negatively regulate either neutrophil or monocyte transmigration under flow.

Cell Adhesion↗

PubMed2001

Rho kinase regulates tight junction function and is necessary for tight junction assembly in polarized intestinal epithelia.

BACKGROUND & AIMS: Tight junctions are crucial determinants of barrier function in polarized intestinal epithelia and are regulated by Rho guanosine triphosphatase. Rho kinase (ROCK) is a downstream effector of Rho. METHODS: A specific inhibitor of ROCK, Y-27632, was used to examine the role of ROCK in the regulation of tight junctions in model intestinal (T84) cells by electrophysiologic, biochemical, morphologic, and molecular biologic approaches. RESULTS: ROCK inhibition induced reorganization of apical F-actin structures and enhanced paracellular permeability but did not alter the distribution or detergent solubility of tight junction proteins. Confocal microscopy showed colocalization of a subpool of ROCK with the tight junction protein zonula occludens 1. Inhibition of ROCK function by a dominant negative mutant of ROCK also produced reorganization of apical F-actin structures without disruption of tight junctions. ROCK inhibition in calcium switch assays showed that ROCK is necessary for the assembly of tight and adherens junctions. Upon calcium repletion, occludin, zonula occludens 1, and E-cadherin failed to redistribute to the intercellular junctions; assembly of the apical F-actin cytoskeleton was prevented; and barrier function failed to recover. CONCLUSIONS: We suggest that ROCK regulates intact tight junctions via its effects on the F-actin cytoskeleton. ROCK is also critical for assembly of the apical junctional proteins and the F-actin cytoskeleton organization during junctional formation.

Actins↗

PubMed2001

Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins.

The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021-16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting "raft-like" membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis.

Actins↗

PubMed2001

Neutrophil transepithelial migration: regulation at the apical epithelial surface by Fc-mediated events.

Neutrophil (PMN) transepithelial migration is a major effector of epithelial defense in inflammatory diseases involving mucosal surfaces. However, major receptor-ligand interactions between epithelial cells and PMN remain incompletely characterized. To better define the molecular events involved in PMN interactions with epithelial cells, we produced a monoclonal antibody called g82 that inhibited PMN transepithelial migration in the physiological basolateral-to-apical direction. The g82 antigen localized to the apical surface of human colonic epithelium and was significantly upregulated under inflammatory conditions. Immunoprecipitation revealed two polypeptides of M(r) 207 and 32 kDa. F(ab')(2) fragments from g82 IgG had no effect on transmigration, suggesting Fc dependence. Further experiments confirmed dependence on the PMN Fc receptor CD32A and that the observed effects were secondary to a failure of PMN to detach from the apical epithelial surface. These Fc-mediated events were epitope specific since binding, isotype-matched antibodies did not affect detachment. These results identify a new mechanism for retention of PMN at the apical epithelial surface following transepithelial migration. This pathway may be important in pathogen clearance and mucosal pathophysiology associated with autoimmunity.

Animals↗

PubMed2000

The coiled-coil domain of occludin can act to organize structural and functional elements of the epithelial tight junction.

Occludin is an integral membrane protein that has been suggested to play a role in the organization and dynamic function of the epithelial tight junction (TJ). A number of other proteins have also been described to localize to the TJ. We have used a novel bait peptide method to investigate potential protein-protein interactions of the putative coiled-coil domain of occludin with some of these other TJ proteins. A 27-amino acid peptide of the human occludin sequence was synthesized, biotinylated at the N terminus, and modified to contain a photoactive moiety at either its hydrophobic or hydrophilic surface. These bait peptides were alpha-helical in solution, characteristic of coiled-coil structures. Photoactivation studies in the presence and absence of control peptides were used to assess the potential interactions in polarized sheets of a human intestinal cell line T84. Although a large number of proteins associated with the TJ or that are known to be involved in regulatory events of epithelial cells failed to be specifically labeled, occludin itself, ZO-1, protein kinase C-zeta, c-Yes, the regulatory subunit of phosphatidylinositol 3-kinase, and the gap junction component connexin 26 were specifically labeled. Our data demonstrate the potential of one specific domain of occludin, contained within 27 amino acids, to coordinate the binding of proteins that have been previously suggested to modulate TJ structure and function.

Amino Acid Sequence↗

PubMed2000

Modulation of tight junction structure and function by cytokines.

Dynamic regulation of tight junction function is fundamental to many physiologic processes. Disruption of tight junction function drastically alters paracellular permeability and is a hallmark of many pathologic states. Recently, an increasing number of cytokines have been shown to influence tight junction function both in vitro and in vivo. Cytokine-induced effects on tight junction barrier function have also been correlated with effects on intrinsic tight junction proteins and the associated actin cytoskeleton. The aim of this article is to review studies relating to the effects of cytokines on tight junction function and structure.

Animals↗

PubMed2000

Modulation of tight junction function by G protein-coupled events.

Small G proteins or GTPases comprise a growing family of signal transduction molecules with inducible properties dependent upon reversible interactions with guanine nucleotides. Activation status of the proteins is characterized by preferential affinity for triphosphorylated guanine nucleotides, initiating signaling events that control fundamental processes involved in cell migration and contraction. Termination of small G protein signaling activity is in part achieved through intrinsic GTPase activity, which catalyzes the removal of GTP and its replacement with functionally inactive GDP. Recent investigations have implicated various small G proteins as messengers that control cell-cell contact between scaffold proteins and the actin cytoskeleton, suggesting an intrinsic mechanism for the regulation of paracellular permeability in polarized epithelial and endothelial cells. This review will examine current evidence for the control of tight junction permeability by small G proteins, and speculate upon future directions that may be of value in further exploring the biological importance of these key mediators.

Animals↗

PubMed2000

Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells.

The epithelial lining of the gastrointestinal tract forms a regulated, selectively permeable barrier between luminal contents and the underlying tissue compartments. Permeability across the epithelium is, in part, determined by the rate-limiting barrier of the paracellular pathway-the most apical intercellular junction referred to as the tight junction (TJ). The TJ is composed of a multiprotein complex that affiliates with the underlying apical actomyosin ring. TJ structure and function, and therefore epithelial permeability, are influenced by diverse physiological and pathological stimuli; here we review examples of such stimuli that are detected at the cell surface. For example, luminal glucose induces an increase in paracellular permeability to small molecules. Similarly, but by other means, cytokines and leukocytes in the vicinity of the epithelium also regulate TJ structure and paracellular permeability by influencing the TJ protein complex and/or its association with the underlying actin cytoskeleton.

Cytokines↗

PubMed2000

Tight junctions are membrane microdomains.

Tight junctions (TJ) of polarized epithelial cells regulate barrier function at mucosal surfaces. Structural proteins of TJs include hyperphosphorylated occludin (HO) and the peripheral membrane protein, ZO-1. Since TJs are dynamically regulated, and lipid-modified signal transduction proteins localize to TJs, we considered the possibility that the TJ itself is composed of microdomains with unique structure. Differential detergent extraction and isopycnic sucrose density gradients were utilized to isolate TJ-enriched membranes from a polarized intestinal epithelial cell line, T84. Here we report that major pools of hyperphosphorylated occludin (HO) and ZO-1 are found in raft-like membrane microdomains with characteristics of the previously described detergent-insoluble glycolipid rafts (DIGs). Properties of such gradient fractions included Triton X-100 (TX-100) insolubility, light scattering at 600 nm, buoyant density of approximately 1.08 g/cm(3) and increased cholesterol content compared to high density fractions. Similar results were obtained using natural epithelium. Unlike the TJ proteins HO and ZO-1, other basolateral transmembrane proteins including E-cadherin, c-met and &bgr; 1 integrin were not increased in DIG-like fractions. Immunoprecipitation studies revealed coprecipitation of a pool of occludin with caveolin-1, a scaffolding protein abundant in DIGs. Coprecipitation results were supported by immunofluorescence and immunogold labeling studies demonstrating caveolin-1 localization in the apical membrane and focal colocalization with occludin in TJs. TJ disassembly by calcium chelation resulted in displacement of TJ proteins from the 'raft-like' compartment. Our findings suggest that raft-like compartments play an important role in the spatial organization of TJs and probably in regulation of paracellular permeability in epithelial cells.

Caveolin 1↗

PubMed2000

Human junction adhesion molecule regulates tight junction resealing in epithelia.

Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35-39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.

Amino Acid Sequence↗

PubMed1999

Cell-specific peptide binding by human neutrophils.

Analysis of peptide binding to human neutrophils (PMN) using phage display techniques has revealed cell-specific motifs reactive with the PMN surface. Phage libraries displaying either linear 9-mer or cyclic 10-mer and 6-mer peptides were incubated with normal human neutrophils followed by elution of bound phage with low pH (pH 2.2) and non-ionic detergent. Three rounds of selection generated several related peptide sequences that bound with high avidity to PMN. Using the linear 9-mer library, PMN-binding phage expressed peptides with the motif (G/A)PNLTGRW. The binding of phage bearing this motif was highly specific since no binding was observed on lymphocytes, fibroblasts, epithelial, or endothelial cells. Functional assays revealed that phage bearing the sequence FGPNLTGRW induced a pertussis toxin-sensitive increase in PMN cytosolic calcium analogous to that observed with Galphai coupled receptors. Other prominent motifs identified included phage bearing the consensus DLXTSK(M/L)X(V/I/L), where X represents a non-conserved position. Phage with this motif bound exclusively to a sub population of human PMN that comprised approximately 50% of the total and did not elicit a calcium response. The binding of such phage to PMN was prevented by co-incubation with competing peptides displaying identical or similar sequences (IC50 range from 0.6 micromol/L to 50 micromol/L for DLXTSK and GPNLTG, respectively). We speculate that these techniques will be useful in identifying functional cell-specific binding motifs and contribute to the development of new therapeutic and diagnostic strategies in human disease.

Amino Acid Sequence↗

PubMed1999

Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta2-microglobulin-associated form.

In order to better understand the role of intestinal CD1d, we sought to define the cellular localization and further characterize the biochemical structure of CD1d in human intestinal epithelial cells (IEC). Using a CD1d-specific rabbit anti-gst-CD1d antibody, immunoprecipitation of radiolabeled cell surface proteins detected a previously identified 37 kDa protein as well as a 48-50 kDa protein which were confirmed by Western blotting with a CD1d-specific mAb, D5. Immunoprecipitation of protein lysates with the CD1d-specific mAb, D5 and 51.1.3, and the beta2-microglobulin (beta2m)-specific mAb, BBM.1, followed by N-glycanase digestion and Western blotting with the D5 mAb showed that the 48-50 kDa protein was a beta2m-associated, CD1d glycoprotein. CD1d was immunolocalized to the apical and lateral regions of native small and large intestinal IEC as defined by confocal laser microscopy using the D5 mAb and the rabbit anti-gst-CD1d antibody. In addition, a large apical intracellular pool of CD1d was identified. Identical observations were made with polarized T84 cells. Selective biotin labeling of apical and basolateral cell surfaces followed by immunoprecipitation with the D5 mAb, N-glycanase digestion and avidin blotting confirmed the presence of glycosylated CD1d on both cell surfaces and immunolocalization of the 37 kDa non-glycosylated form of CD1d to the apical cell surface. These studies show that CD1d is located in an ideal position for luminal antigen sampling and presentation to subjacent intraepithelial lymphocytes.

Antibody Specificity↗

PubMed1998

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells.

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl- secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl- conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I- > Cl-; for forskolin, Cl- > I-). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I- > Cl-). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Neutrophil migration across model intestinal epithelia: monolayer disruption and subsequent events in epithelial repair.

BACKGROUND & AIMS: Acute inflammation of the intestine is associated with transepithelial migration of polymorphonuclear leukocytes (PMNs) and epithelial wounds that rapidly reseal. The aim of this study was to determine mechanisms by which such PMN-induced epithelial wounds reseal. METHODS: Epithelial wound closure was modeled in vitro using T84 intestinal epithelial cells and PMNs. Wound closure was analyzed by confocal microscopy and by determination of barrier function. Wounds were highlighted by apical labeling with antibody to a basolaterally restricted ligand, beta1-integrin. RESULTS: High-density PMN transepithelial migration for 70-110 minutes produced multifocal epithelial wounds that were 1-120 microm in diameter and markedly diminished epithelial barrier function that returned to baseline within 12-20 hours. Large wound closure was initiated by cell flattening and extension of F-actin/vinculin/paxillin-enriched lamellipodia at the leading edge. As wounds became small (approximately <30 microm), epithelial cells at the wound edges assumed columnar phenotype with poorly formed or absent lamellipodia. Apical localized circumferential, dense F-actin/myosin II rings were found to encircle such wounds, suggesting final closure by a sphincter-like contraction. CONCLUSIONS: These data model mucosal repair in acute inflammatory conditions and, for the first time, show sequential early and late mechanisms by which epithelial discontinuities repair.

Cell Movement↗