Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein, are mitogenic and motogenic

2003 ◽  
Vol 285 (2) ◽  
pp. G344-G353 ◽  
Author(s):  
F. Gary Toback ◽  
Margaret M. Walsh-Reitz ◽  
Mark W. Musch ◽  
Eugene B. Chang ◽  
John Del Valle ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Cell ◽  
Primary Cultures ◽  
Gastric Antrum ◽  
Functional Domain ◽  
Antral Mucosa ◽  
Intestinal Epithelial ◽  
Peptide Fragments ◽  
Gel Layer ◽  
Mucosal Protein

Antrum mucosal protein (AMP)-18 is a novel 18-kDa protein synthesized by cells of the gastric antrum mucosa. The protein is present in secretion granules of murine gastric antrum epithelial cells and is a component of canine antrum mucus, suggesting that it is secreted into the viscoelastic gel layer on the mucosal surface. Release of the protein appears to be regulated because forskolin decreased the amount of immunoreactive AMP-18 in primary cultures of canine antrum mucosal epithelial cells, and indomethacin gavaged into the stomach of mice reduced AMP-18 content in antrum mucosal tissue before inducing histological injury. A functional domain of the protein was identified by preparing peptides derived from the center of human AMP-18. A 21-mer peptide stimulated growth of gastric and intestinal epithelial cells, but not fibroblasts, and increased restitution of scrape-wounded gastric epithelial monolayers. These functions of AMP-18 suggest that its release onto the apical cell surface is regulated and that the protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating restitution and proliferation after injury.

Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

1995 ◽  
Vol 108 (1) ◽  
pp. 369-377 ◽  
Author(s):  
K.L. Soole ◽  
M.A. Jepson ◽  
G.P. Hazlewood ◽  
H.J. Gilbert ◽  
B.H. Hirst
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Gpi Anchor ◽  
Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

Bovine pancreatic duct cells express cAMP- and Ca(2+)-activated apical membrane Cl- conductances

1997 ◽  
Vol 273 (1) ◽  
pp. G204-G216 ◽  
Author(s):  
L. al-Nakkash ◽  
C. U. Cotton
Keyword(s):  
Epithelial Cells ◽  
Cell Membrane ◽  
Pancreatic Duct ◽  
Apical Membrane ◽  
Apical Cell ◽  
Primary Cultures ◽  
Apical Cell Membrane ◽  
Anion Secretion ◽  
Duct Epithelium ◽  
Cl Permeability

Secretion of salt and water by the epithelial cells that line pancreatic ducts depends on activation of apical membrane Cl- conductance. In the present study, we characterized two types of Cl- conductances present in the apical cell membrane of bovine pancreatic duct epithelial cells. Primary cultures of bovine main pancreatic duct epithelium and an immortalized cell line (BPD1) derived from primary cultures were used. Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ in intact monolayers of duct epithelium induced sustained anion secretion. Agonist-induced changes in plasma membrane Cl- permeability were accessed by 36 Cl- efflux, whole cell current recording, and measurements of transepithelial Cl- current across permeabilized epithelial monolayers. Elevation of intracellular cAMP elicited a sustained increase in Cl- permeability, whereas elevation of intracellular Ca2+ induced only a transient increase in Cl- permeability. Ca(2+)- but not cAMP-induced increases in Cl- permeability were abolished by preincubation of cells with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). N-phenylanthranilic acid (DPC; 1 mM) and glibenclamide (100 microM), but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 500 microM), inhibited the cAMP-induced increase in Cl- permeability. In contrast, DPC and DIDS, but not glibenclamide, inhibited the Ca(2+)-induced increase in Cl- permeability. We conclude from these experiments that bovine pancreatic duct epithelial cells express at least two types of Cl- channels, cAMP and Ca2+ activated, in the apical cell membrane. Because the Ca(2+)-activated increase in Cl- permeability is transient, the extent to which this pathway contributes to sustained anion secretion by the ductal epithelium remains to be determined.

scholarly journals Role of Intestinal Epithelial Cells in Immune Effects Mediated by Gram-Positive Probiotic Bacteria: Involvement of Toll-Like Receptors

2005 ◽  
Vol 12 (9) ◽  
pp. 1075-1084 ◽  
Author(s):  
Gabriel Vinderola ◽  
Chantal Matar ◽  
Gabriela Perdigon
Keyword(s):  
Small Intestine ◽  
B Cells ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Primary Cultures ◽  
Clonal Expansion ◽  
Probiotic Bacteria ◽  
Intestinal Epithelial ◽  
Toll Like Receptors

ABSTRACT The mechanisms by which probiotic bacteria exert their effects on the immune system are not completely understood, but the epithelium may be a crucial player in the orchestration of the effects induced. In a previous work, we observed that some orally administered strains of lactic acid bacteria (LAB) increased the number of immunoglobulin A (IgA)-producing cells in the small intestine without a concomitant increase in the CD4+ T-cell population, indicating that some LAB strains induce clonal expansion only of B cells triggered to produce IgA. The present work aimed to study the cytokines induced by the interaction of probiotic LAB with murine intestinal epithelial cells (IEC) in healthy animals. We focused our investigation mainly on the secretion of interleukin 6 (IL-6) necessary for the clonal expansion of B cells previously observed with probiotic bacteria. The role of Toll-like receptors (TLRs) in such interaction was also addressed. The cytokines released by primary cultures of IEC in animals fed with Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 were determined. Cytokines were also determined in the supernatants of primary cultures of IEC of unfed animals challenged with different concentrations of viable or nonviable lactobacilli and Escherichia coli, previously blocked or not with anti-TLR2 and anti-TLR4. We concluded that the small intestine is the place where a major distinction would occur between probiotic LAB and pathogens. This distinction comprises the type of cytokines released and the magnitude of the response, cutting across the line that separates IL-6 necessary for B-cell differentiation, which was the case with probiotic lactobacilli, from inflammatory levels of IL-6 for pathogens.

scholarly journals A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 471-479 ◽  
Author(s):  
M J Rindler ◽  
M G Traber
Keyword(s):  
Epithelial Cells ◽  
Secretory Pathway ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Plasma Membrane ◽  
Intestinal Epithelial ◽  
Polarized Secretion ◽  
Sorting Signals

Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

Cell-specific involvement of HNF-1β in α1-antitrypsin gene expression in human respiratory epithelial cells

2002 ◽  
Vol 282 (4) ◽  
pp. L757-L765 ◽  
Author(s):  
Chaobin Hu ◽  
David H. Perlmutter
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cell Line ◽  
Primary Cultures ◽  
Synergistic Action ◽  
Intestinal Epithelial ◽  
Hep G2 ◽  
Respiratory Epithelial Cells ◽  
Human Respiratory Epithelial Cells ◽  
Respiratory Epithelial

The synergistic action of hepatocyte nuclear factor (HNF)-1α and HNF-4 plays an important role in expression of the α1-antitrypsin (α1-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the α1-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that α1-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1α and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that α1-AT gene expression in H441 cells is predominantly driven by HNF-1β, even though HNF-1β has no effect on α1-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of α1-AT and HNF-1β was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on α1-AT gene expression in H441 cells, even when it is cotransfected with HNF-1β or HNF-1α. HNF-3 by itself has little effect on α1-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1β in α1-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.

scholarly journals Expression of Human Decay-Accelerating Factor on Intestinal Epithelium of Transgenic Mice Does Not Facilitate Infection by the Enteral Route

2015 ◽  
Vol 89 (8) ◽  
pp. 4311-4318 ◽  
Author(s):  
Jieyan Pan ◽  
Lili Zhang ◽  
Matthew A. Odenwald ◽  
Le Shen ◽  
Jerrold R. Turner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transgenic Mice ◽  
Intestinal Epithelium ◽  
Intestinal Epithelial Cells ◽  
Apical Cell ◽  
Oral Route ◽  
Intestinal Epithelial ◽  
Human Intestinal Epithelial Cells ◽  
Cvb3 Infection

ABSTRACTIn vitro, infection of polarized human intestinal epithelial cells by coxsackievirus B3 (CVB3) depends on virus interaction with decay-accelerating factor (DAF), a receptor expressed on the apical cell surface. Although mice are highly susceptible to CVB3 infection when virus is delivered by intraperitoneal injection, infection by the enteral route is very inefficient. Murine DAF, unlike human DAF, does not bind virus, and we hypothesized that the absence of an accessible receptor on the intestinal surface is an important barrier to infection by the oral route. We generated transgenic mice that express human DAF specifically on intestinal epithelium and measured their susceptibility to infection by a DAF-binding CVB3 isolate. Human DAF permitted CVB3 to bind to the intestinal surfaceex vivoand to infect polarized monolayers of small-intestinal epithelial cells derived from DAF transgenic mice. However, expression of human DAF did not facilitate infection by the enteral route either in immunocompetent animals or in animals deficient in the interferon alpha/beta receptor. These results indicate that the absence of an apical receptor on intestinal epithelium is not the major barrier to infection of mice by the oral route.IMPORTANCECVB3 infection of human intestinal epithelial cells depends on DAF at the apical cell surface, and expression of human DAF on murine intestinal epithelial cells permits their infectionin vitro. However, expression of human DAF on the intestinal surface of transgenic mice did not facilitate infection by the oral route. Although the role of intestinal DAF in human infection has not been directly examined, these results suggest that DAF is not the critical factor in mice.

scholarly journals Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation

2014 ◽  
Vol 207 (1) ◽  
pp. 123-137 ◽  
Author(s):  
Michael C. Burke ◽  
Feng-Qian Li ◽  
Benjamin Cyge ◽  
Takeshi Arashiro ◽  
Heather M. Brechbuhl ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Basal Body ◽  
Membrane Trafficking ◽  
Apical Cell ◽  
Primary Cultures ◽  
Physical Interaction ◽  
Vesicle Formation ◽  
Nucleotide Exchange ◽  
Apical Cell Membrane ◽  
Tracheal Epithelial Cells

Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

scholarly journals A Novel Strategy to Study the Invasive Capability of Adherent-Invasive Escherichia coli by Using Human Primary Organoid-Derived Epithelial Monolayers

2021 ◽  
Vol 12 ◽  
Author(s):  
Aida Mayorgas ◽  
Isabella Dotti ◽  
Marta Martínez-Picola ◽  
Miriam Esteller ◽  
Queralt Bonet-Rossinyol ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Cross Talk ◽  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Infection Model ◽  
Intestinal Epithelial ◽  
Epithelial Monolayers ◽  
The Cross

Over the last decades, Adherent-Invasive Escherichia coli (AIEC) has been linked to the pathogenesis of Crohn’s Disease. AIEC’s characteristics, as well as its interaction with the gut immune system and its role in intestinal epithelial barrier dysfunction, have been extensively studied. Nevertheless, the currently available techniques to investigate the cross-talk between this pathogen and intestinal epithelial cells (IECs) are based on the infection of immortalized cell lines. Despite their many advantages, cell lines cannot reproduce the conditions in tissues, nor do they reflect interindividual variability or gut location-specific traits. In that sense, the use of human primary cultures, either healthy or diseased, offers a system that can overcome all of these limitations. Here, we developed a new infection model by using freshly isolated human IECs. For the first time, we generated and infected monolayer cultures derived from human colonic organoids to study the mechanisms and effects of AIEC adherence and invasion on primary human epithelial cells. To establish the optimal conditions for AIEC invasion studies in human primary organoid-derived epithelial monolayers, we designed an infection-kinetics study to assess the infection dynamics at different time points, as well as with two multiplicities of infection (MOI). Overall, this method provides a model for the study of host response to AIEC infections, as well as for the understanding of the molecular mechanisms involved in adhesion, invasion and intracellular replication. Therefore, it represents a promising tool for elucidating the cross-talk between AIEC and the intestinal epithelium in healthy and diseased tissues.

scholarly journals Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

2005 ◽  
Vol 7 (8) ◽  
pp. 1109-1116 ◽  
Author(s):  
Rafika Athman ◽  
Maria-Isabel Fernandez ◽  
Pierre Gounon ◽  
Philippe Sansonetti ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Shigella Flexneri ◽  
Primary Cultures ◽  
Intestinal Epithelial ◽  
Mouse Intestine
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