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

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.107.2.471 ◽

1988 ◽

Vol 107 (2) ◽

pp. 471-479 ◽

Cited By ~ 61

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.

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Echovirus 7 Entry into Polarized Intestinal Epithelial Cells Requires Clathrin and Rab7

mBio ◽

10.1128/mbio.00304-11 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 17

Author(s):

Chonsaeng Kim ◽

Jeffrey M. Bergelson

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Intestinal Epithelial Cells ◽

Low Ph ◽

Dominant Negative ◽

Viral Rna ◽

Apical Surface ◽

Intestinal Epithelial ◽

Decay Accelerating Factor ◽

Late Endosomes

ABSTRACTEnteroviruses invade the host by crossing the intestinal mucosa, which is lined by polarized epithelium. A number of enteroviruses, including echoviruses (EV) and group B coxsackieviruses (CVB), initiate infection by attaching to decay-accelerating factor (DAF), a molecule that is highly expressed on the apical surface of polarized epithelial cells. We previously observed that entry of DAF-binding CVB3 into polarized intestinal epithelial cells occurs by an unusual endocytic mechanism that requires caveolin but does not involve clathrin or dynamin. Here we examined the entry of a DAF-binding echovirus, EV7. We found that drugs, small interfering RNAs (siRNAs), and dominant negative mutants that target factors required for clathrin-mediated endocytosis, including clathrin and dynamin, inhibited both EV7 infection and internalization of virions from the cell surface. Once virus had entered the cell, it colocalized with markers of early endosomes (EEA1) and then late endosomes (LAMP-2). Inhibition of endosomal maturation—with siRNAs or dominant negative mutants targeting Rab5 and Rab7—inhibited infection and prevented release of viral RNA into the cell. These results indicate that EV7 is internalized by clathrin-mediated endocytosis and then moves to early and late endosomes before releasing its RNA. Trafficking through endosomes is known to be important for viruses that depend on low pH or endosomal cathepsin proteases to complete the entry process. However, we found that EV7 infection required neither low pH nor cathepsins.IMPORTANCEThe results demonstrate that echovirus 7 (EV7), after binding to decay-accelerating factor (DAF) on the cell surface, enters cells by clathrin-mediated endocytosis; this entry mechanism differs markedly from that of another DAF-binding enterovirus, coxsackievirus B3 (CVB3). Thus, after attachment to the same cell surface receptor, these closely related viruses enter the same cells by different mechanisms. The cellular cues required for release of viral RNA from the enterovirus capsid (“uncoating”) remain poorly defined. We found that EV7 moved to late endosomes and that release of RNA depended on endosomal maturation; nonetheless, EV7 did not depend on the endosomal factors implicated in uncoating and entry by other viruses. The results suggest either that an unidentified endosomal factor is essential for uncoating of EV7 or that trafficking through the endosome is an essential step in a pathway that leads to another intracellular organelle where uncoating is completed.

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Adhesion of uric acid crystals to the surface of renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.6.f989 ◽

2000 ◽

Vol 278 (6) ◽

pp. F989-F998 ◽

Cited By ~ 44

Author(s):

Rima M. Koka ◽

Erick Huang ◽

John C. Lieske

Keyword(s):

Uric Acid ◽

Epithelial Cells ◽

Cell Surface ◽

Hydrophobic Interactions ◽

Calcium Phosphates ◽

Apical Cell ◽

Apical Surface ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Renal Tubular Cells

Adhesion of microcrystals that nucleate in tubular fluid to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones, 12% of which contain uric acid (UA) either alone or admixed with calcium oxalates or calcium phosphates. UA crystals bind rapidly to monolayer cultures of monkey kidney epithelial cells (BSC-1 line), used to model the surface of the nephron, in a concentration-dependent manner. The urinary glycoproteins osteopontin, nephrocalcin, and Tamm-Horsfall glycoprotein had no effect on binding of UA crystals to the cell surface, whereas other polyanions including specific glycosaminoglycans blocked UA crystal adhesion. Specific polycations also inhibited adhesion of UA crystals and appeared to exert their inhibitory effect by coating cells. However, removal of anionic cell surface molecules with neuraminidase, heparitinase I, or chondroitinase ABC each increased UA crystal binding, and sialic acid-binding lectins had no effect. These observations suggest that hydrogen bonding and hydrophobic interactions play a major role in adhesion of electrostatically neutral UA crystals to renal cells, unlike the interaction of calcium-containing crystals with negatively charged molecules on the apical cell surface via ionic forces. After adhesion to the plasma membrane, subsequent cellular events could contribute to UA crystal retention in the kidney and the development of UA or mixed calcium and UA calculi.

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Cell-density-dependent changes in cell-surface glycopeptides and in adhesion of cultured intestinal epithelial cells

Biochemical Journal ◽

10.1042/bj2010359 ◽

1982 ◽

Vol 201 (2) ◽

pp. 359-366 ◽

Cited By ~ 18

Author(s):

Wlodzimierz Sasak ◽

Annette Herscovics ◽

Andrea Quaroni

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Cell Density ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Stages Of Growth ◽

Culture Cells ◽

Significant Difference ◽

Before And After ◽

High Mannose

We studied mannose-containing glycopeptides and glycoproteins of subconfluent and confluent intestinal epithelial cells in culture. Cells were labelled with d-[2-3H]mannose for 24h and treated with Pronase or trypsin to release cell-surface components. The cell-surface and cell-residue fractions were then exhaustively digested with Pronase and the resulting glycopeptides were fractionated on Bio-Gel P-6, before and after treatment with endo-β-N-acetylglucosaminidase H to distinguish between high-mannose and complex oligosaccharides. The cell-surface glycopeptides were enriched in complex oligosaccharides as compared with residue glycopeptides, which contained predominantly high-mannose oligosaccharides. Cell-surface glycopeptides of confluent cells contained a much higher proportion of complex oligosaccharides than did glycopeptides from subconfluent cells. The ability of the cells to bind [3H]concanavalin A decreased linearly with increasing cell density up to 5 days in culture and then remained constant. When growth of the cells was completely inhibited by either retinoic acid or cortisol, no significant difference was observed in the ratio of complex to high-mannose oligosaccharides in the cell-surface glycopeptides of subconfluent cells. Only minor differences were found in total mannose-labelled glycoproteins between subconfluent and confluent cells by two-dimensional gel analysis. The adhesion of the cells to the substratum was measured at different stages of growth and cell density. Subconfluent cells displayed a relatively weak adhesion, which markedly increased with increased cell density up to 6 days in culture. It is suggested that alterations in the structure of the carbohydrates of the cell-surface glycoproteins are dependent on cell density rather than on cell growth. These changes in the glycopeptides are correlated with the changes in adhesion of the cells to the substratum.

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Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00552.2004 ◽

2005 ◽

Vol 289 (1) ◽

pp. G36-G41 ◽

Cited By ~ 64

Author(s):

Hua Xu ◽

Rongji Chen ◽

Fayez K. Ghishan

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Early Life ◽

Intestinal Epithelial Cells ◽

Protein Localization ◽

Basolateral Membrane ◽

Gene Expression Pattern ◽

Intestinal Epithelial ◽

Sodium Hydrogen ◽

Apical Localization

Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na+ absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.

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Trafficking of Shigella Lipopolysaccharide in Polarized Intestinal Epithelial Cells

The Journal of Cell Biology ◽

10.1083/jcb.145.4.689 ◽

1999 ◽

Vol 145 (4) ◽

pp. 689-698 ◽

Cited By ~ 42

Author(s):

Wandy L. Beatty ◽

Stéphane Méresse ◽

Pierre Gounon ◽

Jean Davoust ◽

Joëlle Mounier ◽

...

Keyword(s):

Epithelial Cells ◽

Tight Junctions ◽

Apical Membrane ◽

Intestinal Epithelial Cells ◽

Fluid Phase ◽

Apical Surface ◽

Intestinal Epithelial ◽

Electron Microscopic ◽

Basolateral Side ◽

Endocytic Compartments

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395–4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

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Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells

Molecular Biology of the Cell ◽

10.1091/mbc.e13-09-0530 ◽

2014 ◽

Vol 25 (20) ◽

pp. 3095-3104 ◽

Cited By ~ 24

Author(s):

Keiko Saegusa ◽

Miyuki Sato ◽

Katsuya Sato ◽

Junko Nakajima-Shimada ◽

Akihiro Harada ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Epithelial Cells ◽

Apical Membrane ◽

Intestinal Epithelial Cells ◽

Intestinal Cells ◽

Intermediate Filament Protein ◽

Apical Surface ◽

Intestinal Epithelial ◽

Microtubule Network

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.

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Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor

AJP Cell Physiology ◽

10.1152/ajpcell.00637.2005 ◽

2006 ◽

Vol 291 (6) ◽

pp. C1258-C1270 ◽

Cited By ~ 54

Author(s):

George M. Verghese ◽

Michael F. Gutknecht ◽

George H. Caughey

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Collecting Duct ◽

Critical Role ◽

Lung Epithelial Cells ◽

Peptidase Activity ◽

Apical Surface ◽

Gpi Anchor ◽

Epithelial Monolayer ◽

Lung Epithelial

Prostasin, a trypsinlike serine peptidase, is highly expressed in prostate, kidney, and lung epithelia, where it is bound to the cell surface, secreted, or both. Prostasin activates the epithelial sodium channel (ENaC) and suppresses invasion of prostate and breast cancer cells. The studies reported here establish mechanisms of membrane anchoring and secretion in kidney and lung epithelial cells and demonstrate a critical role for prostasin in regulating epithelial monolayer function. We report that endogenous mouse prostasin is glycosylphosphatidylinositol (GPI) anchored to the cell surface and is constitutively secreted from the apical surface of kidney cortical collecting duct cells. Using site-directed mutagenesis, detergent phase separation, and RNA interference approaches, we show that prostasin secretion depends on GPI anchor cleavage by endogenous GPI-specific phospholipase D1 (Gpld1). Secretion of prostasin by kidney and lung epithelial cells, in contrast to prostate epithelium, does not depend on COOH-terminal processing at conserved Arg322. Using stably transfected M-1 cells expressing wild-type, catalytically inactive, or chimeric transmembrane (not GPI)-anchored prostasins we establish that prostasin regulates transepithelial resistance, current, and paracellular permeability by GPI anchor- and protease activity-dependent mechanisms. These studies demonstrate a novel role for prostasin in regulating epithelial monolayer resistance and permeability in kidney epithelial cells and, furthermore, show specifically that prostasin is a critical regulator of transepithelial ion transport in M-1 cells. These functions depend on the GPI anchor as well as the peptidase activity of prostasin. These studies suggest that cell-specific Gpld1- or peptidase-dependent pathways for prostasin secretion may control prostasin functions in a tissue-specific manner.

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Tu1846 Flagellin Response via TLR5 on Basolateral Membrane of Primary Intestinal Epithelial Cells is Regulated by Notch Signaling

Gastroenterology ◽

10.1016/s0016-5085(12)63333-2 ◽

2012 ◽

Vol 142 (5) ◽

pp. S-859-S-860

Author(s):

Kiichiro Tsuchiya ◽

Xiu Zheng ◽

Yoshihito Kano ◽

Nobukatsu Horita ◽

Ryuichi Okamoto ◽

...

Keyword(s):

Epithelial Cells ◽

Notch Signaling ◽

Intestinal Epithelial Cells ◽

Basolateral Membrane ◽

Intestinal Epithelial

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The function of macromolecular complex of CFTR-NHERF2-LPA2 in inflammatory responses of intestinal epithelial cells

10.1101/186023 ◽

2017 ◽

Author(s):

Shanshan Kong ◽

Weiqiang Zhang

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Cell Line ◽

Human Cell ◽

Inflammatory Responses ◽

Intestinal Epithelial Cells ◽

Human Cell Line ◽

Macromolecular Complex ◽

Apical Surface ◽

Intestinal Epithelial

AbstractCFTR is a cAMP-regulated chloride channel located in the apical surface of intestinal epithelial cells; where it forms a macromolecular complex with NHERF2 and LPA2. CFTR has been shown to play a role in the pathogenies of several types of secretory diarrheas. Inflammatory bowel disease (IBD) is a chronic condition of intestine characterized by severe inflammation and mucosal destruction, genetic analysis has shown that LPA contribute to IBD and patients of cystic fibrosis also display the phenotype of diarrhea. The purpose of this study is to investigate if this complex plays a role in the inflammatory responses of intestinal epithelium.We then explored the role of this complex in maintaining the integrity of tight junction and inflammatory responses in these cells. In vitro assays show that inhibiting CFTR or LPA2 in the intestinal epithelial cell could disrupt the epithelial cell junction, and reduce the TER of intestinal epithelial cells in both mouse and human cell line. EƯSA assay show that intriguing LPA2 through LPS or LPA can increase the secretion of IL-8, while inhibiting or SiRNA knockdown of LPA2 can decrease the secretion of IL-8 in mouse or human intestinal epithelial cells. The CFTR inhibitor can reduce the IL-8 secretion in both mouse and human cell line, the deletion of CFTR in mouse intestine does not affect the IL-8 level, but the knockdown of CFTR in human cell line reduced the IL-8 protein level. The deletion of CFTR in human also reduced the IL-8 mRNA level. This indicates the CFTR-LPA complex is necessary for the expression of IL-8.

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