A polarized human endometrial cell line that binds and transports polymeric IgA

Summary We have demonstrated that a human endometrial cell line, HEC-1, maintains a high transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surfaces of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.     

Microtubules are involved in the secretion of proteins at the apical cell surface of the polarized epithelial cell, Madin-Darby canine kidney

Microtubule-disrupting drugs (nocodazole, colchicine) and cytochalasin D, which inhibits the polymerization of the actin microfilaments, were used to study the role of the cytoskeleton in protein secretion in the polarized Madin-Darby canine kidney (MDCK) epithelial cells. Two proteins were analyzed. The gp 80 glycoprotein complex, which in untreated cells is sorted into the apical pathway and lysozyme, which is released randomly at both cell surfaces in transfected MDCK cells. Our results show that cytochalasin D has no influence on the transport of the gp 80 complex and lysozyme to either cell surface. However, in the presence of nocodazole or colchicine the secretion of both proteins at the apical cell surface is reduced by 50% with a concomitant increase in the basolateral release. These data suggest that microtubules are necessary for an efficient secretion of proteins at the apical cell surface of MDCK cells. In regard to the yet unresolved discrepancy concerning the involvement of microtubules in the transport of membrane proteins to the apical surface of MDCK cells, our results are consistent with the data of Rindler et al. (Rindler, M. J., Ivanov, I. E., and Sabatini, D. D. (1987) J. Cell. Biol. 104, 231-241) who observed a nonpolarized delivery of the influenza virus hemagglutinin in the presence of nocodazole or colchicine.     

Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2)

The polarized delivery of membrane proteins to the cell surface and the initial secretion of lysosomal proteins into the culture medium were studied in the polarized human intestinal adenocarcinoma cell line Caco-2 in the presence or absence of the microtubule-active drug nocodazole. The appearance of newly synthesized proteins at the plasma membrane was measured by their sensitivity to proteases added either to the apical or the basolateral surface of cells grown on nitrocellulose filters. Nocodazole was found to reduce the delivery to the cell surface of an apical membrane protein, aminopeptidase N, and to lead to its partial missorting to the basolateral surface, whereas the drug had no influence on the delivery of a basolateral 120-kD membrane protein defined by a monoclonal antibody. Furthermore, nocodazole selectively blocked the apical secretion of two lysosomal proteins, cathepsin D and acid alpha-glucosidase, whereas the drug had no influence on their basolateral secretion. These results suggest that in Caco-2 cells an intact microtubular network is important for the transport of newly synthesized proteins to the apical cell surface.     

The influence of pH on the vesicular traffic to the surface of the polarized epithelial cell, MDCK

The effect of pH on the secretion of the gp 80 glycoprotein complex and lysozyme from MDCK cells was examined by treatment of the cells with either NH4Cl, chloroquine or monensin. In untreated cells gp 80 is sorted with approximately 75% efficiency into the apical pathway. Lysozyme is secreted in a nonpolar fashion at both cell surfaces. Treatment of the cells with the drugs had nearly identical effects on the transport kinetics and on the ratio of the proteins released at the two plasma membrane domains. At increasing drug concentrations, the transport of both proteins to the apical and the basolateral cell surface was equally retarded. Furthermore, we observed a dose-dependent decrease in the amount of gp 80 and lysozyme released at the basolateral cell surface, which was accompanied by a nearly equivalent increase in the secretion of the two proteins at the apical plasma membrane domain. A twofold rise in the apical to basolateral ratio was already found at drug concentrations which only marginally affected the kinetics of transport. These results show that an increase in intravesicular pH not only redirects secretory proteins sorted into the basolateral pathway (Caplan et al. Nature, 329, 632 (1987] but also secretory proteins devoid of sorting information for that pathway, presumably by modulating the vesicular traffic to the basolateral cell surface.     

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.     

1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside blocks the apical biosynthetic pathway in polarized HT-29 cells

In previous work we reported that long term treatment of polarized HT-29 cells by 1-benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside (GalNAcalpha-O-bn) induced undersialylation and intracellular distribution of apical glycoproteins such as dipeptidyl peptidase IV (DPP-IV), and we suggested therefore that sialylation could act as an apical targeting signal. In this work, the apical direct biosynthetic route was studied after transfection of polarized enterocyte-like HT-29 5M12 cloned cells with a murine cDNA coding for a soluble form of DPP-IV, which was secreted into the apical medium. A 24-h treatment of transfected cells by GalNAcalpha-O-bn markedly inhibited the apical secretion and the sialylation of this soluble murine DPP-IV, which became blocked inside the cell. A similar short GalNAcalpha-O-bn treatment also induced an intracellular distribution of both endogenous transmembrane DPP-IV and proteins involved in the regulation of the apical trafficking such as the apical t-SNARE syntaxin-3 and the raft-associated protein annexin XIIIb, whereas the basolateral t-SNARE syntaxin-4 kept its normal localization. These apical membrane proteins moved efficiently from trans-Golgi network to apical carrier vesicles but failed to be transported from carrier vesicles to the apical plasma membrane. Isolation of membrane microdomains showed that GalNAcalpha-O-bn induced the formation of abnormal lipid-rich microdomains in comparison to normal rafts, as shown by their lower buoyant density and their depletion in annexin XIIIb. In conclusion, GalNAcalpha-O-bn blocks the anterograde traffic to the apical surface of polarized HT-29 cells at the transport level or docking/fusion level of carrier vesicles.     

Acidification slows the transport but does not influence the polarity of secretion of gp80 in the polarized epithelial cell MDCK.

Madin-Darby canine kidney (MDCK) cells stably transfected with a recombinant chicken lysozyme gene were used to examine the effect of cytoplasmic acidification on the secretion of the endogenous gp80 glycoprotein complex and lysozyme. The effect was examined by pulse-chase and immunofluorescence analysis. If filter-grown monolayers were incubated at pH 5.8 the secretion of gp80 was retarded, and the transport of lysozyme was completely inhibited. The immunofluorescence analysis revealed an enrichment of both proteins in the endoplasmic reticulum. The transport block was reversible for both proteins upon transferring the filter-grown monolayer back into medium at pH 7.4.     

Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells

Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells.     

Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2)

We studied the postsynthetic sorting of endogenous plasma membrane proteins in a polarized epithelial cell line, Caco-2. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the arrival of three apical and one basolateral protein at the apical and basolateral cell surface. Apical proteins were inserted simultaneously into both membrane domains. The fraction targeted to the basolateral domain was different for the three apical proteins and was subsequently sorted to the apical domain by transcytosis at different rates. In contrast, a basolateral protein was found in the basolateral membrane only. Thus, sorting of plasma membrane proteins occurred from two sites: the Golgi apparatus and the basolateral membrane. These data explain apparently conflicting results of earlier studies.     

Development of the Na(+)-dependent hexose carrier in LLC-PK1 cells is dependent on microtubules

The Na(+)-dependent hexose carrier, an endogenous apical marker, develops during differentiation of LLC-PK1, an established cell line with characteristics of the proximal tubule. This development was inhibited by the microtubule-disrupting drugs, colchicine and nocodazole, while it was insensitive to lumicolchicine. This strongly suggests that microtubules are involved in the plasma membrane expression of the Na(+)-dependent hexose carrier. We also analyzed the increase in activity of endogenous apical and basolateral membrane proteins during the polarization process. The development of three apical (Na(+)-dependent hexose carrier, gamma-glutamyltransferase and alkaline phosphatase) and one basolateral membrane protein (Na+/K(+)-ATPase) was studied during the reorganization of LLC-PK1 cells into a polarized epithelium. Colchicine inhibited the rapid, transient increase in the expression of the Na(+)-dependent hexose carrier during this polarization process. A similar result was observed for the development of the other apical proteins, while the development of Na+/K(+)-ATPase seemed to be largely insensitive to colchicine. Our results are in agreement with the model that the vesicles containing the apical membrane proteins use microtubules as tracks to reach the plasma membrane. The transport of vesicles containing basolateral membrane proteins clearly occurs by a different pathway which is independent on an intact microtubular network. Since the inhibition by the microtubule-disrupting drugs was complete, it can be concluded that after disruption of microtubules, the apical vesicles do not use the basolateral pathway by default.     

Lipopolysaccharide-binding protein is vectorially secreted and transported by cultured intestinal epithelial cells and is present in the intestinal mucus of mice

Lipopolysaccharide-binding protein (LBP) is an important modulator of the host's response to endotoxin. In a previous study, we found evidence for the synthesis of LBP by intestinal epithelial cells. In this study, we explored the polarity of LBP secretion by these cells. Polarized monolayers of Caco-2 cells were used as intestinal mucosa model. Cells were stimulated apically or basally with cytokines, and LBP secretion was analyzed. Furthermore, the presence of LBP in intestinal mucus of healthy and endotoxemic mice was studied using a mucus-sampling technique. The constitutive unipolar LBP secretion from the apical cell surface was markedly enhanced when cells were exposed to cytokines at their apical surface. However, bioactive LBP was secreted from both cell surfaces after basolateral stimulation of cells. Cytokines also influenced the secretion of the acute phase proteins serum amyloid A, apoA-I, and apoB from both surfaces of Caco-2 cells. Furthermore, transport of exogenous LBP from the basolateral to the apical cell surface was demonstrated. In line with these in vitro data, the presence of LBP in intestinal mucus was strongly enhanced in mice after a challenge with endotoxin. The results indicate that LBP is present at the mucosal surface of the intestine, a phenomenon for which secretion and transport of LBP by intestinal epithelial cells may be responsible.     

A new stable epithelial cell line (RK-L) from normal rat kidney

Summary A stable epithelial cell line has been established from the kidneys of a normal Sprague-Dawley rat. This line, termed RK-L, has a high proliferative capacity (minimal doubling time 12.3 h) and can be grown in medium containing 1% fetal bovine serum. Thus far, the line has been carried through more than 60 serial passages. The RK-L cells were found to display similarities with kidney tubule cells. Using light microscopy, confluent cultures were seen as pavement-like monolayers forming domes, which are thought to result from transepithelial fluid transport. Electron microscopy revealed polarized cells that had microvilli on the apical surface, junction complexes in the apical part of the lateral cell membrane, and a basal lamina-like layer. Pinocytotic activity was indicated by infoldings of the apical plasma membrane and the formation of vesicles. The RK-L line should prove useful for investigations of kidney tubule transport mechanisms.     

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.     

The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin-Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.     

The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.     

Polarized secretion of an ectopic protein in Drosophila salivary glands in vivo.

Epithelial cells can secrete specific proteins in a polarized manner, either from the apical or basolateral surface. Intracellular protein sorting which results in polarized secretion has previously been studied using epithelial tissue culture cells. We describe here the use of Drosophila larval salivary glands for the study of polarized secretion by epithelia in vivo, and address whether an ectopically synthesized secretory protein can be sorted and targeted to the correct cell surface for secretion. Larval cuticle proteins (LCPs) and salivary gland secretion (Sgs) proteins of Drosophila melanogaster are apically secreted proteins that are produced respectively by the epidermis and salivary glands. We have transformed Drosophila with a hybrid gene consisting of the sgs-4 promoter sequence and the coding sequence for a variant (LCP-f2) of LCP-2. We have found that transgenic late third instar larvae produce LCP-f2 only in the salivary glands and that LCP-f2 is properly secreted in vivo in a polarized manner from only the apical surface of the cells into the gland lumen. The results indicate that apical secretion does not depend on a tissue-specific targeting signal contained within the protein.     

Zinc transport and metallothionein secretion in the intestinal human cell line Caco-2

Caco-2, a human cell line, displays several biochemical and morphological characteristics of differentiated enterocytes. Among these is the ability to transport zinc from the apical to the basal compartment. This process was enhanced following exposure by the apical compartment to increasing concentrations of the metal. High pressure liquid chromatography fractionation of the media obtained from cells labeled with radioactive zinc showed that metallothioneins (MTs), small metal-binding, cysteine-rich proteins), were present in the apical and basal media of controls as well as in cells grown in the presence of high concentrations of zinc. Following exposure to the metal, the levels of Zn-MTs in the apical medium increased, while in the basal compartment the greatest part of zinc appeared in a free form with minor changes in the levels of basal MTs. Metabolic labeling experiments with radioactive cysteine confirmed the apical secretion of MTs. A stable transfectant clone of Caco-2 cells (CL11) was selected for its ability to express constitutively high levels of the mouse metallothionein I protein. This cell line showed an enhanced transport of the metal following exposure to high concentrations of zinc and a constitutive secretion of the mouse metallothionein I protein in the apical compartment. Together, these findings strongly support the hypothesis of a functional role between the biosynthesis and secretion of MTs and the transport of zinc in intestinal cells.     

Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes

Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells.     

Basolateral sorting signals differ in their ability to redirect apical proteins to the basolateral cell surface

Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting.     

Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells

MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114- kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.