Spatiotemporal expression of catenins, ZO-1, and occludin during early polarization of hepatic WIF-B9 cells

C. Decaens and D. Cassio

Institut National de la Santé et de la Recherche Médicale U442, Signalisation Cellulaire et Calcium, Université Paris-Sud, Centre Universitaire, 91405 Orsay Cedex, France


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WIF-B9 is a suitable model for in vitro studies of hepatocyte polarity. To better understand polarity establishment, we have localized key proteins of the adhesion system, cytoskeleton, and tight junctions soon after plating, when most cells are isolated or in doublets. In isolated attached cells, only cytoskeletal proteins (tubulin, cytokeratins) displayed a precise localization. As soon as two cells formed a doublet, E-cadherin, alpha -, beta -, and gamma -catenins, and p120 protein were present at the doublet contiguous membrane. Actin, ezrin, and zonula occludens-1 (ZO-1) colocalized at this membrane, but not in all doublets: ezrin was present only at contiguous membrane expressing ZO-1, and ZO-1 was present only at membrane expressing actin. In contrast, occludin was spread throughout the doublet cytoplasm. With time in culture, these proteins localized transiently, as in cells expressing simple epithelial polarity, and finally, as in hepatocytes. We conclude that during WIF-B9 early polarization, key proteins are settled according to a hierarchy, as has been shown for Madin-Darby canine kidney cells. Cytoplasmic complexes of E-cadherin-catenin were detected during the whole polarization process; they were more abundant in fully polarized cells.

hepatoma hybrid WIF-B9; polarity establishment; cell-cell adhesion; cytoskeleton; tight junctions; zonula occludens-1


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CELL POLARITY is at the end of a multistage process that establishes and maintains functionally specialized domains in the plasma membrane (PM) and the cytoplasm (17). The pathway for polarity development in epithelial cells starts with cell adhesion to the matrix and to the contiguous cells. Cell-cell adhesion is mediated by homotypic Ca2+-dependent binding between E-cadherin molecules (44, 58, 65); this type of interaction is at the top of a molecular cascade of protein interactions leading to the development of epithelial cell structure and function (45). Intracellular reorganization involves interactions of E-cadherin with the cortical cytoskeleton components (32, 49), mediated by catenins (22, 51). Ozawa and Kemler (52) have shown that beta -catenin first associates with E-cadherin, while alpha - and gamma -catenin join the complex afterward. alpha -Catenin, by direct interaction with F-actin (57), links the E-cadherin-catenin complex to the actin cytoskeleton and thereby increases cell adhesion (3). Previous studies have indicated that the cadherin-mediated adhesion is a prerequisite for the formation of all intercellular junctions in Madin-Darby canine kidney cells (MDCK): tight junction (TJ), zonula adherens, and desmosomes (25). Recent studies demonstrate that alpha -catenin, by direct interaction with vinculin, plays a role in the assembly of the apical junctional complex in epithelia (67). After the formation of intercellular junctions, structurally and functionally distinct PM domains are settled, leading to the development of cellular polarity.

Moreover, dynamic complexes of E-cadherin and catenins have been described (27, 60); the steps of assembly and the exchange of proteins in these complexes are potential targets for regulation. Indeed, recent studies have indicated that catenins are at the convergence of several transduction pathways (1, 6); accumulating evidence suggests that junctional integrity is regulated by tyrosine phosphorylation, with catenins being target molecules (7), as well as the p120 protein (p120), a substrate of the Src tyrosine kinase and classified as a new catenin (56). For example, beta -catenin mutations and changes in level are implicated in colonic tumorigenesis (35, 47) via regulation by the adenomatous polyposis coli (APC) protein, the product of a tumor suppressor gene (29, 64). These changes in the pool size of beta -catenin may affect the APC-microtubule interactions (62). beta -Catenin is also a key effector in the pathway of Wnt/Wingless (26), one of the major families of developmentally signaling molecules in mouse and Drosophila (13). By moving to the nucleus, beta -catenin can regulate gene expression by direct interaction with the transcription factor LEF-1 (7). Thus catenins are key molecules connecting cell-cell adhesion and signaling pathways with the cytoskeleton; consequently, the interplay between cytoskeletal components and signaling pathways may regulate morphogenesis (6).

Established cell lines such as renal MDCK (38) or intestinal HT29 and Caco-2 (53) have been widely used to study cell polarity. However, no polarized line derived from hepatocytes was available until we isolated and characterized WIF 12-1 cells (14) and their improved derivatives, WIF-B and WIF-B9 (16, 59). These rat hepatoma-human fibroblast hybrid cells are differentiated and polarized (30); in particular, they form bile canalicular-like structures (BC) that are equivalent to the apical pole of a hepatocyte. This polarity is not only structural but also functional, because efficient vectorial transport of a bile acid analog was demonstrated (12).

We have previously shown that the establishment of WIF-B9 polarity is a biphasic phenomenon going from a simple epithelial polarized phenotype to a hepatic polarized one, with the expression of these two phenotypes being mutually exclusive (16). Here we have studied how the cell surface domains, previously evidenced in WIF-B9 cells, are generated. Because the generation and regulation of these domains in polarized epithelial cells were shown to be mediated by the adhesion system components, the cytoskeleton, and the TJ proteins, we have analyzed WIF-B9 early polarity establishment according to the expression of these key components. Three questions were addressed. Are the proteins considered to be key proteins in polarity establishment for MDCK cells also present in WIF-B9 cells? What is their localization, and do they exhibit a hierarchy in their settlement at the very beginning of polarity establishment? Are soluble cytoplasmic complexes of E-cadherin and catenins present, and does their amount vary when cells polarize? To answer these questions, we have 1) studied the expression and localization of components of the adhesion system (alpha -, beta -, and gamma -catenins and p120) in relation to E-cadherin; 2) performed experiments to detect complexes of E-cadherin with each of the four catenins; and 3) analyzed in parallel the localization of cytoskeletal components (actin, actin-associated ezrin, alpha -tubulin, and cytokeratins) and the TJ-associated complex (occludin and ZO-1). The study was done on WIF-B9 cells at 24 h of culture by using a plating cell density such that most cells were isolated and in doublets. The results obtained at that time were compared with those found after several days in culture, when WIF-B9 cells transiently expressed a simple polarized phenotype, and later, when cells expressed the typical hepatic polarity. We have shown here that, at the very beginning of polarity establishment, there is a hierarchy in the settlement of the key proteins examined. Those of the adhesion system are localized very early, whereas those of the TJ, in particular, occludin, are the last to settle. Cytoplasmic complexes of E-cadherin with each of the catenins and with p120 were detected all over the polarization process; they were more abundant when cells expressed a fully polarized phenotype.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Antibodies and Materials

Ascitic mouse monoclonal antibodies raised against E-cadherin, alpha -, beta -, and gamma -catenin, and p120 were purchased from Transduction Laboratories (Lexington, KY); antibodies against alpha -tubulin, alpha -catenin, pan-cytokeratin, and anti-Golgi 58K protein (p58 Golgi) were from Sigma Chemical (St. Louis, MO); cytokeratin 8/18 antibody was from Novocastra Laboratories (Newcastle-upon-Tyne, UK). Rat monoclonal antibody R40-76 raised against ZO-1 was a generous gift from B. Stevenson (University of Alberta, Edmonton, Canada). Rabbit polyclonal antibodies against DPPIV and HA321 were gifts from A. Hubbard (Johns Hopkins University, Baltimore, MD). Rabbit polyclonal antibodies against ezrin were gifts from M. Arpin and D. Louvard (Institut Curie, Paris, France). Rabbit polyclonal antiserum against occludin was a gift from M. Balda (Université de Genève, Geneva, Switzerland). Fluorescent secondary antibodies and phalloidin-rhodamine were from Sigma, Molecular Probes (Eugene, OR), and Diagnostics Pasteur (Marnes la Coquette, France). Horseradish peroxidase-labeled antibodies were from Amersham France (Les Ulis, France), and alkaline phosphatase-labeled antibodies were from Promega France. Buffered glycerin for immunofluorescence was from Diagnostics Pasteur; the SlowFade Antifade kit was from Molecular Probes; DC (detergent compatible) protein assay was from Bio-Rad France; protein A-Sepharose was from Pharmacia Biotech (Orsay, France); and Hybond ECL (enhanced chemiluminescence) nitrocellulose membranes, ECL reagent, and Hyperfilm MP were from Amersham France. CELLocate coverslips were from Eppendorf (Hamburg, Germany).

Cell Culture

Cells were grown as described previously (14) on plastic petri dishes (Falcon Plastics, Cockeysville, MD) or on glass coverslips for immunofluorescence. Cells were regarded as confluent at 1.1-1.5 × 105 cells/cm2. Cells of polarized subconfluent cultures were detached with trypsin-EDTA and plated at 2.5 × 103 or 1.5 × 104 cells/cm2. At different times, from day 1 to days 8-10, the total cell number and the number of cells engaged in BC formation were counted on phase-contrast micrographs (Leitz inverted microscope with a ×20 lens).

Cell Examination and Counting at Day 1

After trypsin-EDTA treatment, well-isolated WIF-B9 cells were plated at densities between 2.5 × 103 (low density) and 1.5 × 104 cells/cm2 (high density). To determine the best conditions from which to obtain isolated cells and cells in doublets, we used three different methods. The first method comprised counting on CELLocate coverslips (5 experiments) that permit easy relocalization of individual cells with time. Five hours after cells were plated, as soon as they were fixed on the support, four areas per CELLocate coverslip with adherent cells were mapped on two or three different coverslips, and cells were counted on phase-contrast micrographs; the same areas were analyzed 24 h later. Each area contained 7-30 cells. The second method entailed counting in culture flasks (1 experiment) of 27 areas of 5 × 5 mm, automatically scanned in Hoffman ×20 on an inverted Nikon Diaphot-300 microscope with a ×20 lens by using a computerized stage controller system (Alliance-Vision, Mirmande, France) based on LabView 4.0VI (National Instruments, Austin, TX), operating a Märzhauser scanning stage, with a resolution digital camera. The positions of adherent cells (5-14 cells/area) were mapped 4 h after they were plated and then were stored in memory to allow subsequent analysis at 24 h of culture. Images were processed with Photoshop (Adobe Systems). Finally, the third method consisted of videomicroscopy experiments in which observation and counting occurred in culture flasks maintained at 37°C in a heated Plexiglas chamber and was recorded during the 24 h following plating on a phase-contrast micrograph with a Zeiss inverted microscope equipped with a video camera connected to a television monitor and a time-lapse recorder, with a ×10 lens.

At the low plating density, the distribution of the 1,000 counted cells was as follows: 44% were isolated, 41% were in doublets, and 15% were in small islets (3-6 cells). At the high plating density, the distribution of the 600 counted cells was as follows: 23% were isolated, 24% were in doublets, and 53% were in small islets (3-10 cells). As determined in two videomicroscopy experiments, cell doublets were the result of either mitosis (30% of isolated cells underwent mitosis in 24 h) or a union between two isolated cells. These cell unions occurred more frequently at the higher plating density, as can be inferred from the higher percentage of cells in small islets.

Immunofluorescence

Cells were fixed with 3% paraformaldehyde-phosphate-buffered saline (PBS) and permeabilized in cold methanol or in acetone (16). Cells were then incubated in primary antibody(ies) (1:100-500 in PBS, undiluted culture supernatant for monoclonal antibodies against ZO-1) or in TRITC-conjugated phalloidin (100 ng/coverslip) for 1 h at room temperature. After PBS rinses, cells were incubated for 30 min at room temperature with fluorescent secondary antibodies (1:100-500). In double-labeling experiments, the cells were incubated with both primary and then secondary antibodies at the same time. Cell nuclei were then stained with Hoechst 33258 and counted in parallel to cell counting in phase contrast. After being mounted in buffered glycerin or the SlowFade Antifade kit, cells were viewed on a Zeiss Axioskop fluorescence microscope. Confocal images were collected on a Bio-Rad MRC 600 confocal microscope with a ×63 objective in z series at 0.5- to 1-µm increments. Optical sections were collected by Kalman averaging of eight images. The images were processed with the use of SOM and CoMOS software (Service d'Imagerie, Orsay, France).

Cell Extraction and Immunoprecipitation

In three different experiments, WIF-B9 cells were isolated by trypsinization and plated at 1.3 × 104 cells/cm2 on 10-cm petri dishes and cultured. Cell extraction was done at days 1, 4, and 8 after cells were plated by using six to eight, three, and one petri dish, respectively. After three PBS washes, cells were extracted by scraping on ice with 500 µl per petri dish of immunoprecipitation buffer [1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.5% Nonidet P-40 in 20 mM Tris · HCl (pH 7.4) with a cocktail of protease inhibitors]. The crude extracts were centrifuged, and the supernatants (soluble fractions) and pellets (insoluble fractions) were separated. Protein amount was estimated in the soluble fractions by DC protein assay.

For immunoprecipitation, 300- to 400-µg proteins of soluble fractions were rotated for 4 h at 4°C with 5 µg of anti-E-cadherin in 1 ml of immunoprecipitation buffer, followed by 1 h of incubation with 40 µl of a 50% protein A-Sepharose solution; after four washes, immunoprecipitates (IP) were eluted from protein A beads by being boiled for 7 min in 2× SDS-electrophoresis buffer with 10% beta -mercaptoethanol.

Western Blotting

Cell lysates (soluble and insoluble parts), after being boiled for 8 min in 5% beta -mercaptoethanol-SDS sample buffer, and E-cadherin-IP were separated by SDS-PAGE on 4-15% polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes. After being blocked in 0.2% Tween 20 in PBS containing 10% low-fat dried milk, the membranes were incubated with primary antibodies for 3 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h, and were subsequently developed with the ECL reagent and exposed to Hyperfilm MP. For colorimetric detection, nitrocellulose membranes were blocked with 2% bovine serum albumin in 0.1% Triton X-100 in PBS and incubated with primary antibodies for 12 h at 4°C and then with alkaline phosphatase-conjugated secondary antibodies for 1 h; nitroblue tetrazolium/bromochloroindolyl phosphate substrate was used for detection. Densitometric analysis of protein band intensities was done on the scanned ECL hyperfilms or nitrocellulose sheets by using the NIH Image program.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Key Proteins Involved in Cell Polarity Exhibit a Hierarchy in Their Localization at Early Stages of Polarity Establishment

We wanted to know which components were present as soon as cells were fixed on the support and as soon as two cells associated. We have determined the conditions in which most cells were either single or in doublets at the very beginning of the culture. Comparing the low and high plating densities, using different methods (see MATERIALS AND METHODS for details), we observed that, at day 1, 85% of cells at the low density vs. 50% of those at the high density were either isolated or in doublets.

According to these observations, we decided to analyze the expression and localization of E-cadherin, catenins, and cytoskeletal and TJ-associated proteins at day 1 after cells were plated, at the low density, when 44% cells were isolated, 41% were in doublets, and 15% were in small cell islets. The results are summarized in Table 1.

                              
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Table 1.   Localization at day 1 

Isolated cells. In isolated cells, alpha -tubulin (Fig. 1A) and cytokeratins (Fig. 1B) formed cytoplasmic networks. p58 Golgi was located near the nucleus (Fig. 1C). The other proteins were spread in the cytoplasm as shown for p120 (Fig. 1D), except for ezrin, which also was located in a cap at the cell apex (Fig. 1E); when present, these caps might be surrounded by apical ZO-1 and alpha -catenin.


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Fig. 1.   Isolated WIF-B9 cells at day 1 after cells were plated: immunolocalization of alpha -tubulin, cytokeratin, p58 Golgi, p120, and ezrin. alpha -Tubulin (A) and cytokeratins (B) form cytoplasmic networks. p58 Golgi protein (C) is located near the nucleus. p120 (D) is spread in the cytoplasm. Ezrin, spread in the cytoplasm, is also located in a cap at the cell apex (E). A'-E' are phase-contrast micrographs corresponding to A-E; in D', the nucleus is stained with Hoechst. Image in E is at the cell apex level, and that in E' is at the middle cell level. Bar, 24 µm.

Doublets. In doublets, alpha -tubulin and cytokeratin formed cytoplasmic networks as in isolated cells, and p58 Golgi was located near the nucleus. E-cadherin, alpha -, beta -, and gamma -catenins and p120 were always localized at the contiguous membranes between the two cells of the doublet (Fig. 2, A-E). Actin, ZO-1, and ezrin were present in the cytoplasm but also were located at the contiguous membranes in a fraction of the doublets: 90% of doublet contiguous membranes for actin (Fig. 2F), 9% for ezrin (Fig. 2G), and 63% for ZO-1 (Fig. 2H). Ezrin was also sometimes present in a cap at the cell apex (Fig. 2I'), surrounded by apical ZO-1 (Fig. 2I), and in focal contacts at the cell periphery (Fig. 2G). Occludin was spread in the cytoplasm (Fig. 2J). Using double-labeling experiments, we have established that ezrin was present only at contiguous membranes expressing ZO-1 and that ZO-1 was present at contiguous membranes expressing actin, thereby showing an order in expression (see Fig. 2, middle).


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Fig. 2.   WIF-B9 doublets (top) and small cell islets (bottom) at day 1 after cells were plated. Top: immunolocalization and expression diagram of adhesion system, cytoskeletal, and tight junction (TJ)-associated proteins in doublet cells. E-cadherin (A), alpha -catenin (B), beta -catenin (C), gamma -catenin (D), and p120 (E) are always concentrated at the contiguous membranes between the 2 cells in doublets; they are also present in the cytoplasm. Actin (F), ezrin (G), and zonula occludens-1 (ZO-1) (H) also can be observed at the contiguous membranes between 2 cells. Ezrin also is present at focal contacts at the cell periphery (G, arrows) and sometimes locates in caps at the cell apex (I') surrounded by ZO-1 (I). Occludin (J) shows only cytoplasmic localization. A'-H' and J' are phase-contrast micrographs corresponding to A-H and J; in B' and E', nuclei are stained with Hoechst. The confocal image I-I' is a double localization and corresponds to a partial compiled z series taken in 1-µm steps between 6 and 8 µm from the bottom of the cells, i.e., at the cell apex level. Bar, 24 µm. Middle: diagram showing order of expression of proteins as determined by double-labeling experiments. Bottom: immunolocalization of TJ-associated proteins in small cell islets: ZO-1 forms apical networks (L), often appearing as dotted lines (K), while occludin is still expressed in the cytoplasm (L'). Image in L is at the cell apex level, and that in L' is at the middle cell level. K' and L'' are corresponding phase-contrast micrographs.

Surprisingly, some doublets had a BC; these doublets accounted for 14% of total doublets. Doublets with BC could be obtained by mitosis of an isolated cell, as was observed by videomicroscopy. We investigated to see whether these cells have the same features as cells with the hepatic phenotype. According to the localization of DPPIV and HA321, apical and lateral pole-specific protein markers of hepatocytes, respectively (results not shown), the BC membrane of these doublets corresponded to the apical pole and the contiguous membranes corresponded to the lateral pole. We observed (Table 1 and Fig. 3) that the cytoskeletal components alpha -tubulin (Fig. 3A) and cytokeratin (Fig. 3B) formed cytoplasmic networks, reinforced near this BC membrane. The p58 Golgi was most often located between the nucleus and the BC (Fig. 3C). The cell-cell adhesion proteins E-cadherin, alpha -catenin, and beta -catenin (Fig. 3D) and gamma -catenin and p120 (Fig. 3E) were always expressed at the contiguous membranes, excluded from the BC, and were sometimes present at the BC-lateral frontier (Fig. 3E). Actin was expressed at the BC membrane (Fig. 3F) and at the BC-lateral frontier, where it colocalized with ZO-1 (Fig. 3F''), and sometimes at the lateral membranes. Occludin (Fig. 3G) colocalized with ZO-1 (Fig. 3G'') at the BC-lateral frontier and was also cytoplasmic. Ezrin was expressed at the BC membrane (Fig. 3H).


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Fig. 3.   WIF-B9 doublet cells with bile canalicular-like structures (BC) at day 1 after cells were plated: immunolocalization of adhesion system, cytoskeletal, and TJ-associated proteins. alpha -Tubulin (A) and cytokeratins (B) make cytoplasmic networks reinforced at the BC. p58 Golgi protein (C) is usually located between the nucleus and the BC. The various catenins are expressed at the lateral pole, as shown for beta -catenin (D) and p120 (E), and excluded from the BC membrane (D), but they are sometimes present at the BC-lateral frontier (E). Actin (F) is strongly expressed at the BC membrane where ezrin (H) is present, too. Actin colocalizes with ZO-1 (F'') at the BC-lateral frontier. ZO-1 (G'') and occludin (G) make a belt in these BC at the apicolateral frontier. Confocal images A-C are single optical x-y sections taken at 6 µm from the bottom of the cells, i.e., at the BC level. D'-H' are phase-contrast micrographs corresponding to D-H. In E', nuclei are stained with Hoechst. Arrows point to the BC. Bar, 24 µm.

Small cell islets. In small cell islets, the adhesion system proteins and actin were always located at the contiguous membranes (not shown). ZO-1 was expressed at apical networks (Fig. 2L), often as dotted lines (Fig. 2K). Occludin was still mostly spread in the cytoplasm (Fig. 2L') and rarely colocalized with ZO-1 at the apical networks.

In summary, 1) as soon as WIF-B9 cells were fixed on the support, some cytoskeletal organization was observed, with the adhesion and TJ-associated proteins still spread in the cytoplasm; and 2) as soon as two cells associated, the cell-cell adhesion proteins were localized at the contiguous membranes between them. Actin, ZO-1, and ezrin located later at the contiguous membranes according to a hierarchy. Occludin, never located at these contiguous membranes, was the last to settle. The diagram in Fig. 2 summarizes these results. 3) In doublets forming BC, proteins of the cytoskeleton, of the adhesion system, and associated with the TJ are localized according to a hepatic phenotype. 4) In small cell islets, the localization of these proteins indicates that the simple epithelial phenotype had not yet been achieved, because ZO-1 apical networks were sometimes absent or discontinuous in some cells of the islets, even if these islets contained 10-20 cells, and because occludin was still cytoplasmic.

With Time in Culture, Key Proteins in Cell Polarity Localize Transiently in a Simple Epithelial Cell-Like Pattern and Finally in a Hepatocyte-Like Pattern

Previously, we have defined apical, lateral, and basal poles in WIF-B9 cells, according to the localization of membrane domain protein markers, by immunofluorescence studies and by analysis of freeze-fracture replicas of TJ. We also have observed that WIF-B9 cells express a simple epithelial phenotype before they express the final hepatic phenotype (16, 30). This study was enlarged to determine the localization of the adhesion system proteins, in particular, that of catenins, and of an intrinsic protein of the TJ, occludin.

WIF-B9 cells with simple epithelial phenotype. A few days after they were plated, WIF-B9 cells formed islets wherein 70-80% of the cells displayed a simple epithelial phenotype with the apical pole at the cell apex, that is, at the zone opposite to the substratum attachment and the lateral pole at the contiguous membranes between cells. The localization of cell-cell adhesion proteins and of occludin in these cells, determined with the use of specific antibodies, is summarized in Table 2 and shown in Fig. 4. All the catenins, alpha -, beta -, and gamma -catenin (Fig. 4A) and p120 (Fig. 4B), were always expressed at the contiguous membranes between cells, that is, at the lateral pole; E-cadherin was previously shown to be located there (16), while sometimes also being present at the apicolateral boundary. The TJ protein occludin (Fig. 4C'') was concentrated as honeycomb networks at the cell apex, colocalizing with ZO-1 (Fig. 4C) at the apicolateral frontier; occludin was also present in the cytoplasm. The actin-associated protein ezrin was associated to the apical pole and to the apicolateral boundary (Fig. 4D). These localizations are very similar to those described for MDCK cells, typical of the simple epithelial phenotype (42).

                              
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Table 2.   Protein localization according to expressed phenotype



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Fig. 4.   WIF-B9 cells expressing the simple epithelial phenotype: immunolocalization of adhesion system and TJ-associated proteins. The adhesion system proteins are expressed at the lateral membrane as shown for gamma -catenin (A) and p120 (B). ZO-1 (C) and occludin (C'') colocalize at the apicolateral frontier; occludin is also present in the cytoplasm. Ezrin (D) is present at the apical pole and at the apicolateral frontier. The confocal image in D is a compiled z series taken in 0.5-µm steps between 5 and 11 µm from the bottom of the cells, i.e., at the apex and upper lateral pole. Images in C and C'' are at the cell apex level. A'-C' are phase-contrast micrographs corresponding to A-C. Bar, 24 µm.

WIF-B9 cells with hepatic phenotype. From day 8 after they were plated, 60-99% (according to plating density) of WIF-B9 cells expressed a hepatic phenotype with the formation of BC structures between two and sometimes three cells, with the apical pole at the BC membrane and the lateral pole at the contiguous membranes between cells. The localization of the adhesion system catenins and of the TJ protein occludin is summarized in Table 2 and shown in Fig. 5. The alpha - and beta -catenins (Fig. 5A) and gamma -catenin and p120 were always at the lateral pole and excluded from the BC membrane, like E-cadherin; alpha - and beta -catenin were also present at the apicolateral boundary, although to a lesser extent than E-cadherin (70% of cells). Occludin (Fig. 5D) was concentrated at the apicolateral frontier, making a belt in the BC, where it colocalized (Fig. 5E') with ZO-1 (Fig. 5E); occludin was also present in the cytoplasm.


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Fig. 5.   WIF-B9 cells expressing the hepatic phenotype: immunolocalization of cytoskeletal, adhesion system, and TJ-associated proteins. The adhesion system proteins are expressed at the lateral pole and excluded from the BC membrane as shown for beta -catenin (A). Ezrin (B) is at the BC membrane. Cytokeratin (C) makes cytoplasmic networks reinforced at the BC. Occludin (D) is expressed at the BC-lateral frontier, making belts at the BC where it colocalizes (E') with ZO-1 (E); the E-E' micrographs show examples of BC formed by 3 cells. A', B', and D' are phase-contrast micrographs corresponding to A, B, and D. The confocal image in C is a compiled z series taken in 1-µm steps between 5 and 7 µm from the bottom of the cells, i.e., at the BC level. Arrows point to the BC. Bar, 24 µm.

Ezrin was localized at the apical pole, associated to the BC membrane (Fig. 5B); although ezrin is not expressed by hepatocytes in vivo, many cells in culture express this apical marker (4). The localization of cytokeratins revealed strong cytoplasmic networks of intermediate filaments, reinforced near the apical pole, namely, at the BC (Fig. 5C). Expressed cytokeratins were cytokeratins 8 and 18, typical of the hepatocyte (results of Western blotting experiments, not shown). These localizations are very similar to those described for hepatocytes (40).

Thus the adhesion system proteins E-cadherin, alpha -, beta -, and gamma -catenins, and p120, as well as the TJ-associated proteins occludin and ZO-1, were all present in WIF-B9 cells and localized according to the polarized phenotype that they expressed.

Analysis of the Amount and Distribution of E-Cadherin and Catenins During WIF-B9 Polarity Establishment

We were interested to know the cytoplasmic distribution of the adhesion system proteins according to the polarized phenotype that is expressed by WIF-B9 cells. Hence, we studied their relative distribution at times corresponding to the best expression of the different phenotypes: 1) at day 1 after plating, when most cells were isolated or in doublets; 2) at day 4, when 75% of the cells expressed a simple epithelial phenotype; and 3) at days 8-10, when more than 75% of the cells expressed a hepatic phenotype.

After cell extraction in 1% Triton X-100 buffer, the soluble and insoluble fractions were studied by Western blotting with antibodies against E-cadherin, the various catenins, and actin (Fig. 6A). To compare these fractions, we analyzed the intensities of the bands (5 times more protein was loaded for the insoluble fractions). On the basis of the results of several Western blotting experiments, we found that the total amount of E-cadherin (Triton X-100 soluble + insoluble) was very similar at days 1, 4, and 8 of culture; it was the same for the amounts of the three catenins, p120, and actin. The major part of these proteins was present in the Triton X-100-soluble fraction whatever the time in culture: 94 ± 3% for E-cadherin, 91 ± 5% for alpha -catenin, 89 ± 3% for beta -catenin, 78 ± 3% for gamma -catenin, 90 ± 3% for p120, and 77 ± 6.5% for actin. Moreover, no significant difference in the insoluble vs. soluble ratio was found whatever the time in culture; for example, the insoluble beta -catenin accounted for 11% of total beta -catenin at day 1, 13% at day 4, and 11% at day 8.


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Fig. 6.   A: amounts and distribution of adhesion system proteins and actin according to the phenotype expressed by WIF-B9 cells. The image is an example of several Western blotting experiments. Each lane was loaded with a cell lysate aliquot corresponding to 30 µg of protein for the soluble fractions (3-4 × 104 cells) and to 5 times more in volume for the corresponding insoluble fractions. After SDS-PAGE and detection of the various proteins with specific corresponding antibodies was completed, the relative amount of proteins was estimated by comparing the intensities of the bands with the use of NIH Image after scanning. Results show that the total amount of each protein is very similar at days 1, 4, and 8 (D1, D4, and D8, respectively). Moreover, the majority of each protein is present in the Triton X-100-soluble (sol) fraction (soluble fraction/total fraction = 77-94% according to the considered protein) whatever the day in culture, that is, whatever the expressed phenotype. The molecular mass of each protein corresponds well to the theoretical value indicated at left, as determined using molecular mass markers. Two isoforms of p120 are present at 100 and 115 kDa. insol, Triton X-100-insoluble fraction. B: presence of cytoplasmic soluble E-cadherin-catenin complexes. The Triton X-100-soluble fractions (300 µg of total protein) were immunoprecipitated with anti E-cadherin. Immunoprecipitates (IP) were then examined for the presence of catenins-p120 reactivity by Western blotting using specific corresponding antibodies. The whole E-cadherin IP (E-cad-IP) fractions were loaded in lanes D1, D4, and D8. Lanes 1 and 2 were loaded with 30 and 15 µg of total protein, respectively, from a Triton X-100-soluble fraction at day 8 before immunoprecipitation. The amount of E-cadherin IP are similar at days 1, 4, and 8 as shown by their reactivity with anti E-cadherin. Soluble complexes of E-cadherin-beta -catenin, E-cadherin-alpha -catenin, E-cadherin-gamma -catenin, and E-cadherin-p120 were present at each culture time. Compared with controls (lanes 1 and 2), soluble complexes of E-cadherin-alpha -catenin were present to a lesser extent than the other soluble complexes. Whatever the catenin, the amount of corresponding E-cadherin soluble complex is similar at days 4 and 8, being nearly twice the amount detected at day 1.

In summary, the total amount and the distribution in soluble-insoluble fractions of E-cadherin, all the catenins, and p120 showed little change whatever the phenotype expressed by WIF-B9 cells, with most of the proteins being Triton X-100 soluble.

Presence of Soluble E-Cadherin-Catenin Complexes During WIF-B9 Polarity Establishment

For MDCK cells, a model has been proposed in which dynamic E-cadherin-catenin complexes play an important role during initiation and maintenance of structural and functional organization (48). Spatial distribution of these complexes in the cell is correlated with the differentiation state: E-cadherin-beta -catenin and E-cadherin-gamma -catenin complexes incorporate alpha -catenin coincident with both their arrival to the PM and their link to the actin cytoskeleton as shown by their entry into the Triton X-100-insoluble fraction (39).

We have looked for the presence of such soluble complexes of E-cadherin with the various catenins in WIF-B9 cells. The Triton X-100-soluble fractions of three independent cell extracts at days 1, 4, and 8-10 of culture were immunoprecipitated with anti-E-cadherin in six separate experiments. These E-cadherin-IP were probed in several Western blotting experiments with antibodies against the various catenins and p120. As controls, two lanes were loaded with 30 and 15 µg of proteins of a soluble fraction at day 8 before immunoprecipitation. We found that the E-cadherin-IP reacted with antibodies directed against alpha -, beta -, and gamma -catenins and p120 (Fig. 6B). This indicates the presence of E-cadherin-alpha -catenin, E-cadherin-beta -catenin, E-cadherin-gamma -catenin, and E-cadherin-p120 soluble complexes in WIF-B9 cells at each time of culture. E-cadherin-alpha -catenin complexes were present in a smaller amount than the other E-cadherin-catenins complexes as shown by the lower intensity of reaction compared with that of the control before immunoprecipitation. As estimated by the intensities of the bands, a similar reactivity with anti-E-cadherin was observed in the IP at days 1, 4, and 8, which is consistent with the similar amount of E-cadherin found in the soluble fractions before immunoprecipitation. The amounts of each soluble complex were rather similar at days 4 and 8. In comparison, the amounts at day 1 were lower, corresponding to 50% of the amount at day 8 for E-cadherin-beta -catenin, 55% for E-cadherin-alpha -catenin, 65% for E-cadherin-gamma -catenin, and 45% for E-cadherin-p120. Thus E-cadherin-catenin complexes are present in WIF-B9 cells and are more abundant when cells express a fully polarized phenotype.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Development of polarity in epithelial cells requires specialized localization of proteins to distinct PM domains. Increasing evidence has been gathered concerning the important role of adhesion system and cytoskeletal components in the various processes leading to this organization (41). Using immunofluorescence and biochemical analysis, we have studied the expression and localization of proteins of the adhesion system (E-cadherin, alpha -, beta -, and gamma -catenins, and p120), proteins of the cytoskeleton (actin-associated ezrin and cytokeratins), and a protein specific to the TJ (occludin) during polarity establishment in WIF-B9 cells.

Polarization: Early Events

We wanted to know the spatial distribution of proteins as soon as WIF-B9 cells were fixed on the support and as soon as two cells associated. Therefore, we studied WIF-B9 cells at 24 h of culture after they were plated, when a majority of cells were isolated and in doublets.

Isolated WIF-B9 cells. In isolated WIF-B9 cells, alpha -tubulin and cytokeratins were organized in cytoplasmic networks and p58 Golgi was located near the nucleus. The other studied proteins were spread in the cytoplasm, showing no organization. As soon as WIF-B9 were fixed on a support, they displayed some architectural order, due in part to the structural properties of alpha -tubulin and cytokeratin. Bacallao et al. (5) made similar observations in isolated MDCK cells 12 h after they were plated: the Golgi complex and microtubules assumed an organization even when E-cadherin and ZO-1 were still spread in the cytoplasm. Ezrin was sometimes expressed in a cap at the WIF-B9 cell apex, consistent with its involvement in the organization of cell membrane structures (66) and cell shape (37).

WIF-B9 doublets. WIF-B9 doublets presented a more organized state. The striking observation was that E-cadherin, all the catenins, and p120 were always highly concentrated at the contiguous membranes between the two cells; in addition, some proteins were still present in the cytoplasm. Therefore, the chief actors of epithelial cell-cell adhesion (51, 65) colocalize at a precise membrane domain as soon as two WIF-B9 cells share contiguous membranes. Actin, ezrin, and ZO-1 were also located at these contiguous membranes, but not in all WIF-B9 doublets. This could indicate that they arrive at the membrane after E-cadherin-catenin components. The presence of actin at the submembrane plaque of cell-cell adhesion is in accordance with its role in functional adhesion by complexing with cadherin/catenins (24). Recent studies have shown the coordinate roles of E-cadherin and actin cables in initial remodeling of cell-cell contacts (2). Ezrin is also involved in the regulation of cell morphology, acting as a linker between the plasma membrane and the cytoskeleton (66). Ezrin was also shown to colocalize with actin at the apical surface of many epithelial cells and at membrane ruffles in cultured cells (10). In WIF-B9 cell doublets, ezrin colocalized with actin at the contiguous membranes. It was also expressed in a cap at the cell apex and in focal contacts at the cell periphery, where no actin was present. ZO-1 is a peripheral membrane protein enriched at the TJ of epithelial and endothelial cells (63). Linkage to a variety of proteins was shown, including colocalization with actin filaments in many cell types (28) and complex formation with catenins during TJ early state assembly in MDCK cells (55). These ZO-1-catenin complexes are localized at the adherens junctional complex before moving to the newly developed TJ (69). In WIF-B9 doublets, ZO-1 colocalized with all the above described partners responsible for the first stages in polarity establishment. Occludin, the first described protein constitutive of the TJ (20), has direct association with ZO-1, with the association with cytoskeleton through ZO-1 being required for occludin to be localized to TJ (19, 21). In WIF-B9 doublets, occludin was not associated to a particular domain of the membrane but was spread in the cytoplasm, indicating that complete TJ formation had not occurred yet.

In summary, we have shown that cell-cell or cell-substratum adhesion was sufficient to generate an initial level of asymmetry in WIF-B9 cells, which is the first critical event in the generation of epithelial polarity (68). We have evidence of a hierarchy in the localization of some of the key components during the early steps of polarity establishment: first, the adhesion proteins E-cadherin, all the catenins, and p120 localized at the site of cell-cell contact, followed by actin, then by ZO-1, and, finally, by ezrin, with occludin remaining cytoplasmic. However, these contiguous membranes could not be considered as lateral poles because the TJ were not completed; even in small cell islets at day 1, occludin was, most often, not precisely localized.

Among WIF-B9 doublets formed 24 h after cells were plated, a minority had BC. In these doublets with BC, all studied proteins had a precise localization and the PM poles were formed, with occludin and ZO-1 colocalizing in belts at the TJ level. Moreover, cytoplasmic networks of alpha -tubulin, cytokeratin, and actin were reinforced near the BC membrane. In summary, these WIF-B9 doublets with BC look very much like isolated hepatocyte couplets, which were shown to keep their polarity: the canalicular lumen is sealed by TJ where ZO-1 localizes, and reinforced pericanalicular sheaths of tubulin and actin are present (11, 23).

Polarization: Later Events

When the majority of WIF-B9 cells expressed a simple epithelial phenotype, we showed that all the catenins and the p120 were always localized at the lateral pole, as is usually described in cells with simple epithelial polarity (22). These proteins of the adhesion system are not always restricted to the so-called adherens junction sites (25); in WIF-B9 cells, we observed that E-cadherin could localize at the apicolateral boundary, that is, at the TJ level. Ezrin was present at the apical pole and at the apicolateral boundary, colocalizing with actin as described in a variety of epithelial cells (10). Finally, we found occludin at the TJ level, colocalizing with ZO-1 as described for human intestinal and bovine renal epithelial cells (21).

After 10 days in culture, the majority of WIF-B9 cells expressed a hepatic phenotype, with formation of BC structures between cells. Our present study indicates that WIF-B9 cells express alpha -, beta -, and gamma -catenins and p120 at the lateral pole (where E-cadherin is localized, too) and, also, sometimes at the apicolateral boundary, namely, at the TJ level where occludin and ZO-1 always colocalized. These observations confirm the close positional relationship between zonula occludens and zonula adherens previously observed in electron microscopic studies (16), an observation already made in hepatocytes (31). Cytokeratins showed reinforced localization at these BC levels. Previous studies on WIF-B cells have already evidenced a highly concentrated network of actin filaments and alpha -tubulin around the BC (30). In rat hepatocytes, colocalizing microtubules, actin bundles, and cytokeratin filament sheaths at the BC level play an essential role in the formation and maintenance of these BC (33, 50); consequently, the cytoskeleton is important for hepatic functions that are correlated with the polarity state of hepatocytes (18, 46). Thus the major cytoskeleton proteins present at the BC level of WIF-B9 cells could contribute to their structural but also functional polarity as demonstrated by the efficient vectorial transport of a bile acid analog (12) and by the functional specialization of stable and dynamic microtubules in protein traffic (54).

In summary, our results concerning cell-cell adhesion proteins, cytokeratin, and occludin reinforce previous studies in which other protein markers for cell polarization were used, which showed that WIF-B9 cells express a simple epithelial phenotype followed by a hepatic phenotype.

Cell-Cell Adhesion Protein Distribution and E-Cadherin-Catenin Complexes During WIF-B9 Polarity Establishment

In hepatocytes, Kim et al. (34) showed that the key components in cell-cell adhesion are also key elements in restoration of differentiated functions; rat hepatocytes induced to differentiate in Matrigel showed increased amounts of E-cadherin and beta -catenin and changes in their relative Triton X-100 solubility. We previously reported that WIF-B and WIF-B9 cells express a higher level of E-cadherin than their less differentiated nonpolarized Fao parent; moreover, rare nonpolarized WIF-B subclones are all characterized by a lower level of E-cadherin (8, 9).

Here we analyzed the distribution of the adhesion system proteins in WIF-B9 cells according to the expressed phenotype. We found that the total amount of E-cadherin was very similar whatever the time in culture, which means that, whatever the expressed phenotype, this protein is mainly present in the Triton X-100-soluble fraction. We obtained the same distribution with the three catenins and p120. Our results are in agreement with those of Kim et al. (34), who showed that E-cadherin and beta -catenin are mostly present in the Triton X-100-soluble fraction in rat hepatocytes. Similar distribution was observed in other cell types: HT29 cells (60) and A6 cells (43). On the contrary, in MDCK cells 5 days after the induction of cell-cell contact, Shore and Nelson (61) observed a conversion of E-cadherin from a soluble to an insoluble 0.5% Triton X-100 pool. This discrepancy is not due to different cell lysis conditions because we found the same relative distribution of E-cadherin using 0.5% or 1% Triton X-100 extractions (data not shown); however, cell culture conditions were not the same, with MDCK cells being plated on hydrated collagen gels. In WIF-B9, another type of cadherin could be responsible in part for adhesion; WIF-B9 cells express a cadherin different from E-cadherin [as evidenced by reactivity with antibodies against pan-cadherin (9)]. They could also express LI-cadherin, a cadherin specific to liver and intestinal epithelia, the adhesive function of which is independent of any interaction with cytoplasmic components (36). Finally, the difference in distribution between MDCK and WIF-B9 cells could be due to the difference in cell lines.

Dynamic cadherin-catenin complexes play an important role in structural and functional organization (48). Looking for their presence in WIF-B9 cells, we found soluble complexes of E-cadherin that contained alpha -, beta -, and gamma -catenins or p120. Interestingly, these complexes were more abundant when WIF-B9 expressed a fully polarized phenotype. As described in MDCK cells, these complexes could be potential targets on signaling pathways in WIF-B9 cells.

In conclusion, our study here shows that the spatiotemporal expression pattern of adhesion system proteins, cytoskeletal proteins, and junction-associated proteins correlates with morphogenetic events in WIF-B9 cells as described for other epithelial cell systems. Some of these proteins are known to take part in signal transduction pathways. It would be interesting to study the level of tyrosine phosphorylation of the cell-cell adhesion proteins and of ezrin in WIF-B9 cells. Ezrin is a substrate for the tyrosine kinase HGF (hepatic growth factor) receptor and is able to convey signals to the actin cytoskeleton machinery (15). Because the molecular mechanisms involved in hepatocyte differentiation are far from being elucidated, we believe that WIF-B9 cells will contribute to the understanding of these mechanisms during the establishment and maintenance of hepatic cell polarity and BC neoformation.


    ACKNOWLEDGEMENTS

We thank A. Forchioni for help with confocal analysis, V. Favaudon for experiments of cell counting with automatic scanning, C. Cruttwell for critical review of the manuscript, and H. Tourbez for invaluable help.


    FOOTNOTES

This work was supported in part by Association pour la Recherche sur le Cancer Grant 6551, the Institut Curie (PIC Signalisation Cellulaire Grant 914), the Centre National de la Recherche Scientifique, and the Institut National de la Santé et de la Recherche Médicale (contract PRISME 98-09).

Address for reprint requests and other correspondence: C. Decaens, INSERM U442, Signalisation Cellulaire et Calcium, Université Paris-Sud (Bat 443), Centre Universitaire, 91405 Orsay Cedex, France (E-mail: catherine.decaens{at}ibaic.u-psud.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 September 2000; accepted in final form 23 October 2000.


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Am J Physiol Cell Physiol 280(3):C527-C539
0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society




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