Cell Adhesion to the Apical Pole of Epithelium: a Function of Ce 11 Polarity

180 European Journal of Cell Biology 66, 180-191 (1995, February) © Wissenschaftliche Verlagsgesellschaft . Stuttgart

Cell adhesion to the apical pole of epithelium: a function of ce 11 polarity

Michael Thie1)a, Bärbel Harrach-Ruprechtb, Heinrich Sauere, Petra Fuchsa, Anja Albersa, Hans-Werner Denkera a Institute of Anatomy, University of Essen, Medical School, Essen/Germany b Institute für Arteriosclerosis Research, University of Münster, Münster/Germany

C Max-Planck-Institute for Molecular Physiology, Dortmund/Germany

ReceivedJuly 6, 1994 Accepted September 16, 1994

Uterine epithelium - epithelial phenotype - polarization • Introduction adhesiveness The epithelial cells lining the uterine cavity are structurally Human uterine epithelinm displays a distinct polarized organization with apical, lateral, and basal plasma membrane domains. Although and functionally polarized cells with distinct basal, lateral, and non-adhesive throughout most of the menstrual cyde, epithelial cells apical membrane domains. As is typical für simple epithelia, allow attachment of trophoblast cells to their apical pole during the apical surface of uterine epithelial cells (UECs) is free and embryo implantation. Arecent hypothesis postulates that epithelial non-adhesive for opposing UECs or embryonic cells such as cells turn off genes for apical-basal polarity and turn on genes for a trophoblast. Nevertheless, the uterine epithelium is not a pas• more mesenchyme-Iike phenotype allowing cell-cell interaction with sive surface, and UECs can be functionally reprogrammed to trophoblast. contribute actively to trophoblast adhesion. When appropri• Using an in vitro assay human uterine celllines (RL95-2, HEC-I-A, ately conditioned with steroid hormones, these epithelial cells AN3-CA) were selected on the basis of adhesiveness for trophoblast• enter astate of so-called receptivity and switch from a non• type cells (JAR). Subsequently, uterine cells were examined for epithelium-specific ultrastructure using transmission electron micros• adhesive state to a potentially adhesive state. When exhibiting copy, and for the expression of E-cadherin, «6-, ßl-, ß4-integrin sub• the adhesive state, the apical membrane domain of UECs units and cytokeratin using immunocytochemistry, confocallaser scan• allows the attachment of trophoblast (für review, see [22]). ning microscopy, and surface replication technique. HEC-I-A mono• The processes involved in modulating adhesiveness of UECs layers are non-adhesive for JAR cells and appear highly polarized für trophoblast, however, have not been identified so far. expressing E-cadherin, «6-, ßl-, ß4-integrin subunits, and cytokeratin. A mechanism to achieve UEC adhesiveness für trophoblast Both, integrins and E-cadherin, are present at the lateral membrane. is recently being discussed postulating that UECs modulate RL95-2 monolayers which are adhesive for JAR cells appear non• their apical-basal polarity [6, 7]. This plasticity in the pheno• polarized. Like HEC-I-A cells, RL95-2 cells express E-cadherin, «6-, ßl-, and ß4-integrin subunits, and cytokeratin. In contrast to HEC-I-A type of adult UECs may involve some of the elementary pro• cells, integrins and E-cadherin are distributed at the entire cell surface. cesses that playa rale in embryology during transformation of AN3-CA monolayers are non-adhesive for JAR cells and appear non• epithelium to mesenchyme. A characteristic of that latter pro• polarized. Cells lack epithelial-specific markers such as keratin and E• cess is, likewise, that apical-basal polarity is lost, and adhesion cadherin. They show only low expression of «6-, ßl-integrin subunits molecules are redistributed and/or newly acquired [12, 13]. In and lack ß4-integrin subunit. Conversely, they express vimentin. analogy, in UECs, part of the master gene pragram for the Thus, modulation of the epithelial phenotype of uterine cells, i. e. epithelial phenotype including genes for apical-basal polarity loss of apical-basal polarity, might prepare the apical cell pole for cell• may be turned off and, vice versa, certain genes for the cell interaction with trophoblast. However, loss of cell polarity would mesenchymal program may be turned on thus enhancing not lead to enhancement of adhesiveness for trophoblast if accom• adhesiveness of UECs for trophoblast. The activation of the panied by a loss of epithelium-specific adhesion molecules. mesenchymal pro gram in definitive epithelia occurs not only during development in vivo [13] but also in vitro, e. g. in lens epithelium [11] and Madin-Darby canine kidney cells [40]. Sig• nals such as tumor-promoting phorbol esters [21], oncoprote• ins [31], growth factors [26], and/or signals generated by cell•

1) Dr. Michael Thie, Institut für Anatomie, Universitätsklinikum, cell and cell-matrix interactions [30] appear to contral gene Hufelandstr. 55, D-45122Essen/Germany. activation in these systems. Loss of polarized epithelial phenotype 181

In this study we characterize parameters of the epithelial and spheroids were washed two times with RPMI 1640 medium prior phenotype of certain human endometrial ceIl lines (RL95-2, to the experiment. After 1 h, spheraid adhesion to the endometrial HEC-I-A, AN3-CA) and correlate these with adhesive or monolayers was quantified by cenrifuging coverslips with cell-spheroid non-adhesive behavior for trophoblast-type ceIls (JAR) in an surface facing down at 12g for 5 min. Attached spheroids were counted attempt to gain insight into the pro gram underlying UEC and expressed as the percentage of the nu mber of spheroids seeded. adhesiveness. We examine ultrastructural features as weIl as Immunofluorescence expression of markers associated to the epithelial phenotype, Cells grown on gJass coversJips were rinsed twice in phosphate• i. e. E-cadherin [35], a6-, ßl-, ß4-integrin subunits [33, 34], buffered saline (PBS), fixed and permeabilized by incubation in 96 % and keratin intermediate filaments [24]. methanol-water for 10 min at -20°C. After severaJ washings with PBS On the basis of our data we postulate that modulation of the and a finaJ wash in PBS supplemented with 0.5 % bovine serum albu• epithelial phenotype of UECs, specificaIly loss of apical-basal min (BSA) , cells were incubated for 1 h at room temperature with the polarity, prepares the apical ceIl pole for ceIl-ceIl interaction primary antibody. Thereafter, cells were rinsed in PBS/0.5 % BSA (4 x with trophoblast. Loss of ceIl polarity, however,' would not 10 min) and incubated with the corresponding fluorescein iso• lead to enhancement of UEC adhesiveness for trophoblast if thiocyanate-conjugated secondary antibody for 1 h at room temper• ature. In controJ experiments the primary antibody was omitted. After accompanied by a loss of epithelium-specific adhesion mole• rinsing with PBS, specimens were mounted with 90 % gJycerol-PBS, cules. This suggests that specific modulation of polarity• supplemented with 1.0 % p-phenyJenediamine as an antiquenching related parameters rather than down-regulation of the entire agent and examined with a Zeiss Axiophot microscope equipped with epithelial pro gram is a key event in this type of adhesive epi• epiillumination (450-490 nm excitation; fiJterset 487909). Photographs thelial interactions . were taken on Neopan 1600 film (Fuji, Tokyo/Japan).

Antibodies Rat monocJonal antibody to a6-integrin subunit (GoH3; [32]) was pro• Materials and methods vided by Dr. A. Sonnenberg (The Netherlands Cancer Institute, Divi• sion of Cell Biology, Amsterdam/The Netherlands), and diJuted 1:3 Routine cell culture with PBS/0.5 % BSA before use. Rat monocJonal antibody to ßl• Human endometrial carcinoma cell lines were purchased from the integrin subunit (AllB2; [38]) was pravided by Dr. C. Damsky American Type Culture Collection (ATCC), Rockville, MDIUSA, i. e. (Department of Anatomy, University of California, San Francisco/ RL95-2 cells (CRL 1671; [37]), HEC-I-A cells (HTB 112; [19]), and USA), and was diJuted 1:3 with PBS/0.5 % BSA before use. Mouse AN3-CA cells (HTB 111; [5]). For routine culture, cell lines were monocJonal antibody to ß4-integrin subunit (3El) was purchased fram grown in plastic flasks in 5 % C02"95 % air at 37°C. In brief, RL95-2 BiomoJ, Hamburg/Germany, and was diluted 1: 100 with PBS/0.5 % cells were seeded out in a 1+1 mixture of Dulbecco's modification of BSA before use. Mouse monocJonal antibody to E-cadherin (6F9; [8]) Eagle's medium and Ham's F12 (Gibco-Life Technologies, Eggenstein/ was donated by Dr. J. Behrens (Max-DeJbrück-Centrum, Berlin/Ger• Germany) supplemented with 10% fetal calf serum (Gibco), 10 mM many), and was diJuted 1:5 with PBS/0.5% BSA before use. Mouse HEPES (Gibco), and 0.5 flg/ml insulin (Gibco), HEC-I-A cells in monocJonal antibody to cytokeratin No 8 (4.1.18) was purchased fram McCoy's 5A medium (Gibco) supplemented with 10 % fetal calf Boehringer Biochemica, Mannheim/Germany, and was diJuted 1:20 serum, and AN3-CA cells in Eagle's minimum essential medium with with PBS/0.5 % BSA before use. Mouse monocJonal antibody to Earle's salts and non-essential amino acids (Gibco) supplemented with vimentin (V9) was obtained fram Sigma-Aldrich, Deisenhofen/Ger• 10 % fetal calf serum. All media were additionally supplemented with many, and was diJuted 1:40 with PBS/0.5 % BSA before use. penicillin (100 lU/mi; Gibco) and streptomycin (100 flg/ml; Gibco). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse The growth medium was changed every 2 to 3 days, and cells were sub• secondary antibodies (F232) and FITC-conjugated rabbit anti-rat sec• cultured by trypsinization (trypsin-EDTA solution; Gibco) when they ondary antibodies (F234) were obtained from Dako Diagnostika, became confluent. HamburgiGermany. Gold (15 nm)-conjugated goat anti-mouse sec• ondary antibodies and goJd (15 nm)-conjugated goat anti-rat second• Cell culture on coverslips ary antibodies were obtained from Biotrend, KäJn/Germany. Anti• Cells were harvested by trypsinization from confluent cultures, bodies were diJuted with PBS/0.5 % BSA according to manufacturers counted, and adjusted to the desired concentration, i. e. RL95-2 instructions. 700000 cells, HEC-I-A 200000 cells, and AN3-CA 300000 cells each in 2.0 ml of their respective culture medium. Subsequently, cell sus• Confocallaser scanning microscopy pension was poured out on POlY-D-lysine-coated glass coverslips (12 Confocal microscopy was carried out using a confocaJ Jaser scanning mm in diameter) situated in 4 cm2 Falcon multiwells. Cells were grawn micrascope (MRC 600, BioRad, Heme! Hampstead/UK) equipped in medium to confluent monolayers and used for experiments within 3 with an argon ion Jaser (Ion Laser TechnoJogy, Salt Lake City, UT/ days after start of cultures. USA) for 488 nm excitation. The confocaJ Jaser scanning unit was coupled to a standard micrascope (Diaphot, Nikon, Düsseldorf/Ger• Attachment assay many). For the experiments a 60-fold oiJ immersion objective (Nikon, Adhesiveness of endometrial cell monolayers for human JAR chorio• DüsseJdorf/Germany) with a numericaJ aperture of 1.4 was chosen. carcinoma cells (ATCC: HTB 144) was measured using a centrifugal The theoretical value for the z-resolution of the microscape was calcu• force-based adhesion assay. The establishment of the adhesion assay lated to be 0.4 flm [39]. Images of horizontal optical sections were has been described previously [17]. Briefly, JAR cell suspension recorded in the x-y plane with 256 Jines/image. For vertical optical sec• (100000 cells per mJ RPMI 1640 medium (Gibco) suppJemented with tions scans were performed in the x-z plane with 200 Jines/image. Pho• 10 % fetal calf serum and penicillin-streptomycin) was incubated on a tographs were taken with a Ilford APX-I00 fiJm (Mobberley, Cheshire/ gyratory shaker at 110 x rpm obtaining multicelluJar spheroids 72 h UK) from a black and white monitor. after initiation of culture. Subsequently, JAR spheroids were har• vested, counted, and gently deJivered onto a confluent monoJayer of Surface replication technique human endometrial celliines grawn on coversJips. Confrantation cul• For surface replication cells grown on coverslips were fixed and incu• tures were grown in RPMI 1640 medium with or without 10 % fetal calf bated with primary and goJd-conjugated secondary antibodies as serum supplementation in a humidified 5 % C02"95 % air incubator at described above. Thereafter, cells were washed, postfixed with 2.5 % 37°C. For confrontation cultures in serum-free medium, monolayers gJutaraJdehyde in PBS for 2 h at raom temperature, dehydrated with 182 M. Thie, B. Harrach-Ruprecht, H. Sauer, P. Fuchs et 01.

graded ethanol and air dried. Platinum-carbon surface replicas of cells JAR cells attached with high efficiency to RL95-2 cells. were made in a Balzers BA 300 apparatus (Balzers/Liechtenstein) Attachment was more effective in confrontation cultures sup• equipped with an electron gun evaporator and a quartz crystal thick• plemented with 10% fetal calf serum (FCS) (RL: 82.4 % ± ness monitor. Replicas were obtained by shadowing the cell surface 16.0 attachment) compared with cultures lacking FCS (RL: with platinum-carbon at an angle of 38°, followed by carbon at 90°. 37.3 % ± 29.5 attachment). Values of JAR cell attachment to The replicas were cleaned overnight in sodium hypo chloride (12 % RL95-2 cells were significantly high er than values of JAR cell active chloride; Hedinger, Stuttgart/Germany) and washed in distilled water. They were picked up on 200 mesh copper grids and examined in attachment to POlY-D-lysine-coated glass coverslips both in the a Philips EM 410 at 60 kV presence (CO: 36.4 % ± 29.6 attachment) and absence of serum (CO: 0 % attachment) pointing to a degree of specific• Electron microscopy ity of attachment between RL95-2 cells and JAR cells. Cells were grown as monolayers on thermanox coverslips (13 mm in Compared to RL95-2 cells, JAR cells attached with low effi• diameter; Nunc, Naperville, IL/USA) as described above. For subse• ciency to HEC-1-A cells. As above, confrontation cultures in quent electron microscopy, samples were rinsed twice in PBS and fixed the presence of FCS showed high er values of attachment in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 30 min (HEC: 40.2 % ± 23.4 attachment) than cultures in the at room temperature. After repeated washings in cacodylate buffer, absence of FCS (HEC: 6.5 % ± 4.4 attachment). However, sampIes were postfixed with 1 % OS04 in cacodylate buffer, dehy• this was comparable to JAR cell attachment to POlY-D-lysine drated with graded ethanol and propylene oxide, and embedded in coated glass coverslips either in the presence (CO: 36.4 % epoxy resin [4]. The embedded monolayers were separated from the ± thermanox coverslip by snap freezing in liquid nitrogen. Ultrathin sec• 29.6 attachment) or absence of serum (CO: 0% attachment). tions were mounted on 200-mesh copper grids, double-stained with This suggests that JAR cell attachment to HEC-1-A cells was uranyl acetate and lead citrate, and examined with a Philips EM 400 at due to serum and JAR cell-specific molecules rather than spe• 80 kV cific for HEC-1- A cells. According to this, HEC-1- A cells can be considered non-adhesive for JAR cells unless serum• derived bridging molecules are provided. JAR cells did not attach to AN3-CA neither in the presence Results of FCS (AN: 1.8 % ± 4.1 attachment) nor in the absence of FCS (AN: 0 % attachment). Thus, AN3-CA cells are intrinsi• Adhesiveness of monolayers cally non-adhesive for JAR cells and lack bin ding sites for The adhesiveness of RL95-2, HEC-1-A, and AN3-CA mono• serum constituents that may act as bridging molecules. layers for JAR cells was measured in the presence of serum and the absence of serum to consider mediating effects of Ultrastructural features serum molecules in cell-cell interaction (Fig. 1). To determine whether differences in the adhesiveness of cell lines were associated with differences in the epithelial mor• phology, we analyzed RL95-2, HEC-1-A, and AN3-CA cells by transmission electron microscopy (Figs. 2-4). RL95-2 cells grew predominantly as monolayers but cells showed a tendency of piling up. Cells were varying in size. c C 0 10050182.4I HECCOAN140.2 (/) ..c'(jj.D:J0>""0

Fig. 2. Photomicrographs of ultrathin sections of RL95-2 cells culti• vated on a POlY-D-lysine-coated coverslip. - a. Cells grow as a mono• layer but show a lack of structural polarization, no regular microvilli and no subplasmalemnal filament network at the apical plasma mem• brane. - b. Only primitive focal adherens junctions are seen at the lateral plasma membranes (arrows). - cl Cell 1. - c2 Cell 2. - cs Coverslip. - me Growth medium. - N Nucleus. - Bars 2 [-Lm(a), 0.25 Fig. 1. Adhesiveness of human endometrial celliines (RL = RL95-2; [lm (b). HEC = HEC-1-A; AN = AN3-CA) for human JAR choriocarcinoma spheroids as determined in the centrifugal force-based spheroid adhe• Fig. 3. Photomicrographs of ultrathin sections of HEC-1-A cells cul• sion assay. Spheroids were delivered onto endometrial monolayers, tivated on a polY-D-lysine-coated coverslip. - a. Cells grow as an and confrontation cultures were grown in medium supplemented with ordered monolayer exhibiting a highly polarized epithelial phenotype either 10% fetal calf serum (black bars) or without fetal calf serum with numerous microvilli at the apical cell pole (arrowheads). - b. (hatched bars) for 1 h. Adhesiveness is expressed as the percentage of Adjacent cells show closely apposed plasma membranes with proper the number of spheroids seeded. For comparison, adhesiveness of formation of tight junctions (Zarge arrows), adherens junctions (small polY-D-lysine-coated glass coverslips (CO) for spheroids is shown. The arrows) and desmosomes (asterisks). - cl Cell 1. - c2 Cell 2. - cs values are mean ± SEM of 25 confrontation pairs counted, from deter• Coverslip. - me Growth medium. - N Nucleus. - Bars 2 [lm (a), 0.25 minations from five separate cultures. [lm (b). EJCB Loss of polarized epithelial phenotype 183

I 184 M. Thie, B. Harrach-Ruprecht, H. Sauer, P. Fuchs et al. EJCB

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Fig. 4. Photomicrographs of ultrathin sections of AN3-CA cells cul• show only primitive focal membrane contacts. Organized intercellular tivated on a polY-D-lysine-coatcd coverslip. - a. Cells grow as a mono• junctions are lacking. - cl Cell 1. - c2 Cell 2. - cs Coverslip. - me laycr but show a lack of structural polarization. - h. Adjacent cells Growth medium. - N Nucleus. - Bars 2 flm (a), 0.25 [.lm(b).

Cells formed small primitive adherens junctions. Regions of loose attachment to one anothcr. The lateral membranes of interacting plasma membran es were alternating with regions adjacent cells ran parallel without forming organized intercel• of large intercellular spaces (Fig. 2b). Occasionally, desmo• lular junctions (Fig. 4b). somes could be observed (data not shown). Tight junctions Thus, RL95-2 cells which are adhesive for trophoblast-type were lacking as proved by freeze fracture electron microscopy cells showed morphological features indicating lack of struc• (data not shown). tural polarization while I-lEC-l-A cells being non-adhesive HEC-I-A cells formed ordered monolayers in which the displayed in highly polarized phenotype with respect to the single cells were more or less uniform in size and shape grow• distribution of organelles and to membrane organization. ing in close contact to one another and to the substrate (Fig. AN3-CA cells appeared non-polarized, althollgh being non• 3a). Cells had a cylindrical shape, nuclei were predominantly adhesive. situated at the base of the cells. Mitochondria, Golgi appara• tus, and endoplasmic reticulum were mostly located at the Epithelial marker expression supranuclear region of the cel!. The apical surface was covered To determine whether differences in epithelial morphology of with numerous microvilli which were relatively short. The cells cell lines were associated with loss of epithelium-associated showed closely apposed plasma membranes at their lateral molecllles, we analyzed the expression of E-cadherin, 0.6-, faces with tight junctions in the subapical region and adherens ßl-, ß4-integrin sllbllnits, and cytokeratin (Figs. 5,6). The cell junctions and desmosomes scattered along the basolateral membranes (Fig. 3b). The tight junctions bctwecn adjaccnt cells were verified using freeze fracture electron microscopy Fig. 5. lmmunostaining of endometrial monolayers (RL = RL95-2; (data not shown). HEC = HEC-l-A; AN = AN3-CA) with monoclonal antibodies to E• AN3-CA cclls formed a regular monolayer in which the cadherin (a-c), cytokeratin No 8 (d-f), and vimentin (g-i). Note that single cells werc round to cylindrical in shape (Fig. 4a). Cclls RL95-2 cells and HEC-l-A cells but not AN3-CA cells are positive for flattcned against the substratum. The cell nuclei were irregu• E-cadherin and cytokeratin. AN3-CA cells but not RL95-2 cells and larly positioncd in thc cclls. Cell organelles piled up prcdom• J-IEC-l-A cclls are positive for vimentin. - Ecd E-cadherin. - ker inantly in thc vicinity of the nucleus. The apical surface Cytokeratin intermecliate filaments. - vim Vimentin intermecliate fila• appeared dome-like lacking microvilli. The cclls showed only ments. - Bar 20 [.lm. EJCB Loss of polorized epithelial phenotype 185

I 186 M. Thie, B. Harrach-Ruprecht, H. Sauer, P. Fuchs et al. EJCB EJCB Lass af palorized epithelial phenatype 187

lines were stained in parallel and the photographic processing was identical thus allowing direct comparison of staining pat• terns. RL95-2 cells and I-lEC-I-A cells were clearly positive for E• cadherin (Figs. 5a, b) and a6-, ~-l-, and r34-integrin subunits (Figs. 6a, b, d, e, g, h), showing characteristic membranc• bound staining. With all these markers, the overall intensity of immunostaining of RL95-2 cells was comparable with that of HEC-I-A cells. AN3-CA cells, on the other hand, were com• pletely negative for E-cadherin (Fig. 5c) and ~4-integrin sub• units (Fig. 6i), and only weakly positive for a6-, and ~1• integrin subunits (Figs. 6c, f). When RL95-2 cells and HEC-I-A cells were labeled with an antibody to cytokeratin No 8, strong intracellular staining appeared (Figs. 5d, e). In RL95-2 cells, staining was most intense in the perinuclear region indicating disorganization of the intermediate filament network, while HEC-I-A cells showed a well-defined network of filaments often associated with regions of ccll-cell contacts. In contrast, cytokeratin No 8 was not expressed by the AN3-CA cclls (Fig. 5f). Whcn RL95-2 cells, HEC-I-A cells and AN3-CA cells were stained for vimentin, AN3-CA cells reacted prominently (Fig. 5i), whereas RL95-2 cells and HEC-I-A cells were devoid of immunoreactivity (Figs. 5g, h). In conclusion, the expression of E-cadherin, a6-, ~1-, ~4• integrin subunits and cytokeratin indicated the epithelial phe• no type of J-lEC-I-A cells and of RL95-2 cells although the lat• tcr exhibited a lack of structural polarization. In contrast, AN3-CA cells showed non-polar morphology corresponding to the lack of E-cadherin, ~4-integrin, and cytokeratin. Fig. 7. Confocal images of endometrial monolayers (RL = RL95-2; According to the expression of vimentin, these cells appeared HEC = HEC-I-A; AN = AN3-CA) after staining with monoclonal to be of mesenchymal phenotype. antibody to E-eadherin. Vertieal seetions reveal that RL95-2 eells are labeled along the entire plasma membrane (a). HEC-I-A eells are Apico-basolateral distribution of E-cadherin and labeled laterally (b), while AN3-CA eells are not labeled at all; note autofluoreseenee of nllclells (e). Arrows mark the position of eell-eell integrin subunits (u.6, ß1, ß4) contaets in monolayers. - es Coverslip. - Bar 10 [.lm. In order to visualize the domain-specific localization of E• cadherin and integrins, monolayers were examined by confo• callaser scanning microscopy (Figs. 7, 8). Images of vertical optical sections were individually processed to visualize pro• tein distribution in cells. Thus, intensity of staining differs labeled rather uniformly at the entire plasma membrane (Figs. from the original data given in Figures 5 and 6. 8a, d, g), while HEC-I-A cells were labeled mainly at sites of RL95-2 cells were labeled by the anti-E-cadherin antibody cell-cell contacts (Figs. 8b, e, h). AN3-CA cells showed stain• along thc cntire plasma membrane (Fig. 7a). However, fluo• ing at sites of cell-cell contacts using anti-a6-, ~ l-integrin anti• rescence was not evenly distributed over the whole cell mem• bodies (Figs. 8c, f), and no staining using anti-~4-integrin anti• brane as staining was often pronounced at regions of cell-cell bodies (Fig. 8i). contacts. In I-IEC-I-A cells (Fig. 7b), in contrast, fluorescent In summary, the random distribution of E-cadherin and staining was largely confined to the sites of cell-cell contacts integrin subunits along the entire plasma membrane in RL95-2 and was absent from the apical surface. As anticipated, the cells corroborated the morphological observation that cclls plasma membrane was not labeled at all by the antibody lacked a defined polarization. In contrast, the lateral distribu• against E-cadherin in AN3-CA cells (Fig. 7c). tion of proteins in HEC-I-A cells confirmed the epithelial A similar staining pattern was observed with antibodies to polarization of these cells. a6-, r31-, r34-integrin subunits. Again, RL95-2 cells were Antigen expression at the apical cell surface The light microscopical analysis of RL95-2 cells showed a spe• cific fIuoroescence of E-cadherin and integrins at the apical cell surface. However, confocallight microscopy did not allow to gain more details about distribution within the plane of the Fig. 6. Immunostaining of endometrial monolayers (RL = RL95-2; membrane or about membrane confinement. Therefore, we HEC = HEC-I-A; AN = AN3-CA) with monoclonal antibodies to a6• searched for microdomains using whole-mount preparations integrin subllnit (a-c), ßl-integrin subllnit (d-C), ancl ß4-integrin sub• and a surface replication technique (Fig. 9). unit (g-i). RL95-2 cclls and l-IEC-I-A cclls are positive for a6-, ßl-, r34-integrin subunits. AN3-CA eells are only weakly positive for a6-, Antibodies for E-cadherin as weIl as for the integrin sub• ßl-integrin sllbllnits and eompletely negative for ß4-integrin subunits. units showed an identical staining pattern. Single immunogold - Bar 20 fun. particles indicating reactivity were evenly distributed over the I 188 M. Thie, B. Harrach-Ruprecht, H. Sauer, P. Fuchs et 01. EJCB

Fig. 8. Confoeal images of endometrial monolayers (RL = RL95-2; I-lEC = I-lEC-I-A; AN = AN3-CA) after staining with monoclonal antiboe!y to a6-inlegrin subunil (a-e), ßl-integrin subunit (d-f), ancl r-\4-inlegrin subunit (g-i). Verlieal seetions reveal that RL95-2 cells are label cd at the entire plasma membrane (a, d, g), while I-IEC-I-A eells are labelee! al siles of cell-cell conlacls (b, e, h)_ AN3-CA eells show staining at cell-cell contacts using anti-a6-, ßl-integrin subunits (e, f) and no staining using anti-ß4-integrin subunits; note autofluorescence of nucleus (i). Arrows mark the position of eell-eell eontaets in mono• layers. - es Coverslip. - Bar 10 [.tm.

Discussion

In the present communication, attachment and invasion• competent chüriocarcinüma cells were used as a probe für attachment-permissiveness and non-permissiveness of the investigated endometrial cell lines. Three main conclusions are suggested by our results. (i) Expression of apolar pheno• type (= f-IEC-l-A cells) prevents adhesion of invasive cells to the apical plasma membrane. (ii) Lack of cell polarity (= AN3-CA cells) is not sufficient to allow adhesion. (iii) Expres• sion of adhesion moleeules of the types investigated here is a precondition of attachment, but these mülecules need to be present at the exposed (apical) membrane (= RL95-2 cells), i. e., the adhesion-receptive phenotype is characterized by the expression of the appropriate molecules in the membrane in a non-polar distribution. By extrapolation, one might postulate that, in vivo, modulation of the epithelial phenotype of uter• wh oie cell surface at low density. Occasionally, gold particles ine cells, i. e. loss üf apical-basal polarity, might prepare the were associated in sm all clusters. I-lowever, clusters revealed apical cell pole for cell-cell interaction with throphoblast. no ordered distribution within the plasma membrane. In cor• However, loss of polarity would not lead to receptivity if responding experiments using HEC-l-A cells, virtually no accompanied by a loss of expression of appropriate ac\hesion immunogold particles were detectable (data not shown). molecules. EJCB Lass af palarized epithelial phenatype 189

Fig. 9. Surfaee replieation of RL95-2 eells after staining with mono• ticlcs are associated in small clusters (arrowheads). - es Coverslip. • clonal antibody to aG-integrin subunits. Note single immunogold par• pm Exposcd plasma membrane. - Bar 0.5 [Am. tielcs cvenly distributed ovcr thc wholc ccll surfaee. Oeeasionally, par-

These data extend findings by others who have pointed out involved in adhesiveness for trophoblast-type cells as heparan the importance of polar organization of UECs for receptivity/ sulfate proteoglycans and dermatan sulfate-containing proteo• non-receptivity phenomena [10] and who have observed glycans as well as their corresponding binding sites might par• changes in the apical, lateral and basal plasma membrane ticipate in the adhesiveness of RL95-2 cells [29]. E-cadherin domains and in the organization of the cytoskeleton related to would mediate contact by calcium-dependent homotypic intcr• the adhesive behavior of uterine epithelial cells in vivo [6, 22]. action 19], whereas integrins mediate both homotypic or het• Moreover, the data reported herc put those findings into a erotypic interactions and often require the prescncc of plasma/ new context. The possibility of modulation of the epithelial matrix protein [14]. Binding of JAR cells to RL95-2 monolay• phenotype, i. e. loss of apical-basal polarity by random distri• crs in serum-free medium could be due to cither homotypic or bution of cell-adhesion molendes, like E-cadherin and a6-, heterotypic integrin-integrin. interaction, while enhancement ß1-, ß4-integrin subunits as shown in RL95-2 cells, renders the of JAR cell attachment to RL95-2 cells by serum suggests endometrial cell lines an interesting tool to study control of aclhesion via cross-bridging molecules thus pointing to a possi• adhesiveness in this respect. blc involvcment of integrins. JAR cell attachment to HEC-I• As yet, we do not know whether E-cadherin and integrins A cells in the presence of serum should be due to J AR cell inserted into the exposed membrane of RL95-2 cells are receptor molecules binding to serum malendes which in turn I 190 M. Thie, B. Harrach-Ruprecht, H. Sauer, P. Fuchs et al.

are bound to the HEC-l- A cell surface again via some type of proteins might impair adhesion function of proteins as shown bin ding site. Failure or proper association of serum molecules for E-cadherin [1, 2, 15]. Whether even distribution of E• with the apical surface of AN3-CA cells might lead to a failure cadherin and integrins on the free surface membrane of RL95• of JAR cell attachment to AN3-CA cells. Wh ether an inverse 2 cells demonstrated by surface replication technique might be correlation between cadherin expression and integrin expres• due to disassembled interactions between transmembrane sion which occurs during terminal differentiation of keratino• molecules and cytoskeletal proteins and/or selective targeting cytes [16] also exists in RL95-2 cells will have to be investi• and retention of molecules remains to be seen. gated. In a study of the expression of various integrin subunits in epithelial cells of human endometrium in vivo [20], a6-, ß4• Acknowledgements. The skillful technical assistance of Birgit Nowak integrin subunits were reported to be located at the basolater• and Dorothea Schünke is gratefully acknowledged. We also wish to al surface of the cells in the samples investigated. We have thank Dr. J. Behrens, Dr. C. Damsky, and Dr. A. Sonnenberg for the preliminary data that a6-integrin subunit shows a redistribu• generous gifts of antibodies. We are also obliged to Prof. Dr. R. K. H. tion at the lateral plasma membrane, i. e. along the apical• Kinne (Director of the Department of Epithelial Physiology of the basal axis, at transition from the proliferative to the secretory Max-Planck-Institute for Molecular Physiology, Dortmund) for mak• ing available to us the confocal microscopy unit and his interest in this phase in vivo [36]. Thus, further studies using human samples work. should give new insights into the distribution of integrins and E-cadherin throughout the menstrual cycle. 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