Cdx1 or Cdx2 expression activates E-cadherin-mediated cell-cell adhesion and compaction in human COLO 205 cells

Matthew S. Keller ,* Toshihiko Ezaki,* Rong-Jun Guo, and John P. Lynch

Division of Gastroenterology, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Submitted 18 November 2003 ; accepted in final form 18 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A mature columnar intestinal epithelium develops late in embryogenesis and is maintained throughout the life of the organism. Although the mechanisms driving intestine-specific gene expression have been well studied, those promoting the acquisition of cell-cell junctions, columnar morphogenesis, and polarization have been less studied. The Cdx homeodomain transcription factors (Cdx1 and Cdx2) regulate intestine-specific gene expression and intestinal epithelial differentiation. We report here that Cdx expression induces E-cadherin activity and cell-cell adhesion in human COLO 205 cancer cells. Within days of Cdx1 or Cdx2 expression, a new homotypic cell-cell adhesion phenotype is induced. This is a specific response to Cdx, inasmuch as a Cdx1 mutant failed to elicit the effect. Additionally, Cdx-expressing COLO 205 cells demonstrate a reduced proliferative capacity and an increase in the mRNA expression of differentiation-associated genes. Electron micrographs of these cells demonstrate induction of tight, adherens, and desmosomal junctions, as well as a columnar shape and apical microvilli. Investigations of the adhesion phenotype determined that it was Ca2+ dependent and could be blocked by an E-cadherin-blocking antibody. However, E-cadherin protein levels and intracellular distribution were unchanged. Cdx expression restored the ability of the cell membranes to adhere and undergo compaction. We conclude that Cdx1 or Cdx2 expression is sufficient to induce an E-cadherin-dependent adhesion of COLO 205 cells. This adhesion is associated with polarization and cell-cell membrane compaction, as well as induction of a differentiated gene-expression pattern. Ascertaining the mechanism for this novel Cdx effect may yield insight into the development of mature colonic epithelium.

columnar morphogenesis; desmosal and tight junctions; cell polarity


THE HUMAN SMALL INTESTINE and colon are lined by a columnar epithelium that is continuously renewed. This epithelium is highly specialized and possesses certain features that permit it to function as a barrier that specifically absorbs nutrients, salts, and water (17). These qualities include a polarized morphology, apical microvilli to increase absorptive surface area, specific distribution of apical and basal channels and pumps to aid active absorption, and a great variety of cell-cell adhesive junctions to strengthen the barrier function and retard nutrient losses (17, 48). Although the mechanisms regulating intestinal epithelial proliferation, differentiation, and lineage determination have been largely identified (4, 6, 14, 16), little is known about how these mechanisms promote the development of a polarized, columnar morphology and the variety of adhesive junctions that are essential to the function of the intestinal epithelium.

Adherens junctions are among three primary types of cell-cell junctional complexes; the other two types are desmosomes and tight junctions. Intracellular adhesion in adherens junctions is mediated by cadherins, a large family of transmembrane proteins. E-cadherin is the primary classical cadherin functioning in all types of epithelium, including intestinal epithelium. It has been well established that E-cadherin expression and activity are important factors in the regulation of epithelial cell adhesion, membrane compaction, cell polarity, and proliferation. These myriad effects largely stem from the ability of classical cadherins to interact with the cellular cytoskeleton. E-cadherin is linked to the actin cytoskeleton through the bridging proteins {alpha}- and {beta}-catenin. This connection to the actin cytoskeleton is necessary for the development of strong cell-cell adhesion (9, 35). Moreover, the actin cytoskeletal reorganization prompted by E-cadherin specifies the lateral cell membrane domains and cell polarity (26, 55, 58). Membrane compaction, the process of maximizing cell-cell contact by spreading the adhesion laterally from the point of initial contact, requires E-cadherin activity and actin polymerization as well (1, 35, 52). Epithelial cell motility in normal tissues and that associated with metastasis of neoplastic cells is regulated in part by E-cadherin adhesion (3, 53). Finally, because of its interactions with {beta}-catenin, E-cadherin activity can modulate Wnt/{beta}-catenin nuclear signaling and transformation (8, 31, 44). The importance of E-cadherin activity in intestinal epithelial homeostasis is underscored by studies in vivo, where forced intestinal expression of E-cadherin suppressed proliferation, promoted apoptosis, and delayed cell migration, whereas inhibition of cadherin function resulted in the loss of the polarized, columnar phenotype (10, 11).

Cdx1 and Cdx2 are homeodomain proteins related to the Drosophila caudal gene and are known to regulate pattern formation early in development as well as intestine-specific gene expression in adults (7, 45, 47). Cdx1 and Cdx2 also promote differentiation of intestinal cells and can regulate intestinal and colon cancer cell proliferation (7, 20, 21, 24, 42, 46). In rat IEC-6 cells, an undifferentiated intestinal cell line, Cdx1 and Cdx2 expression induced a polarized, columnar cell morphology as well as tight and desmosomal adhesion junctions (42, 46); however, the mechanisms underlying this effect were not investigated. In another study, Cdx2 overexpression in Caco-2-TC7 cells reportedly increased E-cadherin protein and mRNA levels; however, this increase appeared to be modest and was not associated with any measurable changes in cell-cell adhesiveness or morphology (19). More recently, LI-cadherin and claudin-2, two genes involved in cell-cell adhesion, have been identified as novel transcriptional targets of Cdx2 (12, 40). Thus, although several studies have suggested a role for Cdx1 and Cdx2 in the induction of cell-cell adhesion and adherens junctions, no study has demonstrated significant regulation of E-cadherin function or activity by Cdx. Given the importance of E-cadherin for intestinal epithelial morphology and homeostasis, it would seem likely that regulation by Cdx at some level would exist.

In our present study, we use a retrovirus to express Cdx1 and Cdx2 in a variety of colon cancer cell lines. We identify a cell line that undergoes a marked transformation with the expression of the Cdx transcription factors. COLO 205 cells, a poorly differentiated human adenocarcinoma cell line, become more differentiated and acquire a new homotypic cell-cell adhesion phenotype with Cdx1 or Cdx2 expression. This effect was specific, inasmuch as an NH2 terminally truncated Cdx1 protein was unable to elicit this effect. Importantly, the expression of these homeodomain transcription factors in COLO 205 cells produced a variety of effects associated with more differentiated colonocytes. These cells appeared to have a reduced proliferative capacity, and the gene expression profile was significantly altered. In addition, expression of Cdx1 or Cdx2 induced a morphological change in COLO 205 cells. These cells expressed polarized, columnar features with evidence of new, highly organized cell-cell adhesive junctions such as adherens, desmosomal, and tight junctions. Finally, studies of the cell-cell adhesion mechanism confirm that it is Ca2+ dependent and requires E-cadherin activity, inasmuch as the cells have acquired the ability to undergo membrane compaction. This is not due to changes in E-cadherin protein and mRNA levels, however. Taken together, we conclude that Cdx1 or Cdx2 expression in human COLO 205 cells modulates cell adhesion, polarity, membrane compaction, proliferation, and gene expression patterns, in part by regulation of E-cadherin activity but not its levels. These findings suggest a mechanism whereby the Cdx transcription factors may modulate enterocyte and colonocyte cell adhesion and morphology and regulate intestine-specific gene expression and proliferation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and transfections.

DLD-1, HCT-116, COLO DM, COLO 205, LS174T, LoVo, HT-29, Caco-2, T84, and SW480 colon cancer cells were obtained from the American Type Culture Collection maintained under standard conditions. {Phi}-NX-A (amphotropic) retroviral packaging cells were obtained from the American Type Culture Collection with the approval of Gary Nolan (Stanford University) and maintained as described elsewhere (34). HCT-116 cells were transfected using FuGENE 6 (Roche). Luciferase assays were carried out using the dual-luciferase assay kit and the pRL-CMV vector (both from Promega) as transfection control.

Production of retroviral vectors and retroviral infections.

Murine Cdx1 or Cdx2 cDNAs were subcloned into the multicloning site of the MIGR1 retroviral vector (kindly provided by Warren Pear, University of Pennsylvania), yielding MIGR-Cdx1 and MIGR-Cdx2. These vectors were transfected into {Phi}-NX-A cells using the CellPHhect calcium phosphate precipitation kit (Amersham Bioscience), and infectious retroviral supernatants were isolated at 48 and 72 h, purified as described elsewhere (34), aliquoted, and stored at –70°C. Cdx1 truncation constructs were synthesized by PCR using oligonucleotides to amplify Cdx1 truncations from a murine cDNA template and were described previously (20). They were subcloned into the MIGR1 multicloning site as well, and infectious retroviral particles were isolated.

Retroviral infections were performed as described previously (34). Colon cancer cell lines were initially plated sparsely, 1 x 103 per well in a six-well culture dish. Retroviral supernatants to be used were diluted to 50% with the cell culture medium of the cell line to be infected. Polybrene (Sigma) was added to a final concentration of 8 µg/ml, and the mixture was applied to the cells overnight. The medium was changed on the following morning, and the cells were cultured normally. By 4 days after infection, green fluorescent protein (GFP)-positive cells are clearly visible. At 6 days after retroviral infection, the cells were processed for the various end points. Living unsorted cells were photographed using an inverted fluorescent microscope. Images were obtained using a Photometrix Coolsnap-CF black-and-white charge coupled device (CCD) camera (Roeper Scientific) mounted on a Leica DM IRB fluorescent microscope. For fixed images of unsorted or sorted cells, cells were fixed in freshly prepared 4% paraformaldehyde in PBS for 10 min at room temperature, the cells were examined by fluorescent microscopy, and images were obtained using a Photometrix Coolsnap-CF black-and-white CCD camera mounted on a Nikon E600 fluorescent microscope.

GFP-positive cells were sorted 6 or 7 days after retroviral infection. Unsorted cells were trypsinized and fully dissociated, washed in 1x PBS-1 mM EDTA (PBSE), and resuspended in PBSE before they were sorted on a FACSort cytometer (Becton Dickson). 5 x 104 GFP-positive cells were isolated and pooled for culture. Pooled cells were cultured for 10–14 days before use in various assays. Sorted cells on 100-mm plates were cultured for 4–6 days, with frequent medium changes, and whole cell protein or total RNA was isolated. Immunoblots for Cdx1 and Cdx2 protein were performed exactly as described previously (20).

Electron microscopy.

Sorted GFP-positive cells were cultured for 7 or 14 days after confluency in a Transwell culture chamber (polycarbonate, 0.4-µm pore size; Costar) with daily medium changes. Wells were washed with PBS and then fixed at room temperature in 2.5% glutaraldehyde-3.2% paraformaldehyde buffered with 0.1 M sodium cacodylate (pH 7.4) and postfixed in the same buffer with 2% osmium tetroxide. Samples were then dehydrated and embedded in Epon, and ultrathin sections were examined by transmission electron microscopy (TEM).

Cell accumulation by water-soluble tetrazolium salt assay.

The accumulation of cells in culture was measured using the water-soluble tetrazolium salt (WST) reagent (WST-1, Roche). Sorted cells were fully dissociated in PBSE, washed in PBS, and resuspended in culture medium. Cells (5 x 103) in 100 µl of media were loaded in each of 8 wells of a 96-well plate coated with poly-D-lysine (Biocoat, Becton Dickson). There was one 96-well plate for each day examined, and each plate contained wells with medium alone (background), COLO 205 wild-type, and sorted MIGR1-, MIGR-Cdx1-, and MIGR-Cdx2-treated cells. Cell culture medium was changed on day 4. For the WST-1 assay, 10 µl of WST-1 reagent were added to the cells, and the cells were incubated at 37°C for 4 h (days 1 and 2), 2 h (days 4 and 5), and 1 h (day 6). After the incubation period, the optical density (OD) of the formazan product (at 450 nm) and the plate background (at 630 nm) were measured on an EL 312e Microplate Reader (Bio-Tek Instruments). The medium and plate background values were subtracted from the OD at 450 nm (OD450) values, which were then normalized to 2 h. Each pool of cells was assayed twice by this method, and nine different pools were analyzed (3 each from MIGR1-, MIGR-Cdx1-, and MIGR-Cdx2-infected COLO 205 cells).

RT-PCR analysis.

Total RNA was isolated from sorted GFP-positive cells using RNeasy (Qiagen). Total RNA (5 µg) was used for cDNA synthesis with the first-strand cDNA synthesis kit (Invitrogen), and the final cDNA product was diluted to 50 µl with sterile water. RT negative controls were included initially to assay for genomic DNA contamination. Primer sequences and PCR concentrations are listed in Table 1. Before their use in the SYBRgreen RT-PCR assay, primer sets were confirmed to produce a single amplicon by agarose gel electrophoresis. This amplicon was cloned using the TA cloning kit (Invitrogen) and sequenced to confirm amplification of the correct amplicon. For the RT-PCR, 1 µl of cDNA was mixed with SYBRgreen RT-PCR Master Mix (Applied Biosystems) and forward and reverse primers and then assayed in an ABI Prism 7000 sequence detection system as directed by the manufacturer. After the amplification, a melting-curve analysis was carried out to ensure amplification of a single product. All RT-PCR were performed in duplicate. A ribosomal phosphoprotein, 36B4, was used as the normalization control for this assay. Fold change in RNA levels was calculated from the cycle number at which the fluorescence signal passes a threshold level (Ct values) using the formula previously described (39, 41, 57). The difference in the Ct values of the test and control RNAs after normalization to the control RNA 36B4 ({Delta}{Delta}Ct values) for each gene were averaged across the RNA pools, a standard deviation was calculated, and these values were converted to fold change. This formula and the use of 36B4 were validated for this assay in COLO 205 cells as described elsewhere (39, 41, 57).

Cell adhesion assays.

This protocol was adapted from a dissociation assay described by Nagafuchi et al. (29). Equal numbers of uninfected COLO 205 and MIGR1-, MIGR-Cdx1-, and MIGR-Cdx2-infected and selected cells were plated in six-well culture plates to begin this assay. Six to eight wells of each were started. Cell culture medium was changed every other day. MIGR-Cdx1 and MIGR-Cdx2 cells formed multicellular clumps of adherent cells by day 4 in culture. On day 6, half of the wells were washed briefly with TC buffer (1x HBSS without Ca2+ or Mg2+ and with 20 mM HEPES, pH 7.4, 1 mM CaCl2, and 0.01% trypsin) and the other half with TE buffer (1x HBSS without Ca2+ or Mg2+ and with 20 mM HEPES, pH 7.4, 1 mM EGTA, and 0.01% trypsin) and then incubated with 1 ml of TC or TE buffer for 30 min at 37°C. The cells were scraped from the plate and then dissociated by repipetting 10 times, and total particles were counted on a hemocytometer (a single cell or a clump of cells = 1 particle). Extent of dissociation = NTC/NTE, where N is the number of particles per milliliter counted by hemocytometer.

For the blocking antibody studies, the method of Behrens et al. (3) and Vleminckx et al. (53) was used. On the day after 1 x 10E4 fully dissociated cells were plated per well in a 24-well plate, wells are incubated in 300 µl of medium containing DECMA-1 antibody (Sigma; 12 µg/ml) or control rat IgG antibodies (Sigma) at the same concentration. The cell culture medium was changed every 24 h, with the addition of fresh antibody. After 7 days of culture, representative micrographs were obtained.

E-cadherin Western blots and immunofluorescence.

Several 100-mm plates of our COLO 205 cells were cultured for 4 days, whole cell protein extracts were prepared as described elsewhere (21), and the products were analyzed by SDS-PAGE and electroblotted. The E-cadherin antibody 610182 (BD Transduction Laboratories) was used in the immunoblotting as well as immunofluorescence experiments. For a Western blot loading control, we used the actin A-4700 (Sigma).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Retroviral vector MIGR1 can express transcriptionally active Cdx1 and Cdx2 in human colon cancer cell lines.

To more easily study the effects of Cdx1 and Cdx2 expression on colon cancer cell proliferation, differentiation, and gene expression patterns, we investigated retroviral vectors. We obtained the MIGR1 retroviral vector (33). The MIGR1 retrovirus encodes a polycistronic mRNA due to the presence of an internal ribosomal entry site and a 3' GFP cDNA. Upstream of the internal ribosomal entry site is a multicloning site into which Cdx1 or Cdx2 cDNAs were cloned. Thus all GFP-positive cells will be expressing Cdx1 or Cdx2 as well. We tested the ability of the MIGR1 retrovirus to infect a number of colon cancer cell lines and found that the retrovirus was able to effectively infect nearly all of them (Table 2).

Cells infected with the MIGR1-based retrovirus can be identified and sorted by flow cytometry for GFP expression, permitting the isolation of pure populations of retrovirally infected cells. HCT-116 and COLO 205 cells were infected with MIGR-Cdx1, MIGR-Cdx2, and the control MIGR1 viruses, and GFP-positive cells were isolated and pooled. GFP fluorescence was visually observed in all pooled cell lines (data not shown). Cdx1 protein was observed only in the MIGR-Cdx1-infected HCT-116 and COLO 205 cells (Fig. 1A and data not shown). Similarly, only the MIGR-Cdx2-infected cells expressed greatly increased Cdx2 protein levels (Fig. 1A and data not shown). Transfection of a Cdx-responsive reporter vector sucrase-isomaltase (SI; –183 to +54) luciferase into the HCT-116 cell lines demonstrated activation of the reporter in cells treated with MIGR-Cdx1 or MIGR-Cdx2 but not in MIGR1-infected cells (Fig. 1B). Retroviral expression of Cdx1 or Cdx2 led to a sixfold increase in luciferase activity compared with controls. Therefore, the retroviral vector MIGR1 can express functionally active levels of Cdx1 or Cdx2 protein in HCT-116 and COLO 205 cells.



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Fig. 1. MIGR1 retroviral vector expresses transcriptionally active Cdx1 or Cdx2 in colon cancer cell lines. HCT-116 and COLO 205 cells were infected with control MIGR1 (M) or Cdx-expressing MIGR-Cdx1 (X1) or MIGR-Cdx2 (X2) retroviruses. Green fluorescent protein (GFP)-positive cells were isolated by flow cytometry and expanded, and whole cell extracts were prepared. A: Western immunoblotting of COLO 205 whole cell extracts for Cdx1 and Cdx2 proteins. One of several blots is shown. B: transcriptional activity of virally expressed Cdx1 and Cdx2 proteins in HCT-116 cells. Infected and control cells were transfected with a Cdx-responsive reporter, sucrase-isomaltase (SI, –183 to +54) luciferase, and transfection control (pRL-CMV). Luciferase activity was measured at 48 h and normalized to total protein and Renilla luciferase levels. One of 3 experiments is shown. *Significantly different from wild-type (wt) and MIGR1 control cells (P < 0.0001, by ANOVA and Tukey's rank mean testing).

 
Cdx1 and Cdx2 expression in COLO 205 cells induces a homotypic cell-cell adhesion phenotype.

On expression of Cdx1 or Cdx2 in COLO 205 cells, we observed a striking phenotypic change. COLO 205 cells are a poorly differentiated human colon cancer cell line that adhere only loosely to each other and the culture dish (2). Within several days of retroviral infection, we noted aggregation of cells infected by the MIGR-Cdx1 or MIGR-Cdx2 viruses but not the control MIGR1 (Fig. 2A). This was observed before any GFP-mediated cell sorting and appears to be a homotypic (like adhering to like) adhesion, where only GFP-positive cells are binding (Fig. 2A). GFP-negative cells appeared to be excluded from the clustering (arrows, Fig. 2A). Isolation of GFP-positive cells by flow cytometry yielded cells with significantly different patterns of growth in the tissue culture dish. MIGR1-infected and wild-type COLO 205 cells grew in the same way as single, rounded, and poorly adherent cells (Fig. 2B). Cdx1- or Cdx2-expressing COLO 205 cells grew in clusters, tightly adherent to other cells and the tissue culture dish (Fig. 2B).



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Fig. 2. Cdx1 or Cdx2 expression induces a homotypic cell-cell adhesion in COLO 205 cells. A: COLO 205 cells were infected with retroviruses, fixed, and imaged 5 days later. Arrow indicates uninfected, GFP-negative cells. Cell-cell clustering occurred spontaneously among GFP-positive cells receiving MIGR-Cdx1 or MIGR-Cdx2 retrovirus. B: similar to A, except GFP-positive cells were isolated by flow cytometry and expanded before they were fixed and imaged. C: MIGR retroviruses expressing mutant truncated Cdx1 were used to infect COLO 205 cells. Cdx1-ND, Cdx1-(141–268), in which NH2 terminus was deleted; Cdx1-CD, Cdx1-(1–223), in which COOH terminus was removed. Cdx1-CD, but not Cdx1-ND is transcriptionally active (20).

 
To determine whether the cell adhesion effect was a specific response to Cdx gene expression, we tested Cdx1 truncation mutants for the ability to elicit this behavior. Cdx1 truncation mutants, Cdx1-ND [Cdx1-(141–268)], in which the NH2 terminus was deleted, or Cdx1-CD [Cdx1 Cdx1-(1–223)], in which the COOH terminus was removed, were subcloned into the MIGR1 retrovirus, and infectious retroviral particles were isolated. In a previous study, we determined that Cdx1-CD, but not Cdx1-ND, remained transcriptionally active and able to induce the expression of an SI (–183 to +54) luciferase reporter (20). This was expected given that the NH2 terminus of Cdx1 contains a transcription-activating domain (47). When these mutant constructs were expressed in COLO 205 cells, only Cdx1-CD could elicit the cell-cell adhesion phenotype (Fig. 2C). This suggested that Cdx1's cell-cell adhesion effect in COLO 205 cells was specific and correlated with the ability of the protein to function as a transcription factor.

Cdx1 and Cdx2 expression reduces accumulation of COLO 205 cells in culture.

We and others have established that expression of Cdx1 or Cdx2 in intestinal and colon cancer cell lines can alter cell proliferation. To determine what effect, if any, their expression had on COLO 205 cell accumulation, we utilized a colorimetric assay to measure cell numbers in culture, the WST-1 assay (Roche). COLO 205 cells were infected with each of the retroviral constructs on three different occasions, and pools of GFP-positive cells were isolated by flow cytometry, yielding a total of nine pools of retrovirally infected cells for analysis. Each of the three sets were analyzed using the WST-1 assay. We found that the COLO-MIGR-Cdx1 or COLO-MIGR-Cdx2 cells accumulated more slowly in culture than the wild-type COLO 205 cells or the control infected COLO-MIGR1 cells (Fig. 3). This effect was reproduced in each of the three sets of isolated cells. The differences in accumulation were usually apparent at 3 or 4 days in culture. The Cdx1- or Cdx2-expressing COLO 205 cells demonstrated very similar rates of accumulation in culture: there were no reproducible differences between them. In summary, expression of Cdx1 or Cdx2 in COLO 205 cells led to reductions in their rate of accumulation in culture.

Expression of Cdx1 or Cdx2 induces a polarized, columnar cell morphology.

Expression of Cdx1 or Cdx2 in COLO 205 cells induces marked changes in cell culture phenotype; however, it was not known whether this was associated with the induction of a more differentiated columnar morphology. To investigate for these effects, COLO-MIGR1-, COLO-MIGR-Cdx1-, or COLO-MIGR-Cdx2-infected cells were cultured for 1 or 2 wk on Transwell chambers (Costar). They were then fixed, embedded, and examined by TEM. COLO-MIGR1 cells appeared fairly homogeneous, and there were no obvious differences between the 1- and 2-wk cultures (Fig. 4A and data not shown). The cells were rounded, with a high nuclear-to-cytoplasm ratio. Their few microvilli were poorly organized and randomly distributed. There was no obvious polarization, and no complex cell-cell adhesion structures, such as tight junctions and desmosomes. Although these cells did pile up in culture into stratified, multicellular structures, they did not exhibit any organizing behaviors.

Cdx1- or Cdx2-expressing COLO 205 cells were markedly different from the MIGR1 control cells by TEM. These cells were more heterogeneous in their appearance and tended to have reduced nuclear-to-cytoplasm ratios (Fig. 4A). Cell-cell membranes were more closely approximated and the stratified cells seemed more tightly bound than in the control MIGR1 cells. Columnar-shaped cells were plainly visible throughout the culture. Additionally, the cells demonstrated polarizing characteristics. Microvilli were denser than on control cells and were oriented upward on the surface cells or toward the open lumens of the numerous cystic structures present below the surface (Fig. 4, A and B). Under higher magnification, these cells displayed evidence of advanced cell-cell adhesive structures, including adherens, desmosomal, and tight junctions (Figs. 4C). There were no significant differences between the Cdx1- and Cdx2-expressing cells by TEM, and the 2-wk cultures were marginally more differentiated than the 1-wk cultures (data not shown).



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Fig. 4. Cdx1 or Cdx2 expression induces a polarized, columnar morphology in COLO 205 cells. Sorted GFP-positive cells were cultured for 7 or 14 days after confluency in a Transwell culture chamber (Costar) with daily medium changes. Membranes were washed with PBS and then fixed and processed for transmission electron microscopy. A: COLO-MIGR1, COLO-MIGR-Cdx1, and COLO-MIGR-Cdx2 cells at high magnification 14 days after confluency. COLO-MIGR-Cdx1 and COLO-MIGR-Cdx2 cells are columnar and visibly polarized with apical microvilli. B: cystic lumens organized within the COLO-MIGR-Cdx1 and COLO-MIGR-Cdx2 cell layers. Cells are polarized toward the surface lumen or the cystic lumen. Inset: high magnification of the surface of the cystic lumen demonstrating surface organization of microvilli is shown at magnification x20,000 on left. C: high-power examination of cell-cell junctions of COLO-MIGR-Cdx1 and COLO-MIGR-Cdx2 cells at 14 days. Note presence of apical microvilli and desmosomal and tight junctions.

 
Cdx1 and Cdx2 expression in COLO 205 cells induces markers of colonocyte differentiation.

We next investigated whether Cdx1 and Cdx2 expression in COLO 205 cells induced genes associated with colonocyte differentiation. Using SYBRgreen real-time quantitative PCR (Applied Biosystems), we specifically tested for changes in mRNA levels for known Cdx target genes, such as SI, lactase, guanylyl cyclase C (GC-C), and carbonic anhydrase I (CAI) (32, 45, 49, 51), as well as genes associated with colonocyte differentiation, such as intestinal alkaline phosphatase, villin, downregulated in adenoma (DRA), CAI, cytokeratin 20 (CytoK20), galectin 1, and Na+/H+ exchanger (NHE) isoform 2 (NHE-2) (18, 23, 27, 28, 36, 43, 56). A ribosomal phosphoprotein, 36B4, was used as our loading control (accession no. M17885). As with prior studies, RNA was isolated from multiple pools of retrovirally infected COLO 205 cells. Several RNA sets were isolated from the same pools to determine the variability of the measurements within the same pool of cells.

Several genes were highly induced by Cdx1 or Cdx 2 expression (Fig. 5). DRA and CAI were significantly induced by Cdx1 or Cdx2 compared with the MIGR1 control. Cdx1 expression may induce their expression slightly better than Cdx2; however, this difference did not reach statistical significance. CytoK20 and LPH appeared to be moderately induced by Cdx1 and Cdx2 expression; however, this difference from controls did not reach statistical significance. Of the other classic Cdx-target genes, GC-C levels were unaltered and SI was not detected (data not shown). Villin, galectin 1, and alkaline phosphatase mRNA expression was detected, but the levels were unaltered by Cdx expression (data not shown). Surprisingly, the mRNA levels for NHE-2 were reduced in the Cdx1- and Cdx2-expressing COLO 205 cells (Fig. 5). For all the genes examined, Cdx1 and Cdx2 expression induced similar patterns of mRNA expression. In summary, Cdx1 or Cdx2 expression in COLO 205 cells led to the induction of a more differentiated colonocyte pattern of gene expression.



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Fig. 5. Cdx expression induces a mature colonocyte pattern of gene expression. Total RNA was isolated from sorted GFP-positive cells and used for cDNA synthesis and then subjected to quantitative SYBRgreen RT-PCR analysis. 36B4, a ribosomal phosphoprotein, was used as normalization control. Fold change in RNA levels was calculated from Ct values as described elsewhere (39, 41, 57). Compiled results on selected genes from quantitative RT-PCR studies are shown. CAI, carbonic anhydrase I; DRA, downregulated in adenoma; CytoK20, cytokeratin 20; LPH, lactase; NHE, Na+/H+ exchanger. *Significantly different from controls (P < 0.05); n = 5.

 
Cdx1- and Cdx2-induced cell-cell adhesion is Ca2+ dependent and can be inhibited by antibodies blocking E-cadherin activity.

The homotypic nature of the cell-cell adhesion suggested the involvement of cadherins, a Ca2+-dependent family of proteins involved in cell-cell adherens and desmosomal junctions. To test for this directly, we performed a quantitative cell-dissociation assay in the presence and absence of Ca2+. In the presence of Ca2+, the dissociation index of COLO 205 wild-type or MIGR1-infected cells was 0.37 and 0.38, respectively, and there was minimal clustering of the cells on visual inspection (Fig. 6). The dissociation index of Cdx1- and Cdx2-expressing cells was fourfold lower, ~0.09, reflecting greater Ca2+-dependent cell-cell adhesion than control cells. This difference was statistically significant and was readily apparent on visual inspection (Fig. 6B). These findings support the hypothesis that the Ca2+-dependent cadherin proteins are involved in the cell-cell adhesion phenotype induced by Cdx1 and Cdx2.



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Fig. 6. Cell-cell adhesion induced by Cdx expression is Ca2+ dependent. Sorted GFP-positive COLO-MIGR1, COLO-MIGR-Cdx1, and COLO-MIGR-Cdx2 cells were subjected to a quantitative cell dissociation assay to characterize cell-adhesion phenotype. Equal numbers of cells were plated. On day 6, cells were lightly trypsinized, washed briefly, pelleted, and resuspended in buffers containing 1 mM CaCl2 or 1 mM EGTA and incubated at 37°C. After 30 min, cells were dispersed by repipetting, and the number of particles (N; single cell or cell clumps) was counted on a hemocytometer. Dissociation index was calculated for each pool of cells as ratio of NCa2+ to NEGTA. A: graphic representation of results (n = 14). *Significantly different from controls (P < 0.005). B: dissociation at end of assay. Cells were cultured in each well of a 6-well plate.

 
E-cadherin is a prominent member of the cadherin family that is expressed in epithelial cells, including those in the small intestine and colon. Additionally, COLO 205 cells are known to express E-cadherin, as well as the other factors necessary for E-cadherin-mediated cell-cell adhesion ({alpha}-catenin, {beta}-catenin, pp120-catenin); however, these factors are inactive (2). Inasmuch as E-cadherin mRNA levels were reportedly increased by Cdx2 expression in Caco-2 cells (19), we examined our COLO 205 cells for induction of E-cadherin mRNA or protein with Cdx1 or Cdx2 expression. No changes in E-cadherin mRNA or protein expression levels were noted by Western blot analysis and SYBRgreen real-time quantitative PCR (data not shown). We next examined E-cadherin intracellular distribution by immunofluorescence (Fig. 7). Although there appeared to be no significant differences in E-cadherin distribution, the ability of the Cdx1- or Cdx2-expressing COLO 205 cells to undergo membrane compaction was very obvious. E-cadherin appeared to cluster at cell-cell junctions in COLO-MIGR1 cells; however, these cells tended to remain round and were unable to extend their junctions laterally to close off spaces between cells (white arrows, Fig. 7). COLO-MIGR-Cdx1 and COLO-MIGR-Cdx2 cells were very proficient at compacting their membranes, which distorted their shape and left no empty space between the cells in contact (Fig. 7).



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Fig. 7. Cdx1 or Cdx2 expression induces membrane compaction in COLO 205 cells. Sorted GFP-positive COLO-MIGR1, COLO-MIGR-Cdx1, and COLO-MIGR-Cdx2 cells were cultured for 5 days, fixed, and processed for E-cadherin immunofluorescence. E-cadherin localizes to sites of the cell-cell junction in all the cells. However, COLO-MIGR1 cells tended to remain round and were unable to extend their junctions laterally to close off spaces between cells. COLO-MIGR-Cdx1 and COLO-MIGR-Cdx2 cells left no empty space between the cells in contact. White arrows, empty spaces between cells in contact.

 
To further confirm that E-cadherin activity was required for the Cdx-mediated cell-cell adhesion, we used a mouse monoclonal E-cadherin-blocking antibody. This antibody is known to inhibit E-cadherin cell-adhesion when added to living cells in culture (3, 53). As a positive control, we used COLO 205 cells treated with 7 nM staurosporine. This PKC inhibitor activates E-cadherin-mediated cell-cell adhesion in COLO 205 cells (2). Addition of the blocking antibody to our Cdx1- or Cdx2-expressing COLO 205 cells clearly inhibited the formation of cell-cell adhesions and multicellular "clumps" compared with the control rat IgG antibodies (Fig. 8). Furthermore, this effect was identical to that seen with COLO 205 cells treated with 7 nM staurosporine (data not shown). These findings suggest that induction of cell-cell adhesion and membrane compaction in COLO 205 cells by Cdx1 or Cdx2 requires E-cadherin expression and activity.


    DISCUSSION
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The columnar epithelium of the small intestine and colon of adult humans and mice is continuously renewed. Although great progress has been made in understanding the mechanisms by which this epithelium is renewed, little is known about how the cellular polarity and columnar morphology are established and maintained. Our studies demonstrate that Cdx1 or Cdx2 expression alone is sufficient to induce the E-cadherin cell-cell adhesive activity that is essential for columnar morphogenesis and polarization.

Cdx1 or Cdx2 expression regulates COLO 205 proliferation, morphology, and polarization.

Our initial finding that Cdx expression in a poorly differentiated colon cancer cell line induced an adhesive phenotype was striking. This observation seemed more dramatic than the effects previously described in IEC-6 cells (42, 46). Wild-type COLO 205 cells and those infected with the control retrovirus are unable to engage in normal epithelial cell-cell adhesive interactions (2). Expression of Cdx1 or Cdx2 in these cells completely restored cell-cell adhesion and permitted the development of complex interactions such as desmosomal and tight junctions. This effect of Cdx was robust, inasmuch as it was observed reliably with each retroviral infection. It is also a specific effect of Cdx, one that requires Cdx transcriptional activity, because a Cdx1 mutant with deletion of the 140-amino acid NH2 terminus could not induce this phenotype.

Associated with this adhesion phenotype, Cdx expression altered COLO 205 proliferation and differentiation. Cdx1 and Cdx2 expression modestly reduced the rate of accumulation of COLO 205 cells in culture. This effect was reproducibly observed in entirely separate pools of retrovirally infected cells. There was no obvious increase in cell death with Cdx expression; therefore, we conclude that the reduced accumulation is most likely due to reduced proliferation, rather than increased apoptosis. Additional studies on the growth effects were not performed, because the difference between control and Cdx-expressing cells, although significant and reproducible, was modest compared with other model systems (31, 32).

Studies of COLO 205 differentiation with Cdx expression were of greater interest. The mRNA levels of several genes that are highly expressed in differentiated colonocytes (27, 28, 43) were increased in the Cdx1- and Cdx2-expressing COLO 205 cells. DRA and CAI were induced ~30- and 40-fold, respectively. CytoK20 and lactase appeared to be weakly induced, although additional testing is necessary to confirm this. Unexpectedly, G-CC expression was unaltered (32). GC-C has been reported to be a transcriptional target for Cdx2 in T84 and Caco-2 cells. The reasons for the lack of response are unknown, but perhaps COLO 205 cells lack an essential transcription factor or cofactor that is required for Cdx induction of the GC-C gene.

NHE-2 is a marker for colonocyte differentiation that is detectable in crypt and surface colonocytes, but its levels are greater in crypt cells (5). Moreover, an analysis of its promoter suggested that it may be a Cdx-responsive gene (23). It was surprising, then, that Cdx expression resulted in a ~10-fold reduction in NHE-2 mRNA levels. One possibility is that Cdx expression promotes a differentiated, surface cell phenotype in these cells. One way to confirm this would be to investigate NHE-3 mRNA and protein levels. NHE-3 protein levels are increased in surface colonocytes (5). We predict that NHE-3 levels are increased in our Cdx-expressing COLO 205 cells compared with controls, and we are engaged in studies to confirm this.

In addition to the more differentiated pattern of gene expression, Cdx1 and Cdx2 induced a mature, columnar morphogenesis in COLO 205 cells. This change was associated with the induction of several different types of cell-cell adhesion junctions, including desmosomal and tight junctions. Moreover, this morphogenesis included polarization. It is impressive that this degree of organization can be elicited with the expression of a single gene. Previous reports have described similar effects of Cdx expression in rat small intestine-derived IEC-6 cells (42, 46); however, this is the first report of this effect on human colonocytes. More significantly, we have begun to investigate a mechanism for this effect and have determined that E-cadherin adhesion activity, but not its expression, is induced by Cdx expression and is required for the observed morphogenesis. This agrees with previously published studies that established the importance of E-cadherin activity for intestinal epithelial homeostasis and morphology (10, 11). We believe that this is the first report of a transcription factor regulating E-cadherin activity and function, rather than its expression levels. Elucidating the mechanism for this effect will allow us to directly tie the Cdx factors, known mediators of intestinal cell differentiation, with mechanisms that regulate cell shape and polarity.

Cdx induction of E-cadherin activity promotes cell-cell adhesion, cell membrane compaction, and polarity.

We investigated current models for the induction of epithelial cell adhesion and polarity. These models, although incomplete, are nonetheless complex and depend on contributions from a diverse group of factors (21, 26, 58). An important component in these models is the cell-cell adhesion mediated by E-cadherin (13, 55). COLO 205 cells are known to lack E-cadherin-binding activity, despite fully expressing E-cadherin and other factors required for E-cadherin function (2). We hypothesized that Cdx expression restored this function, promoting strong cell-cell adhesion and polarization. Our studies support this hypothesis. The cell-cell adhesion induced by Cdx expression is dependent on Ca2+, implicating a cadherin in the process. Moreover, the DECMA-1 antibody inhibited cell adhesion in the Cdx1- and Cdx2-expressing cells. DECMA-1 is a well-studied inhibitor of E-cadherin-binding activity (3, 53). In studies designed to elucidate a mechanism for this effect, we noted that Cdx expression did not alter E-cadherin mRNA or protein levels. Nor did Cdx expression promote E-cadherin membrane localization. We found, however, that Cdx1 or Cdx2 expression induced membrane compaction in COLO 205 cells. Membrane compaction is an active process requiring functioning E-cadherin adhesion and actin polymerization (1, 52). It is believed to depend on an "adhesion zipper" phenomenon to seal the membranes together (52). All of these findings establish that Cdx1 and Cdx2 expression in COLO 205 cells induces E-cadherin activity but not levels, and this induction is necessary for the cell adhesion and columnar morphogenesis that is observed.

Many questions remain unanswered. Foremost among them is how does Cdx expression induce E-cadherin activity? Many signaling pathways and regulatory factors have been reported to modulate E-cadherin activity, including tyrosine kinases, such as Src, Fer, and Fyn (37), Rho family GTPases (15), and serine-threonine kinases (2). Often, these factors regulate the protein-protein interactions linking E-cadherin to the actin cytoskeleton (2, 15, 37). It is interesting to note that {beta}-catenin is an important component of the cadherin cell-cell adhesion complex (9), linking E-cadherin to {alpha}-catenin and the actin cytoskeleton. {beta}-Catenin is also a critical regulator of intestinal crypt cell and colon cancer cell proliferation (16, 22, 38, 50, 54) and differentiation (25, 30, 50). It is possible that Cdx1 and Cdx2 expression reduces proliferation and promotes differentiation and columnar morphogenesis by modulating {beta}-catenin and its role in these diverse processes.

Columnar morphogenesis and polarization is a complex process.

The induction of the complete columnar, polarized morphology of fully differentiated colonocytes requires more than E-cadherin activation. Desmosomal and tight junctions are also necessary to further strengthen intracellular bonds and make them an impermeable barrier to salt and fluid transit (17, 48). In addition, there is associated reorganization of cytoskeletal proteins, including actin and intermediate filaments, as well as microtubules. Finally, trafficking of membrane lipids and protein between the Golgi and the cell membrane is more tightly regulated in polarized epithelial cells (17, 48). In our studies, we observe the induction of desmosomal and tight junctions by TEM. Claudin-2 has been reported to be a transcriptional target for Cdx2 (40), so it is possible that Cdx directly induces these junctions. Contributing to membrane apposition could be LI-cadherin, another cell adhesion molecule; the gene is a transcriptional target of Cdx2 (12). It is unlikely that LI-cadherin directly modulates complex processes, such as polarization and membrane compaction, however, because, unlike E-cadherin, LI-cadherin is not known to interact with cytoskeletal proteins or alter cytoskeletal organization. Therefore, the precise role of these various factors in the development of a mature, polarized, columnar morphology is unknown.

In summary, expression of the caudal-related homeodomain proteins Cdx1 or Cdx2 in human colon cancer COLO 205 cells induced E-cadherin cell-cell adhesive activity and was associated with the induction of a mature colonocyte morphology, reduced proliferation, and a more differentiated pattern of gene expression. Our observations further underscore the importance of the caudal-related transcription factors in the induction of the intestinal cell phenotype. The COLO 205 cell adhesion model we have developed here is an ideal system in which to define how Cdx1 or Cdx2 expression induces E-cadherin activity and to promote the development of columnar morphogenesis and polarization. The elucidation of the mechanisms governing Cdx regulation of cell adhesion and morphogenesis is essential for full understanding of colonocyte development and maturation.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-02695-02, DK-047437-09, and DK-062819-01 (to J. Lynch), a Pilot Project award from the Center for Molecular Studies in Digestive and Liver Disease (to J. Lynch), and Morphology, Cell Culture, and Molecular Biology Core Facilities of the NIH/NIDDK Center for Molecular Studies in Digestive and Liver Disease at the University of Pennsylvania Grant P30-DK-50306. This work was also supported in part by American Cancer Society Grant IRG-78-002-25 and a Glaxo Institute for Digestive Health Research Award (to J. Lynch).



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Fig. 3. Reduced accumulation of Cdx-expressing COLO 205 cells in culture. COLO 205 cells were infected with each of the retroviral constructs, and pools of GFP-positive cells were isolated by flow cytometry. A total of 9 different pools of retrovirally infected cells (3 each for MIGR1, MIGR-Cdx1, and MIGR-Cdx2) were studied using water-soluble tetrazolium salt (WST-1) assay (Roche). Increased numbers of cells in culture are indicated by increased optical density at 450 nm (OD450). Each pool was studied on ≥2 occasions; results are from 1 of several experiments (n = 8). {bullet}, Wild-type COLO 205 cells; {triangleup}, MIGR1-infected cells; {blacksquare}, COLO-MIGR-Cdx1 cells; {circ}, COLO-MIGR-Cdx2 cells. *Significantly different from wild-type and control MIGR1 cells (P < 0.0005, by ANOVA and Tukey's rank mean testing).

 


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Fig. 8. E-cadherin activity is required for Cdx-mediated cell-cell adhesion of COLO 205 cells. Sorted GFP-positive COLO-MIGR1, COLO-MIGR-Cdx1, and COLO-MIGR-Cdx2 cells were cultured for 7 days in the presence of a monoclonal antibody (DECMA-1; Sigma) that binds E-cadherin's extracellular domain and inhibits its activity (3, 53). A rat IgG antibody (Sigma) at the same concentration was used as a control. Cell culture medium was changed every 24 h, with fresh antibody added. After 7 days of culture, representative micrographs were obtained.

 

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Table 1. Primer sequences for quantitative RT-PCR analysis

 

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Table 2. MIGR1 retroviral infection rate and relative GFP expression levels

 

    ACKNOWLEDGMENTS
 
We thank Dr. Eun Ran Suh and Anil Rustgi for helpful comments regarding the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. P. Lynch, Div. of Gastroenterology/660 CRB, 415 Curie Blvd., Philadelphia, PA 19104 (E-mail:lynchj{at}mail.med.upenn.edu).

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.

* M. S. Keller and T. Ezaki contributed equally to this work. Back


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