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Am J Physiol Gastrointest Liver Physiol 293: G54-G65, 2007. First published April 26, 2007; doi:10.1152/ajpgi.00533.2006
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MUCOSAL BIOLOGY

The homeodomain transcription factors Cdx1 and Cdx2 induce E-cadherin adhesion activity by reducing beta- and p120-catenin tyrosine phosphorylation

Toshihiko Ezaki,1,2 Rong-Jun Guo,1 Hong Li,1 Albert B. Reynolds,3 and John P. Lynch1

1Division of Gastroenterology Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; 2Division of Gastroenterology, Keio University School of Medicine, Tokyo, Japan; and 3Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee

Submitted 16 November 2006 ; accepted in final form 19 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The homeodomain transcription factors Cdx1 and Cdx2 are regulators of intestine-specific gene expression. They also regulate intestinal cell differentiation and proliferation; however, these effects are poorly understood. Previously, we have shown that expression of Cdx1 or Cdx2 in human Colo 205 cells induces a mature colonocyte morphology characterized by the induction of a polarized, columnar shape with apical microvilli and strong cell-cell adhesion. To elucidate the mechanism underlying this phenomenon, we investigated the adherens junction complex. Cdx1 or Cdx2 expression reduced Colo 205 cell migration and invasion in vitro, suggesting a physiologically significant change in cadherin function. However, Cdx expression did not significantly effect E-cadherin, {alpha}-, beta-, or {gamma}-catenin, or p120-catenin protein levels. Additionally, no alteration in their intracellular distribution was observed. Cdx expression did not alter the coprecipitation of beta-catenin with E-cadherin; however, it did reduce p120-catenin-E-cadherin coprecipitation. Tyrosine phosphorylation of beta- and p120-catenin is known to disrupt E-cadherin-mediated cell adhesion and is associated with robust p120-catenin/E-cadherin interactions. We specifically investigated beta- and p120-catenin for tyrosine phosphorylation and found that it was significantly diminished by Cdx1 or Cdx2 expression. We restored beta- and p120-catenin tyrosine phosphorylation in Cdx2-expressing cells by knocking down the expression of protein tyrosine phosphatase 1B and noted a significant decline in cell-cell adhesion. We conclude that Cdx expression in Colo 205 cells induces E-cadherin-dependent cell-cell adhesion by reducing beta- and p120-catenin tyrosine phosphorylation. Ascertaining the mechanism for this novel Cdx effect may improve our understanding of the regulation of cell-cell adhesion in the colonic epithelium.

CELL ADHESION JUNCTIONS are important regulators of intestinal cell proliferation, apoptosis, migration, and columnar morphology (26, 29, 32). Moreover, these adhesion junctions are necessary for the highly specialized barrier function of normal intestinal epithelium (44). Three different types of junctional complexes contribute to the strength and function of the intestinal epithelium: adherens junctions (AJ), desmosomes, and tight junctions (TJ). While the mechanisms regulating intestinal epithelial proliferation, differentiation, and lineage determination have been explored (11, 15, 30, 37, 82), little is known about how these mechanisms promote the development of a polarized, columnar cell morphology and the variety of adhesive junctions that are essential to the function of the intestinal epithelium.

AJs are protein complexes that support cell to cell binding. They are required for the normal development and function of the epithelium (26, 41, 70). These junctions play critical roles in a number of processes including cell polarization, motility, proliferation, and cell-cell membrane compaction. In intestinal epithelia, AJ function is mediated by the transmembrane protein E-cadherin. E-cadherin must be connected to the actin cytoskeleton for strong cell-cell binding to develop (25, 61). Two proteins serve to bridge E-cadherin to the actin cytoskeleton, {alpha}- and beta-catenin. In addition, another catenin, p120-catenin, is a critical regulator of cadherin protein stability and activity (17, 26, 41, 70).

The connection with the cytoskeleton allows E-cadherin to modulate a variety of cellular processes. Cell-cell contact and E-cadherin engagement prompts a reorganization of the actin cytoskeleton that serves to specify the lateral cell membrane domains and promote cell polarization (55, 84, 93). Epithelial cell motility depends on the ability to relax cell-cell adhesive interactions and cytoskeletal reorganizations, all of which depend in part on the modulation of AJ function (56, 93). Membrane compaction, the process of actively spreading the adhesion laterally from the point of initial contact, requires E-cadherin function and actin polymerization (1, 61, 83). Finally, due to its interactions with beta-catenin, E-cadherin activity can modulate Wnt/beta-catenin nuclear signaling and suppress proliferation (24, 42, 45, 58, 71).

Due to these critical effects on proliferation and migration, E-cadherin expression and activity are tightly regulated in normal epithelial cells (25, 60, 77, 78). We see further evidence for this in the observation that disruption of E-cadherin function is a common late event in the development of epithelial neoplasms. This disruption contributes to carcinogenesis by enhancing cell proliferation, migration, and tissue invasion (34, 60, 85). For these many reasons, the regulation of E-cadherin expression and function has been of great interest. E-cadherin and AJs are regulated at multiple levels including gene expression (60), protein stability (36, 43), intracellular trafficking (14), and function (25, 46). While these studies have illuminated a number of critical regulatory mechanisms common to all epithelium, they have not yielded insight into the regulation by tissue-specific factors, such as those that specify an intestinal epithelial phenotype.

The homeodomain transcription factors Cdx1 and Cdx2 have key roles regulating intestinal epithelial differentiation and proliferation (7, 28). The Cdx homologs modulate a diverse set of processes including proliferation, apoptosis, cell adhesion, and the acquisition of a columnar morphology (28). They are also necessary for the expression of an increasing number of intestine-specific genes (16, 18, 21, 31, 33, 38, 50, 72, 76, 79, 91, 92). By targeting these processes and genes, Cdx homologs promote the appearance of a mature intestinal columnar cell phenotype. Cdx expression in rat IEC6 cells, an undifferentiated intestinal cell line, induces a columnar morphogenesis characterized by the development of polarized columnar cells with apical microvilli (69, 73). Associated with these changes was the induction of new cell-cell adhesive junctions including AJs, desmosomal junctions, and TJs (73). While Cdx2 has been shown to regulate the expression of several cell-cell adhesion proteins including LI-cadherin and claudin-2 (33, 52, 67), no study has mechanistically explored the regulation of E-cadherin function or activity by Cdx1 or Cdx2.

Colo 205 cells are a poorly differentiated human colon cancer cell line that does not form strong cell-cell adhesive junctions despite the expression of all the components of the E-cadherin AJ complex (4). We (40) have previously reported that expression of Cdx1 or Cdx2 in Colo 205 cells induces a mature colonocyte phenotype that is associated with a more differentiated pattern of gene expression, induction of new cell-cell adhesion junctions, and membrane compaction (40). E-cadherin AJs have a key role in this process, as an anti-E-cadherin neutralizing antibody blocked cell-cell binding. In the present study, we analyzed in greater detail the molecular basis for Cdx-mediated induction of Colo 205 cell adhesion. Cdx1 or Cdx2 expression did not alter the protein levels or the intracellular distribution for E-cadherin or its associated AJ factors significantly. Cdx expression appeared to reduced beta- and p120-catenin tyrosine phosphorylation. Short hairpin (sh)RNA-targeted knockdown of protein tyrosine phosphatase (PTP)-1B restored the beta- and p120-catenin tyrosine phosphorylation and was associated with a reversal in the cell-cell adhesion phenotype. We conclude that Cdx expression in Colo 205 cells induces E-cadherin-dependent cell-cell adhesion by reducing beta- and p120-catenin tyrosine phosphorylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Colo 205 colon cancer cells were obtained from the American Type Culture Collection and maintained under the recommended 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 previously described (59). Murine Cdx1 or Cdx2 cDNA was subcloned into the MIGR1 retroviral vector (40, 48) and transfected into {Phi}-NX-A cells using the CellPHect calcium phosphate precipitation kit (Amersham Bioscience). Infectious retroviral supernatants were isolated at 48 h, purified as previously described (59), aliquoted, and stored at –70°C.

For migration and invasion assays, Colo-MIGR1 and Colo-MIGR-Cdx2 cells were serum starved overnight in RPMI with only 0.5% FBS. FluoroBlok inserts (Falcon) with 8.0-µm pore size were used in this assay. A single cell suspension was isolated in PBS after cells were scraped, and 1 x 105cells were then placed into the upper FluorBlok chamber in RPMI with only 0.5% FBS. RPMI with 20% FBS was placed in the lower chamber, and cells were incubated at 37°C. Fresh media were added each day. At 48 and 72 h, green fluorescent protein (GFP)-positive cells that had traversed the membrane were visualized and counted. For invasion assays, cells and media were prepared as before. A Tumor Invasion System (BD Bioscience) coated with Matrigel was utilized, and inserts were rehydrated as directed by the manufacturer. Then, 3 x 105cells were placed in the upper chamber, and cells were incubated at 37°C. GFP-positive cells were again counted at 48 h. Means and SDs were calculated and compared using Student's t-test.

Immunoblots and immunofluoroescence. Whole cell extracts were prepared as previously described (49). Briefly, cells were gently trypsinized from the plate, washed twice in PBS, and then resuspended in 80 µl of buffer A (1x PBS with 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.2 mM PMSF, 1 mM NaF, and 1 mM NaVO4). Two volumes of 2x lysis buffer were added [250 mM Tris·Cl (pH 7.4), 10% SDS, 20% glycerol, and 1 mM DTT), and cells were heated to 100°C for 5 min. Cells were placed on ice briefly, sonicated for 15 s, and then stored at –70°C. Protein concentration was determined by the BCA protein assay (Pierce). For immunoprecipitation-quality extracts, lysates were prepared with M-PER reagent (Pierece Biotechnology), and products were analyzed by SDS-PAGE and electroblotted. E-cadherin, beta-catenin, p120-catenin, {alpha}-catenin, {gamma}-catenin, phosphotyrosine-PY20, and PTP-1B antibodies (610182, 610154, 610134, 610194, 610254, 610000, and 610139, BD Transduction Laboratories) were used in immunoblot analysis. For phosphotyrosine assays, phosphatase activity was inhibited by treatment of the cell lysate with pervanedate as previously described (54). LI-cadherin antibody (sc-6978, Santa Cruz Biotechnology), claudin-2 (18–7363, Zymed Laboratories), and phospho-beta-catenin (Ser33/37/Thr41) antibodies (9561, Cell Signaling Technology) were also used in immunoblot analysis. E-cadherin, beta-catenin, and p120-catenin antibodies (610182, 610154, and 610134, BD Transduction Laboratories) were used in immunofluorescence. For the Western blot loading control, we used actin A-4700 (Sigma). Fluorescence was examined using a QImaging black-and-white charge-coupled device camera mounted on a Nikon E600 fluorescent microscope. Images were captured by the IP Lab Scientific Image processing software (Scanalytics) and deconvoluted using Huygens Essential software (Scientific Volume Imaging) using standard settings and 10 iterations.

Quantitative real-time PCR. Total RNA was isolated from MIGR1-, MIGR-Cdx1-, and MIGR-Cdx2-infected Colo 205 cells using RNeasy (Qiagen). The First-Strand cDNA Synthesis Kit (Invitrogen) was used for cDNA synthesis. Primer sequence and PCR concentrations are shown in Table 1. For RT-PCR, cDNA and primers were mixed with SYBR Green RT-PCR Master Mix (Applied Biosystems) and then assayed in an ABI Prism 7000 sequence detection system as directed by the manufacturer. A ribosomal phosphoprotein, 36B4, was used as the normalization control.


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  		Table 1. Primer sequences for quantitative RT-PCR analysis

 
Immunoprecipitation. Whole cell extracts were prepared with M-PER reagent (Pierece Biotechnology) including pervanadate solution (0.3 mM Na3VO4 and 0.6 mM H2O2) in lysis buffer. Immunoprecipitation was performed with Seize Classic Mammalian Immunoprecipitation Kit (Pierce Biotechnology). Antibodies (20 µg) were incubated with 100 µl of ImmunoPure Plus Immobilized Protein G for 1 h at room temperature and mixed with 1 mg of whole cell extract for 16 h at 4°C. E-cadherin sc-7870 and beta-catenin sc-1496 antibodies (Santa Cruz Biotechnology) were used in immunoprecipitation.

Production of the shRNA vector. The shRNA vector targeting PTP-1B was generated by annealing complementary oligonucleotides encoding a hairpin RNA with a previously reported targeting sequence (75). The annealed oligonucleotides were then cloned into pSUPER.Retro.neo vector (OligoEngine). Retroviral supernatants were made and collected as above, and retrovirally infected Colo-MIGR-Cdx2 cells were isolated by neomycin selection.

Cell dissociation assay. This protocol was adapted from a dissociation assay described by Nagafuchi et al. (57). Equal numbers of Colo-MIGRI, Colo-MIGR-Cdx2, Colo-MIGR-Cdx2-pSuper, and Colo-MIGR-Cdx2-pSUPER-PTP-1B 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. 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 EGTA, and 0.01% trypsin] and the other half were washed briefly 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. 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 crump of cells = 1 particle). The extent of dissociation equaled NTC/NTE, where NTC and NTE are the numbers of particles per milliliter counted by hemocytometer in the TC or TE buffer, respectively. These ratios were transformed using the arcsine transformation prior to statistical analysis with ANOVA and Tukey tests.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cdx2 expression reduces Colo 205 cell migration and invasion. In our previous study (40), we established that Cdx1 or Cdx2 expression induces a mature colonocyte morphology by activating E-cadherin-dependent AJs. In that study, we utilized a retroviral vector to express Cdx1 or Cdx2 in a human colon cancer cell line (Colo 205) that does not express endogenous Cdx genes. The level of Cdx2 expression achieved was similar to that seen naturally in colon cancer cell lines like DLD1 and LoVo and less than that of T84 cells (data not shown). To further explore the functional significance of the Colo 205 cell-cell adhesion changes, we initiated in vitro migration and invasion assays. We directly compared the ability of Cdx2-expressing and control Colo-205 cells to migrate and invade in Boyden chamber assays. Neither cell line rapidly transited the filter membrane in the migration assay; however, migration of Cdx2-expressing Colo 205 cells was reduced by 80% compared with control Colo-MIGR1 cells (P < 0.005; Fig. 1A). Similarly, Cdx2 expression diminished invasion through a Matrigel matrix by 40% (P < 0.02; Fig. 1B). Taken together, these observations suggest that the cell-cell adhesiveness of Cdx1- and Cdx2-expressing Colo 205 cells is physiologically significant and may play an important role in intestinal epithelial cell morphology and function.


Figure 1
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  		Fig. 1. Cdx2 expression reduces Colo 205 cell migration and adhesion activity. Colo-MIGR1 and Colo-MIGR-Cdx2 cells were serum starved overnight in RPMI with only 0.5% FBS, and cells were then placed in Boyden chambers for migration and invasion assays. A: for migration assays, 1 x 105 cells were placed into the upper FluorBlok (Falcon) chamber in RPMI with only 0.5% FBS. RPMI with 20% FBS was placed in the lower chamber, and cells were incubated at 37°C. At 48 and 72 h, green fluorescent protein-positive (GFP+) cells that had traversed the membrane were visualized and counted. *Significantly differs from controls by Student's t-test, P < 0.005 (n = 9), from 3 separate experiments. B: for invasion assays, cells and media were prepared as before. Then, 3 x 105 cells were placed in the upper chamber of a Matrigel-coated Tumor Invasion System (BD Bioscience), and cells were incubated at 37°C. GFP+ cells that had traversed the matrix and membrane were again visualized and counted at 48 h. *Significantly differs from controls by Student's t-test, P < 0.02 (n = 11), from 3 separate experiments.

 
Cdx1 or Cdx2 expression does not significantly alter the expression levels of AJ proteins in Colo 205 cells. Having established that Cdx-induced cell-cell adhesion can effect Colo 205 cell physiology in addition to morphology, we set out next to further explore the molecular basis for the effect. We thus investigated potential mechanisms by which the transcription factors Cdx1 and Cdx2 may exert their influence on E-cadherin-dependent cell adhesion. We first examined the protein expression levels for AJ proteins. It had previously been reported that Colo 205 cells expressed all the required components of an E-cadherin AJ complex (4). As expected, all components were detected (Fig. 2A). Protein levels of E-cadherin, beta-catenin, and {alpha}-catenin were essentially unchanged with Cdx1 or Cdx2 expression (Fig. 2A). {gamma}-Catenin, another armadillo family member that has been reported to substitute for beta-catenin in AJs under certain conditions (10), was similarly unaltered with Cdx expression. Protein levels of p120-catenin were modestly increased by Cdx1 or Cdx1 expression, but the increase was twofold or less and of unclear importance, considering the dramatic phenotype induced by Cdx expression. Of greater significance, a faster migrating band, possibly representing hypophosphorylated p120-catenin, was reliably observed in the Cdx-expressing Colo 205 cells when the electrophoresis time was greatly extended.


Figure 2
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  		Fig. 2. Expression of tight and desmosomal junction, but not adherens junction, proteins is enhanced with Cdx1 or Cdx2 expression in Colo 205 cells. Colo 205 cells infected with the control retrovirus MIGR1 or the viruses to express Cdx1 ore Cdx1 were cultured as described in the text, and total RNA and protein were isolated. A: immunoblot analysis for the adherens junction proteins E-cadherin, {alpha}-, beta-, {delta}-, and p120-catenin. M, control MIGR1-infected cells; X1, MIGR-Cdx1-infected cells; X2, MIGR-Cdx2 cells. *Significantly extended electrophoresis to better separate p120-catenin isoforms; 1 of 3 experiments is shown. B: a similar experiment examining the protein levels of other cell adhesion proteins. DSC2, desmocollin-2. C: 5 µg of total RNA from MIGR1, MIGR-Cdx1, and MIGR-Cdx2 cells were used for cDNA synthesis and then subjected to quantitative SYBR green RT-PCR analysis. 36B4, a ribosomal phosphoprotein, was used as the normalization control. The fold change in RNA levels was calculated from the threshold cycle values as previously described (64, 68, 86). *Significantly differs from controls, P < 0.05 (n = 4).

 
We also analyzed the protein levels for other cell adhesion molecules distinct from AJ factors (Fig. 2, B and C). Claudin-2, a critical component of TJs (3), and LI-cadherin, an unusual member of the cadherin superfamily lacking a significant cytoplasmic domain (23), were both induced by Cdx expression. Both of these genes are reported to be transcriptional targets of Cdx2 (33, 52, 67). In addition, Cdx1 and Cdx2 enhanced desmocollin (DSC)-2 protein levels. DSC-2 is a cadherin superfamily member, and it serves as the transmembrane portion of desmosome junctions. We examined RNA expression levels with quantitative real-time PCR for several of these factors (Fig. 2C). mRNA levels of E-cadherin and claudin-2 were essentially unchanged by Cdx expression. LI-cadherin mRNA levels were induced about threefold in Cdx-expressing Colo 205 cells. These findings suggest that Cdx1 and Cdx2 have important roles in the regulation of various cell-cell adhesion junctions including AJs, desmosomal junctions, and TJs._

Expression of Cdx1 or Cdx2 modulates the interactions between AJ proteins. Previously, we demonstrated that a monoclonal antibody that blocks E-cadherin binding can disrupt Cdx-mediated cell-cell adhesion in Colo-205 cells. Since Cdx expression did not appear to alter the levels of E-cadherin or other AJ proteins, we explored for effects of Cdx upon cadherin and catenin localization or protein-protein interactions. We localized E-cadherin, beta-catenin, and p120-catenin by immunofluorescence. E-cadherin was detected on the cell surface in all cells, and it colocalized with beta- and p120-catenin (Fig. 3). All three proteins colocalized to the cell surface and sites of cell-cell contact in all cells. p120-catenin protein was present at the cell surface in both Cdx1- or Cdx2-expressing and control cells, suggesting that E-cadhein function is not enhanced in Colo 205 cells by increased p120-catenin-mediated stabilization (43, 88). However, one critical difference noted between Cdx-expressing and control Colo-MIGR1 cells was the ability of Cdx-expressing cells to tightly adhere to each other and spread the points of contact laterally. Control Colo-MIGR1 cells remained round and were unable to increase the cell surface areas in contact (Fig. 3). This spreading of cell-cell contact is an active process known as membrane compaction (1, 83). This process requires functioning cadherin-mediated cell-cell adhesion for the contact to spread laterally.


Figure 3
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  		Fig. 3. Cdx2 expression does not alter cell surface expression of cell adhesion proteins. Colo-MIGR1 and Colo-MIGR-Cdx2 cells were cultured for several days, fixed, and then processed for immunofluorescence. E-cadherin, beta-catenin, and p120-catenin localized to the surface of the cells in overlapping distributions in both Colo-MIGR1 and Colo-MIGR-Cdx2. Images were captured by IP Lab Scientific Image processing software (Scanalytics) and deconvoluted using Huygens Essential Software (Scientific Volume Imaging) using 10 iterations with standard settings.

 
To further evaluate E-cadherin function in our Cdx-expressing Colo 205 cells, we investigated the interaction between E-cadherin and the catenins required for AJ function. E-cadherin was immunoprecipitated with an anti-E-cadherin antibody, and the immunoprecipitation products examined for the presence of beta- and p120-catenin by immunoblot analysis (Fig. 4). Cdx2, but not Cdx1, expression modestly reduced the efficiency of beta-catenin coimmunoprecipitation with E-cadherin, possibly because of strengthened linkage of beta-catenin to the insoluable actin cytoskeleton. Most significantly, expression of Cdx1 or Cdx2 reduced p120-catenin coprecipitation with E-cadherin. Thus, while our immunofluorescence data suggested E-cadherin and p120-catenin colocalized in vivo, the strength of the interaction between them appeared to be weakened with Cdx1 or Cdx2 expression (Fig. 4). Moreover, while p120-catenin is required to stabilize E-cadherin protein at the cell membrane, the strength of the E-cadherin-p120-catenin interaction can be significantly altered by posttranslational events without affecting E-cadherin stability (2). This observation is consistent with previously published observations demonstrating that p120-catenin is required to stabilize E-cadherin at the cell surface. In summary, Cdx expression in Colo 205 cells is associated with a significantly weakened interaction between E-cadherin and regulatory p120-catenin.


Figure 4
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  		Fig. 4. Reduced affinity of p120-catenin for E-cadherin in Cdx1- and Cdx2-expressing Colo 205 cells. Immunoprecipitation (IP)-quality protein lysates were prepared as described in the text from Colo-MIGR1, Colo-MIGR-Cdx1, and Colo-MIGR-Cdx2 cells. A: E-cadherin was immunoprecipitated, and the products were subjected to sequential immunoblotting for p120-catenin, beta-catenin, and E-cadherin. B: a small portion of the lysate input was run in parallel to evaluate starting levels of proteins prior to IP. One of three experiments is shown.

 
Cdx2 expression modulates p120-catenin and beta-catenin tyrosine phosphorylation. Phosphorylation of the catenins associated with the cadherin complex has been reported as an important regulator of AJ function. Recently, some investigators have reported that changes in the tyrosine phosphorylation state of beta-catenin and/or p120-catenin can regulate cadherin-mediated cell-cell adhesion (2, 8, 46, 62, 63, 89, 90). Typically, tyrosine phosphorylation of these catenins is associated with disruption of cadherin-actin cytoskeletal interactions and loss of cell-cell adhesive interactions. Moreover, tyrosine phosphorylation of p120-catenin can strengthen its interaction with E-cadherin (2). Given that our Cdx-expressing cells demonstrated active E-cadherin function and weakened p120-catenin/E-cadherin binding in immunoprecipitation experiments, we wondered if Cdx expression altered beta- and p120-catenin tyrosine phosphorylation. Therefore, to test for this, we immunoprecipitated beta-catenin or p120-catenin from Cdx1- or Cdx2-expressing and control cells and analyzed the products by Western blot. We sequentially probed with antibodies for anti-phospho-tyrosine, anti-p120-catenin, and anti-beta-catenin (Fig. 5A). Interestingly, we detected two tyrosine-phosphorylated bands in control Colo-MIGR1 cells immunoprecipitated with anti-beta-catenin antibodies but not in Cdx-expressing Colo 205 cells. By stripping and reprobing the blot for beta-catenin and p120-catenin, and aligning the Western blots based on molecular weight markers, we determined that the upper and lower bands were tyrosine-phosphorylated p120-catenin and beta-catenin, respectively (data not shown). E-cadherin could also be detected (data not shown) and likely served as the bridge linking these two factors. Immunoprecipitation of p120-catenin yielded similar results (Fig. 5B). There was considerable tyrosine-phosphorylated p120-catenin in control cells but none detected in Cdx1- or Cdx2-expressing cells, despite plenty of immunoprecipitated p120-protein. A weak beta-catenin coimmunoprecipitation band could also be detected, as could E-cadherin (Fig. 5B and data not shown). beta-Catenin is also modified by serine-threonine kinases. An immunoblot with anti-phospho-beta-catenin (Ser33/37/Thr41)-specific antibody was performed. Serine/threonine-phosphorylated beta-catenin was detected equally in Colo-MIGR and Cdx-expressing Colo 205 cells (data not shown). In summary, Cdx1 and Cdx2 expression reduces beta- and p120-catenin tyrosine phosphorylation. This hypophosphorylation is associated with weakened p120-E-cadherin protein-protein interactions and enhanced E-cadherin function.


Figure 5
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  		Fig. 5. Cdx expression reduces beta- and p120-catenin tyrosine phosphorylation. IP-quality protein lysates were prepared as described in the text from Colo-MIGR1, Colo-MIGR-Cdx1, and Colo-MIGR-Cdx2 cells. beta-Catenin and p120-catenin were immunoprecipitated, and the products were subjected to immunoblotting with an anti-phosphotyrosine (anti-pY) antibody. Blots were then striped and serially probed for p120-catenin and beta-catenin. A: beta-catenin immunoprecipitate was probed first for {alpha}-phosphotyrosine, and the blot was stripped and reprobed for beta- and p120-catenin. The beta-catenin Western blot is presented to demonstrate successful beta-catenin IP. A small portion of the lysate input was run in parallel to demonstrate starting levels of these proteins. B: p120-catenin immunoprecipitate was probed first for {alpha}-phosphotyrosine, and the blot was stripped and reprobed for beta- and p120-catenin. The p120-catenin Western blot is presented to demonstrate successful p120-catenin IP.

 
Reduction of PTP-1B levels reverses cell adhesion phenotype and restores beta- and p120-catenin tyrosine phosphorylation. The nonreceptor tyrosine phosphatase PTP-1B has been proposed to serve a critical role maintaining continuous beta-catenin dephosphorylation at Tyr654 (46, 89, 90). This supports E-cadherin function by maintaining the E-cadherin-beta-catenin interaction required for linkage to the actin cytoskeleton. We investigated whether Cdx expression was associated with a change in PTP-1B protein levels but found none (Fig. 6). However, given its critical role in maintaining beta-catenin dephosphorylation at Tyr654, we elected to perturb PTP-1B levels in our cells using shRNA to determine the effect it had upon E-cadherin function and catenin tyrosine phosphorylation. We subcloned a shRNA sequence targeting PTP-1B (75) into the retroviral vector pSuper.retro.neo (OligoEngine). We successfully established cell lines expressing the retrovirus that resulted in substantial reductions in PTP-1B protein levels (Fig. 6). Immediately, we noticed a change in cell morphology from the tight-clustering cells apparent with Cdx1 or Cdx2 expression to cells more similar to the MIGR1 and wild-type Colo 205 cells that were loosely associated and less compacted than the Colo-MIGR-Cdx2 cells from which they were derived (Fig. 6). Cdx2 protein levels were unchanged by the shRNA knockdown of PTP-1B protein (data not shown). This effect was specific, as the control empty pSuper vector, as well as pSuper vectors targeting other factors, did not disrupt cell-cell binding in a similar manner ( Fig. 5 and data not shown).


Figure 6
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  		Fig. 6. Reduction of protein tyrosine phosphatase (PTP)-1B protein levels reverses Cdx1- and Cdx2-mediated cell clustering. PTP-1B expression was targeted using the short hairpin (sh)RNA-expressing retrovirus pSuper.retro.neo (Oligoengine). Colo-MIGR-Cdx2 cells were infected with either the empty retrovirus (sup) or the virus containing the PTP-1B targeting sequence (PTP-1B), and neomycin-resistant cells were selected. A: Western blot for PTP-1B protein levels. M, Colo-MIGR1 cells; X2, Colo-MIGR-Cdx2 cells; X2sup, Colo-MIGR-Cdx2-pSuper cells; X2PTP1B, Colo-MIGR-Cdx2-pSup-PTP1B cells. B: phase-contrast microscopy of PTP-1B knockdown and control cells showing changes in cell morphology and cell-cell adhesion with loss of PTP-1B protein levels.

 
To quantify the degree of disruption to the cell adhesion phenotype, we performed a quantitative cell dissociation assay in the presence and absence of Ca2+. In the presence of Ca2+, the Colo-MIGR1 cells' dissociation index was 0.55 (Fig. 7), and there was minimal clustering of the cells on visual inspection (data not shown). The Colo-MIGR-Cdx2 and MIGR-Cdx2-pSuper control cells' dissociation index was sixfold lower, ~0.09, reflecting greater Ca2+-dependent cell-cell adhesion than control cells. However, Colo-MIGR-Cdx2 cells with the pSuper vector targeting PTP-1B had a significant reversion of their measured dissociation index to 0.28, about half of MIGR1 control cells. This quantitative assay measuring adhesion function confirmed the impressions from phase-contrast microscopy showing that cell-adhesiveness is significantly reduced by PTP-1B knockdown. Moreover, it suggests PTP-1B activity is required for the cell-cell adhesion activity induced by Cdx1 and Cdx2 in Colo 205 cells. We also assessed whether the knockdown restored tyrosine phosphorylation to beta-catenin. As before, we immunoprecipitated beta-catenin and probed sequentially with anti-phosphotyrosine and anti-beta-catenin antibodies. We observed that PTP-1B knockdown restored tyrosine phosphorylation to beta- and p120-catenin (Fig. 7). Moreover, p120-catenin, which did not efficiently coimmunoprecipitate with E-cadherin or beta-catenin when Cdx2 was expressed, once again pulled down readily with beta-catenin when PTP-1B levels were reduced and tyrosine phosphorylation of the catenins was reestablished (Fig. 7).


Figure 7
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  		Fig. 7. PTP-1B reduction is associated with increased cell dissociation and beta- and p120-catenin tyrosine phosphorylation. A: Colo-MIGR1, Colo-MIGR-Cdx2, Colo-MIGR-Cdx2-pSuper, and Colo-MIGR-Cdx2-pSup-PTP1B cells were subjected to a cell dissociation assay to quantify Ca2+-dependent adhesion, a measure of cadherin function. Equal numbers of cells were plated. On day 6, cells were lightly trypsinized, washed briefly, pelleted, resuspended in buffers containing either 1 mM CaCl2 (TC buffer) or 1 mM EGTA (TE buffer), and incubated at 37°C. After 30 min, cells were dispersed by repipetting, and the numbers of particles (N; single cell or cell clumps) were counted on a hemacytometer. The dissociation index was calculated for each pool of cells as the ratio of NTC to NTE (n = 12). *Significantly differs from MIGR1, P < 0.001; bullet significantly differs from MIGR-Cdx2 and MIGR-Cdx2-psuper, P < 0.05. B: IP-quality protein lysates were prepared as described in the text from Colo-MIGR1 (MIGR1), Colo-MIGR-Cdx2 (M-X2), and Colo-MIGR-Cdx2-pSup-PTP1B (M-X2PTP1B) cells. beta-Catenin was immunoprecipitated, and the products were subjected to immunoblotting with an anti-phosphotyrosine antibody. Blots were then striped and reprobed for beta-catenin. One of several experiments is shown. C: Colo-MIGR-Cdx2, Colo-MIGR-pSuper, and Colo-MIGR-pSup-PTP1B cells were cultured for several days, fixed, and then processed for E-cadherin localization by immunofluorescence as described in the text. Images were deconvoluted using Huygens Essential Software (Scientific Volume Imaging).

 
Finally, to determine if the PTP-1B knockdown altered E-cadherin localization or function, we performed an immunofluorescence study of E-cadherin. We found that E-cadherin protein remained localized to the cell surface in all the cells whether or not PTP-1B protein was present at control levels. Moreover, we noticed that cells with PTP-1B knockdown had significantly reduced ability to undergo membrane compaction. Membrane compaction is the ability of cells to extend areas of contact laterally, closing off empty spaces between cells. We (40) noted previously that Cdx1 or Cdx2 expression specifically induced membrane compaction in Colo 205 cells. Thus, the knockdown of PTP-1B in Cdx2-expressing Colo 205 cells appeared to specifically reverse Cdx2's effects rather than simply inhibit E-cadherin function through an unrelated mechanism. We therefore conclude that shRNA knockdown of PTP-1B in Colo-MIGR-Cdx2 cells restores beta-catenin tyrosine phosphorylation and partially reverses the cell adhesion phenotype induced by Cdx expression.


   DISCUSSION
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 ABSTRACT
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Cell adhesion junctions are critically important regulators of intestinal cell biology and are necessary for the highly specialized barrier function of the normal intestinal epithelium (44). However, little is known about how the mechanisms that regulate intestinal cell differentiation act to promote the required cell-cell adhesive junctions. In a prior study (40), we established that expression of the intestine-specific homeodomain transcription factors Cdx1 or Cdx2 alone is sufficient to induce E-cadherin cell-cell adhesion activity in Colo 205 cells, a human colon cancer cell line. In the present study, we extended this finding with an exploration of the molecular basis for this effect.

Activation of cell-cell adhesion by Cdx2 is associated with reduced cell migration and invasion. In our original report, we described the induction of cell-cell adhesion by Cdx1 and Cdx2 expression but did not explore further the physiological implications of this effect. Here, we used Boyden chambers and established for the first time that Cdx expression was associated with reduced cell migration and invasion through a Matrigel-coated membrane. This finding was expected, given the literature regarding the role of E-cadherin function in limiting cell migration and tumor invasion; however, this had never before been demonstrated with Cdx1 or Cdx2 expression. This establishes that the cell-cell adhesion induced by Cdx1 or Cdx2 expression was physiologically significant. More importantly, it suggests that these processes may contribute to intestinal cell morphology and barrier maintenance in vivo. Finally, it suggests that restoration of Cdx2 expression may serve to limit the invasiveness and cell migration in some human colon cancers.

AJ dysfunction in Colo 205 cells is not due to loss of cadherin or catenin protein expression. Unlike many epithelial cancers (13, 20, 34, 36, 85), loss of E-cadherin function in Colo 205 cells does not appear to be due to loss of cadherin or catenin protein expression. Colo 205 cells are a poorly differentiated human colon cancer cell line that does not form strong cell-cell AJs despite the expression of all the components of the E-cadherin AJ (4). This unusual circumstance has been of interest to cell biologists, who have utilized inhibitors of protein kinase C and tyrosine kinases to explore the regulation of cadherin function in these cells (4, 51). However, the molecular basis for the E-cadherin dysfunction remains unknown.

Colo 205 cells also do not express their endogenous Cdx1 and Cdx2 genes. When we restored this expression using retroviral vectors, we noted a differentiation of these cells to a more mature phenotype, including activation of several types of cell-cell adhesion junctions and the emergence of a polarized, columnar morphology. E-cadherin function is required for this cell adhesion phenotype. To elucidate the molecular basis for this effect, we looked initially for changes in the levels or localization of factors involved in cell adhesion. Cdx expression was not associated with a significant change in RNA or protein levels for E-cadherin. Moreover, neither {alpha}-, beta-, nor {gamma}-catenin protein levels were significantly altered, either. In addition, we found abundant levels of all these factors at the cell membrane and regions of cell-cell membrane contact in our control Colo-MIGR1 cells, suggesting that subcellular sequestration was not the reason for the E-cadherin dysfunction.

Tyrosine phosphorylation is a potent regulator of E-cadherin function and p120-catenin/E-cadherin interactions in Colo 205 cells. A clue to the regulatory mechanism did emerge from our investigations of p120-catenin. Cdx expression did modestly enhance p120-catenin protein levels, but what was more striking was the increase in faster migrating p120-catenin, possibly due to increases in a hypophosphorylated form of p120-catenin. Moreover, when we examined for coimmunoprecipitation of catenins with E-cadherin, we observed that p120-catenin precipitated strongly with E-cadherin in control Colo-MIGR1 cells but not Cdx-expressing cells (Fig. 3). This suggested to us that Cdx1 or Cdx2 expression induced E-cadherin function by a mechanism that resulted in a weakened E-cadherin/p120-catenin binding strength.

beta-Catenin and p120-catenin are both phosphoproteins, and tyrosine phosphorylation in particular has been associated with enhancing p120-catenin/E-cadherin binding interactions as well as disruption of E-cadherin function (2, 8, 46, 62, 63, 89, 90). We therefore investigated specifically for changes in tyrosine phosphorylation of beta- and p120-catenin associated with Cdx1 or Cdx2 expression. We found that tyrosine phosphorylation of both beta- and p120-catenin was largely lost in Cdx-expressing Colo 205 cells (Fig. 5). This appeared to be specific for tyrosine phosphorylation, as levels of serine-threonine phosphorylation of beta-catenin (Ser33/37/Thr41) were unchanged with Cdx expression (data not shown).

Tyrosine phosphorylation of beta-catenin can disrupt its interactions with E-cadherin or {alpha}-catenin, depending on which site is phosphorylated. This can cause a breakdown of the bridge to the actin cytoskeleton and disrupts strong cell-cell adhesion activity (62, 63) To confirm that these observed changes in tyrosine phosphorylation were functionally responsible for the activation of E-cadherin, we attempted to reverse it. The dephosphorylation of beta-catenin at Tyr654 is maintained in part by the activity of the nonreceptor tyrosine phosphatase PTP-1B (46, 89, 90). We successfully established cell lines with diminished PTP-1B levels in Cdx2-expressing Colo 205 cells using a shRNA sequence targeting PTP-1B (75). There was an obvious reversal in the phenotype when PTP-1B levels were reduced, and cell-cell adhesion appeared to be significantly diminished on inspection by phase-contrast microscopy. This effect was specific, as Cdx2 levels were unchanged in these cells, and the control empty pSuper vector, as well as pSuper vectors targeting other factors, did not disrupt cell-cell binding (Fig. 6 and data not shown).

We quantified this reduction using a cell dissociation assay that measures Ca2+-dependent cell adhesion and found it to be significantly diminished when PTP-1B protein levels were reduced (Fig. 7). Therefore, Cdx-expressing Colo 205 cells require ongoing PTP-1B activity to fully promote Ca2+-dependent cell-cell adhesion. Moreover, when we examined beta- and p120-catenin for tyrosine phosphorylation, we found that the knockdown of PTP-1B protein was associated with a return of tyrosine phosphorylated beta- and p120-catenin. Additionally, the tight association between p120-catenin and E-cadherin, which was weakened by Cdx expression and loss of tyrosine phosphorylation, was also restored (Fig. 7 and data not shown). Taken together, these observations suggest that Cdx1 or Cdx2 expression in Colo 205 cells restores E-cadherin function and cell-cell adhesion by promoting dephosphorylation of beta- and p120-catenin tyrosine residues. Moreover, PTP-1B phosphatase appears to be required to maintain these catenins in a dephosphorylated state. It is presently unclear whether this phosphatase is directly involved in the Cdx-mediated dephosphorylation of beta- and p120-catenin or simply required to reverse an unrelated kinase activity. We were unable to demonstrate a change in PTP-1B protein levels associated with Cdx expression. It remains a possibility that Cdx expression alters PTP-1B activity rather than its levels through an as-yet unidentified mechanism. To prove this, we would need to be able to measure native PTP-1B phosphatase activity in our cells, which we have tried to do but have not yet succeeded. This remains a question to be addressed in future studies.

Regulation of E-cadherin function by beta- and p120-catenin tyrosine phosphorylation. Our findings with Cdx expression in Colo 205 cells agrees with prior reports (8, 47, 63) describing the role of tyrosine phosphorylation in the regulation of cadherin function. beta-Catenin and p120-catenin are well-described substrates for a number of tyrosine kinases including the Src, Fer, Fyn, and Yes kinases (46, 53, 63, 90). Tyrosine phosphorylation by these kinases has long been associated with disruption of cadherin function, but recently a molecular explanation for this effect has been put forward (5, 47, 62, 63, 89). E-cadherin and {alpha}-catenin binding can be disrupted by tyrosine phosphorylation of beta-catenin at specific residues. This disruption severs the link to the cytoskeleton, a link critical to strong E-cadherin-medicated cell-cell adhesion.

Equally important to this regulatory mechanism is the removal of phosphate groups, which can be effected by tyrosine phosphatases, a large family of proteins. A number of these phosphatases have been shown to dephosphorylate beta- and/or p120-catenin tyrosine residues, including PPTP-1B, PTP-1C, PTP-1D, PTPRJ, PTP-µ, PTP-{kappa}, PTP-D2, and LMW-PTP (6, 9, 12, 22, 35, 39, 46, 74). Modulation of the expression levels for these phosphatases is associated with altered cell-cell binding and cadherin function and is marked by changes in tyrosine phosphorylation of beta- and p120-catenin or E-cadherin. One model has suggested that p120-catenin may serve as the scaffolding by which the nonreceptor tyrosine kinases and phosphatases bind and modulate the phosphorylation state of AJ proteins (46, 89, 90).

Previous studies by other investigators have also suggested a possible role for tyrosine phosphorylation in the regulation of Colo 205 cell adhesion. The Src kinase inhibitor herbimycin A was shown to induce Colo 2005 cell aggregation, polarization, and differentiation (51). In another study (4), the broad-spectrum kinase inhibitor staurosporin rapidly induced E-cadherin-mediated cell-cell adhesion in Colo 205 cells within 4–6 h. Staurosporin is a potent inhibitor of Src, Fgr, Lyn, and Syk tyrosine kinases in addition to protein kinases A and C and others. Moreover, these investigators demonstrated that both staurosporin or herbimycin A treatment induced changes in p120-catenin band migration on a Western blot analysis that was consistent with protein dephosphorylation. Finally, one group established that expression of a p120-catenin mutant in which the NH2-terminus is truncated acted in a dominant fashion to induce cadherin-mediated cell adhesion in Colo 205 cells. The NH2-terminus of p120-catenin is where the majority of tyrosine and serine-threonine phosphorylation sites are located (87). Thus, in summary, our findings are consistent with prior observations in Colo 205 cells as well as current models for the regulation of cadherin function in vivo.

Cdx1 and Cdx2 have overlapping functions and gene targets. In some of the earliest studies of Cdx1 and Cdx2, initial immunohistochemical analyses suggested that Cdx2 expression was limited to the differentiated compartment of the intestine (intestinal villi and surface epithelium of the colon). In contrast, Cdx1 was found to be expressed in the crypt compartment, including cells that were actively proliferating. This led some to conclude that Cdx1 and Cdx2 had "opposing" effects on intestinal epithelial cells. Thus, Cdx1 was thought to be an "oncogene" and inhibit differentiation, and Cdx2 was thought to be "a tumor suppressor" and promote differentiation. The data over the years have not supported this rigid conception. We now know Cdx2 is indeed expressed in the crypt compartment (66). Also, in certain contexts, Cdx2 expression can promote cell proliferation and carcinogenesis, in other words, behave as an oncogene (19, 65, 80). Other work by the present authors (27, 48, 49) has demonstrated that both Cdx1 and Cdx2 can both inhibit Wnt signaling and thereby limit colon cancer cell proliferation.

At the amino acid level, Cdx1 and Cdx2 share a great deal of homology. The DNA-binding homeodomain is highly conserved (>90%), suggesting that there should be a considerable overlap in the DNA binding sites they recognize. Outside of the DNA binding region, there are other large regions of homology, suggesting an overlap in function as well. This is supported by studies in our laboratory, where Cdx1 can transcriptionally activate classic Cdx2-targeted promoters like sucrase isomaltase and intestinal phospholipase A/lysophospholipase (data not shown). This is also supported by published genetic studies. The skeletal abnormalities in double-mutant mice (Cdx1–/–/Cdx2+/–) are more severe than those in either mutation alone (Cdx1–/– or Cdx2+/– mice), suggesting that Cdx1 and Cdx2 have overlapping functions with regards to skeletal axis elongation (81).

Therefore, for all of theses reasons, it was not surprising that Cdx1 and Cdx2 elicited similar responses when expressed in Colo 205 cells. They are both primarily transcriptional activators with highly conserved DNA binding domains. They ought to share a large number of target genes and effects. In summary, Cdx1 and Cdx2 expression in Colo 205 cells promotes cell-cell adhesion and columnar morphogenesis by inhibiting tyrosine phosphorylation of beta- and p120-catenin. This allows the development of strong E-cadherin binding, a prerequisite for the development of cell polarity as well as TJs and desmosomal junctions. Future studies will explore the mechanism by which Cdx regulates tyrosine phosphorylation of these critical cell-cell adhesion proteins as well as how Cdx expression coordinates the development of TJs and desmosomal junctions in addition to AJs. The coordinated function of all three junctions is necessary for proper intestinal epithelial function, and their loss during carcinogenesis may contribute to enhanced metastatic ability of colon cancer cells.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-02695, DK-047437, DK-062819, and DK-068366 (to J. Lynch); a Pilot Project award from the Center for Molecular Studies in Digestive and Liver Disease (to J. Lynch); and the Morphology, Cell Culture, and Molecular Biology Core Facilities of the NIDDK Center for Molecular Studies in Digestive and Liver Disease at the University of Pennsylvania (Grant P30-DK-50306).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. P. Lynch, Div. of Gastroenterology/650 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.


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