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Originally published In Press as doi:10.1074/jbc.M103450200 on August 1, 2001

J. Biol. Chem., Vol. 276, Issue 42, 39094-39102, October 19, 2001
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Progressive Changes in Adherens Junction Structure during Intestinal Adenoma Formation in Apc Mutant Mice*

Adelaide M. CarothersDagger , Kurt A. Melstrom Jr.Dagger , James D. Mueller§, Michael J. WeyantDagger , and Monica M. Bertagnolli||

From the Dagger  Department of Surgery, Weill College of Medicine, Cornell University, New York, the Strang Cancer Prevention Center, New York, New York 10021, and the  Departments of Surgery and § Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115

Received for publication, April 18, 2001, and in revised form, July 27, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (ApcMin/-). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (ApcMin/+) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc+/+) or with heterozygous loss of Apc protein (Apc1638N). To study this phenotype further, we determined the effect of the ApcMin mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta -catenin in ApcMin/+ enterocytes. Subcellular fractionation of proteins from Apc+/+, ApcMin/+, and ApcMin/- intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in ApcMin/+, suggesting E-cadherin internalization in these enterocytes. beta -Catenin tyrosine phosphorylation was also increased in ApcMin/+ enterocytes, consistent with its dissociation from E-cadherin. Furthermore, ApcMin/+ enterocytes showed a decreased association between beta -catenin and receptor protein-tyrosine phosphatase beta /zeta (RPTPbeta /zeta ), and ApcMin/- cells demonstrated an association between beta -catenin and receptor protein-tyrosine phosphatase gamma . In contrast to the ApcMin/+ enterocytes, ApcMin/- adenomas displayed increased expression and association of E-cadherin, beta -catenin, and alpha -catenin relative to Apc+/+ controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (ApcMin/+) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The adenomatous polyposis coli (APC)1 protein regulates intestinal cell growth. Loss of APC protein by germ line mutation causes familial adenomatous polyposis, an autosomal dominant cancer syndrome characterized by thousands of lower intestinal adenomas. APC loss is also an initiating event in most sporadic colorectal cancer (1, 2). The best characterized function of APC is its association with a multiprotein complex that facilitates the degradation of the oncoprotein, beta -catenin (3, 4). APC binds to free cytosolic beta -catenin together with the scaffolding protein, axin, and glycogen synthase kinase 3beta (4, 5). This kinase phosphorylates beta -catenin on serine and threonine residues in a domain of the N terminus. These modifications, in turn, initiate the degradation of beta -catenin by the ubiquitin-dependent proteasome (6). Missense mutations in the CTNNB1 gene that eliminate the glycogen synthase kinase 3beta substrate residues of beta -catenin permit tumorigenesis without APC mutation by stabilizing the protein and allowing its downstream signaling activities (7-10). In tumor cells bearing mutant beta -catenin or APC proteins, free cytosolic beta -catenin escapes degradation and binds to Tcf-4, producing a transcriptional regulator that is capable of inducing the expression of growth-promoting genes such as c-Myc and cyclin D1 (8, 11-13).

The largest cellular pool of beta -catenin is membrane-associated and engaged in adhesion-mediated signaling via cell-cell contact at the AJ (14). The AJ is a highly dynamic structure composed of the transmembrane protein, E-cadherin, and several members of the catenin family, including alpha -, beta -, and gamma -catenin, and p120ctn (reviewed in 15). When bound to E-cadherin, beta -catenin links adhesion complexes to the actin cytoskeleton in ways that maintain epithelial cell polarity, preserve barrier function, and facilitate cell migration (15). Studies examining the localization of wild-type and cancer-associated truncated APC proteins suggest the involvement of this tumor suppressor in AJ function (reviewed in Ref. 16). By altering the availability of beta -catenin for engagement with AJ constituent proteins, APC may indirectly modulate epithelial cell adhesion (17). APC was found co-localized with the actin cytoskeleton and with AJs in the epithelial cells of Drosophila embryos consistent with a role in regulating epithelial cell-cell contacts (18, 19). Immunohistochemical studies also showed that APC resides in the lateral cytoplasm of intestinal epithelial cells, in regions containing the catenins (20). Tumor-associated APC mutations generally cause chain termination and result in expression of truncated proteins lacking the C terminus (21). By immunofluorescence microscopy, both full-length and truncated APC proteins were associated with the membrane at the apical borders of murine intestinal cells (22), suggesting that both forms of the protein are available to modulate adhesive interactions.

Epithelial homeostasis depends on the dynamic regulation of cadherin-catenin adhesion complexes at the AJ. The intestinal epithelium is normally maintained by the robust proliferation of stem cells situated in crypts. The progeny of stem cells differentiate as they migrate to the tips of the villi, where they eventually senesce and are exfoliated into the intestinal lumen. To mediate the processes of embryonic development, wound healing, and crypt-villus migration, the AJ must be efficiently disassembled and reassembled (reviewed in Ref. 23). The importance of the AJ to epithelial homeostasis is illustrated by the observation that loss of AJ structure correlates with tumor formation, invasion, and metastasis (24-26).

Targeted gene disruption in mice shows that beta -catenin is essential for maintenance of AJ activity, as this protein is required for the structural organization of the embryonic ectoderm (27). A high expression of E-cadherin reduces the nuclear localization and transcriptional potential of beta -catenin (14, 28). The interactions of beta -catenin and E-cadherin, and consequently downstream signal transduction, are also regulated by tyrosine phosphorylation of AJ constituents including beta -catenin (29-31). beta -Catenin can be phosphorylated via c-Src family tyrosine kinases (SFKs) such as Fer (32) or epidermal growth factor receptor (EGFR) (33). In addition, receptor protein-tyrosine phosphatases (RPTPs) can dephosphorylate beta -catenin, thus promoting cell-cell adhesion and migration (34). The consequence of beta -catenin tyrosine phosphorylation is its dissociation from the AJ (14, 33, 35) which, in turn, can cause loss of AJ structure (36, 37). Under certain conditions of AJ loss, E-cadherin may be internalized and returned to the membrane of epithelial cells as adhesion contacts re-form (36, 37).

The importance of appropriate adhesive interactions to optimal enterocyte migration was demonstrated by the targeted expression of E-cadherin in the murine small intestine (38, 39). Previous studies in our laboratory showed that Apc mutation also affects enterocyte migration in the intestinal mucosa of Min/+ mice. The germ line of the Min/+ mouse contains a chain terminating mutation in Apc, and Min/+ mice develop multiple intestinal adenomas that exhibit loss of the wild-type Apc allele (ApcMin/-). We found that Min/+ non-tumor enterocytes (ApcMin/+) displayed an ~25% reduced migration rate when compared with the Apc+/+ littermate controls and the functionally hemizygous Apc1638N enterocytes (40). This result suggested Apc genotype-dependent alterations in intestinal cell-cell adhesion. To characterize further the effect of the ApcMin allele upon enterocyte adhesion, we examined the changes in AJ structure that occur during the progression from wild-type (Apc+/+) to slowly migrating ApcMin/+ enterocytes and to adenoma cells (ApcMin/-). We extracted enterocytes from the mouse small intestine using a method that preserves cell-cell and cell-ECM contacts and therefore provides an accurate evaluation of the in vivo status of adhesion complexes (41). Here we show that diminished beta -catenin-E-cadherin association and internalization of E-cadherin occurs in the non-tumor enterocytes from Min/+ mice when compared with their wild-type littermates and to ApcMin/- adenomas. These changes suggest that the Min mutation alters the membrane-associated pool of beta -catenin in adult animals in ways that reduce cell-cell adhesion and slow enterocyte migration but that nevertheless maintain the histologically normal appearance of the mucosa. The data further support the view that the Min mutation yields a dominant negative effect on Apc function.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Five-week-old female Min/+ mice were obtained from The Jackson Laboratories (Bar Harbor, ME). AIN-76A chow was prepared by Research Diets (New Brunswick, NJ). Antibodies directed against beta -catenin (clone 14), EGFR (clone 13), RPTPbeta /zeta (clone 12), p120ctn (clone 98), and against an epitope on the intracellular portion of E-cadherin (clone 36) were purchased from BD Transduction Laboratories (San Diego, CA). Anti-RPTP-gamma (M-18), anti-PTP LAR (R-20), and anti-c-Src (SRC2) (sc-18) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A second RPTPbeta /zeta antibody (MAB5210) was generously provided by Chemicon International, Inc. (Temecula, CA). Anti-alpha -catenin (clone alpha CAT-7A4) and anti-E-cadherin (clone ECCD-2) antibodies were obtained from Zymed Laboratories Inc. (South San Francisco, CA). Anti-phosphotyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibody recognizing the extracellular domain of E-cadherin (DECMA-1) and anti-beta -actin antibody (clone AC-40), and the proteasome inhibitor, N-acetyl-Leu-Leu-norleucinal (ALLN), were purchased from Sigma. Anti-v-Src antibody (Ab-1) was purchased from Oncogene Research Products (Cambridge, MA), and anti-Src-p-Tyr416 was obtained from Cell Signaling Technology (Beverly, MA). The LSAB2 detection system was obtained from Dako Corp. (Carpinteria, CA). IPs used the protein G kit of Roche Molecular Biochemicals. Reagents and materials for IB analyses were as described previously (41).

Animal Maintenance and Enterocyte Isolation-- Female Apc+/+ and ApcMin/+ mice were fed AIN-76A diet and tap water ad libidum. Similar growth and food intake occurred in the two groups. At 110 days of age all mice were euthanized by CO2 inhalation, and their intestinal tracts were removed, opened, and washed with cold phosphate-buffered saline (PBS). Tumors were excised, pooled, and frozen in liquid N2. Enterocytes from the ileum were isolated in the following manner. Sections of proximal small intestine were opened longitudinally, washed with PBS, and inspected for evidence of tumor involvement by examination under × 3 magnification. Enterocytes were removed from 1- to 2-cm segments of tumor-free ileum by lightly scraping the mucosal surface with the edge of a microscope slide. The material obtained by scraping was then washed in cold PBS, and the resulting pellet was frozen by immersion in liquid nitrogen. Frozen cell pellets were stored at -70 °C until the time of assay. Enterocytes prepared in this manner also contained some lamina propria and fibroblasts, although the absorptive enterocytes and goblet cells of villi substantially outnumbered these cell types (Fig. 1). Serial sections underestimate the ratio between enterocytes and stromal cells, which were shown by three-dimensional analyses to exceed other cell types by >100-fold (42, 43). These preparations, therefore, provided a favorable signal-to-noise ratio for studying the characteristics of post-mitotic, differentiated enterocytes in their in vivo condition.


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  		Fig. 1.   Enterocytes and adenoma cells isolated from mouse small intestine. Photomicrograph of a hematoxylin and eosin-stained paraffin section (× 100) demonstrates preserved cell-cell and cell-ECM interactions in enterocyte preparations, and the high ratio of histologically normal enterocytes to stromal and hematopoietic cells (A); hematoxylin and eosin-stained paraffin adenoma section (× 100) demonstrates preserved cell-cell and cell-ECM interactions, and the high ratio of adenoma cells to normal enterocytes and stromal cells (B).

Lysate Preparation, Immunoprecipitation (IP), and Immunoblot (IB) Analysis-- Cold lysis buffer (38) was added to the tumors or enterocytes pooled from two mice of the same genotype. Unless otherwise indicated, the proteasome inhibitor, ALLN (10 mM), was added to the lysis buffer. Cells and tumors in lysis buffer were separately homogenized by 10 strokes in chilled Dounces. All subsequent steps for protein isolation were performed at 4 °C. Lysates were centrifuged for 10 min at 12,000 × g, and protein concentrations of supernatants were determined by Lowry assay. Normalized aliquots of each lysate were placed in Laemmli buffer (67 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromphenol blue, and 0.05% beta -mercaptoethanol (v/v)) and stored at -70 °C. In parallel, portions of each lysate were pre-cleared to remove immunoglobulins by mixing with protein G beads at 4 °C for 4 h, followed by centrifugation and removal of the beads. After pre-clearing, the same amount of protein from each lysate was reacted with primary antibody for 1 h. Immune complexes were precipitated for 12 h with protein G beads. The beads were washed, and the bound proteins were released in 50 µl of Laemmli buffer. The entire amount of each IP sample or the total cell lysate samples were resolved by 7.5% SDS-polyacrylamide gel electrophoresis. Procedures for IB analyses were as detailed (42). Washed membranes were stripped by incubation at 65 °C for 20 min in 68 mM Tris-HCl, pH 6.8, 10% SDS, and 0.01% beta -mercaptoethanol before re-probing. All experiments were repeated at least twice using independently prepared lysates.

Immunohistochemistry-- Hematoxylin and eosin staining of formalin-fixed, paraffin-embedded tissue was performed, and tissues were examined by light microscopy to document histology. Serial sections of mid-small intestinal mucosa from Apc+/+ and ApcMin/+ animals were deparaffinized and rehydrated. Endogenous peroxidases were quenched in 3% H2O2, and the slides were rinsed in PBS. The sections were then incubated at 25 °C for 1 h with a rat anti-mouse E-cadherin antibody (clone ECCD-2). Detection was accomplished using the LSAB 2 system using a biotinylated anti-rat antibody at 1:300 dilution.

Membrane Fractionation-- Enterocytes and tumor cells isolated as described above were washed twice in Tris-buffered saline, pH 7.4, containing 1 mM CaCl2. Pelleted cells were frozen in liquid N2 and stored at -70 °C. At the time of assay, cell pellets were placed in 1 ml of a hypotonic lysis buffer without EDTA, NaCl, and detergent (10 mM Tris-HCl, pH 7.8, 1 mM CaCl2, 5 mM KCl, plus proteasome, protease, and phosphatase inhibitors as described (40)). Cells were allowed to thaw and swell on ice for 15 min, before being homogenized with 30 strokes. Cell suspensions were centrifuged for 5 min at 1,700 rpm. The pellet containing debris, nuclei, and unlysed cells were discarded; the supernatants were centrifuged at 100,000 × g for 1 h. The subsequent high speed centrifugation supernatants (S100) were retained; the pellets (P100) were suspended in lysis buffer containing 1% Triton X-100 and centrifuged as above. The supernatants (detergent-soluble P100) were retained; the pellets (detergent-insoluble P100), suspended in Laemmli sample buffer, were also retained. Aliquots of each fraction were removed for protein determination by Lowry assay. Normalized amounts of the fractionated proteins were resolved by 10% SDS-PAGE and IB analyses performed.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

E-cadherin-beta -Catenin Complexes Were Decreased in ApcMin/+ Enterocytes and Increased in ApcMin/- Adenoma Cells-- Because the migration of ApcMin/+ enterocytes is decreased relative to that of their wild-type littermates (Apc+/+) (40), we predicted that the AJ structure and/or function of ApcMin/+ intestinal cells is also altered. Furthermore, we expected that changes in the AJ would also be found in ApcMin/- tumor cells, since these cells may have lost the ability to migrate altogether (14, 15, 40, 44). We examined the overall expression of E-cadherin in Apc+/+, ApcMin/+, and ApcMin/- adenoma cells by IB analysis. As shown in Fig. 2A, equivalent expression of E-cadherin was present in Apc+/+ and non-tumor ApcMin/+ tissues; however, expression of this protein was increased in ApcMin/- adenoma cells. To examine the association between E-cadherin and beta -catenin in the tissue samples, beta -catenin was immunoprecipitated from tissue lysates prepared with the proteasome inhibitor, ALLN. An IB of the precipitated proteins detected the association of E-cadherin with beta -catenin in Apc+/+ cells (Fig. 2B). This experiment showed a significantly increased association of E-cadherin with beta -catenin in the ApcMin/- adenoma cells. In ApcMin/+ enterocytes, however, a decreased association between these proteins was evident.


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  		Fig. 2.   E-cadherin expression and beta -catenin association are altered during Apc-associated tumorigenesis. IB analysis of E-cadherin expression was performed using lysates from Apc+/+, ApcMin/+, and ApcMin/- cells. Proteins (50 µg) were resolved by 10% SDS-PAGE, electroblotted to a membrane. The top portion of the membrane was probed with clone 36 anti-E-cadherin antibody, and the bottom portion was probed with anti-actin antibody (A). B, IP of the same lysates (300 µg of protein) using anti-beta -catenin antibody followed by IB analysis using clone 36 anti-E-cadherin antibody. The band corresponding to 120-kDa E-cadherin in a HeLa cell lysate serves as a standard. Ig heavy chain (HC) bands (~50 kDa) serve as internal loading controls.

These results suggested an effect of the ApcMin allele on the AJ structure of intestinal epithelial cells. To characterize this effect further, we determined the subcellular location of E-cadherin and beta -catenin by fractionating Apc+/+, ApcMin/+, and ApcMin/- cells. The cells were lysed without detergent; nuclei were removed, and then the lysate samples were centrifuged at high speed to obtain cytosolic (S100) and membrane (P100) fractions. The membrane fractions were further separated by treatment with detergent to yield membrane-soluble and membrane-insoluble fractions. IB analyses were separately performed to detect both the intracellular (clone 36) and extracellular (DECMA-1) domains of E-cadherin. As shown in Fig. 3, E-cadherin was detected by both antibodies in the P100 membrane-associated (detergent-soluble) and membrane integral (detergent-insoluble) fractions in all three samples. The localization of E-cadherin in these two epithelial cell compartments was described in previous studies (45-47). Attachment of E-cadherin to the actin cytoskeleton via alpha - and beta -catenin renders the complex insoluble in detergent (45-47). Interestingly, this experiment showed an increase in both the extra- and intracellular components of E-cadherin in the cytosol of ApcMin/+ enterocytes (Fig. 3). Internalization of E-cadherin is promoted in epithelial cells grown under conditions that do not permit stable cell-cell contact, such as low cell density (36, 37). These results are therefore consistent with a significant loss of AJ structure in the ApcMin/+ enterocytes. This result was confirmed using immunohistochemistry to detect the location of E-cadherin in sections obtained from Apc+/+ and ApcMin/+ small intestine (Fig. 4). E-cadherin antibody demonstrates prominent staining of the lateral membranes in the Apc+/+ enterocytes (Fig. 4A) that is lost in the ApcMin/+ tissue (Fig. 4B).


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  		Fig. 3.   Comparison of E-cadherin and beta -catenin localization in Apc+/+, ApcMin/+, and ApcMin/- enterocyte lysates following cell fractionation. Intestinal cell scrapings and adenomas were lysed in hypotonic buffer without detergent. Cell fractionation was performed as detailed under "Experimental Procedures." IB analysis used 50 µg of protein for the E-cadherin blots (clone 36 and DECMA-1) and 10 µg for the beta -catenin (clone 14) one.


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  		Fig. 4.   Decreased membrane E-cadherin in ApcMin/+ enterocytes. Photomicrograph (× 100) of small intestine sections from Apc+/+ and ApcMin/+ mice. Immunohistochemistry performed using anti-E-cadherin antibody shows prominent membrane staining for E-cadherin in Apc+/+ enterocytes (A) that is substantially decreased in ApcMin/+ tissue (B).

Cell fractionation also showed that beta -catenin was located primarily in the membrane detergent-soluble P100 fraction of all three cell types (Fig. 3). The ApcMin/- adenomas lack functional Apc and are therefore unable to degrade free cytosolic beta -catenin (48, 49); therefore, we expected to find increased amounts of cytosolic beta -catenin in the ApcMin/- cells. Because the fractionation shown in Fig. 3 was performed using cells treated with a proteasome inhibitor, the increase in cytosolic beta -catenin in the ApcMin/- adenomas could not be detected. To compare the effect of Apc-mediated degradation on beta -catenin levels in Apc+/+, ApcMin/+, and ApcMin/- tissues, we measured beta -catenin expression by IB using whole cell lysates prepared with and without ALLN (Fig. 5). Bands of 92 kDa, the size of beta -catenin, were present in the ApcMin/+ and Apc+/+ samples at similar low intensities (Fig. 5, arrows, left). In the ApcMin/- tumor sample, however, a broad intense band was seen in this location, indicating a lack of beta -catenin degradation following loss of the wild-type Apc allele. As a control, the activity of the proteasome was blocked with ALLN. In this experiment, Apc+/+, ApcMin/+, and ApcMin/- cells contained similar high levels of beta -catenin (Fig. 5, right), consistent with the results seen in Fig. 3. Thus, while we did not assess the nuclear pool of beta -catenin in our samples, we conclude that in these normal tissues and benign adenomas, beta -catenin is mainly associated with the membrane.


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  		Fig. 5.   IB analysis of beta -catenin using total cell lysates prepared in the presence and absence of the proteasome inhibitor, ALLN. Without inhibition of the proteasome, full-length beta -catenin was present at low levels in Apc+/+ and non-tumor ApcMin/+ tissue (3rd and 4th lanes from left), but this protein was greatly increased in the ApcMin/- cells (5th lane from left). With ALLN, the amount of beta -catenin was the approximately the same in each cell type (6th to 8th lanes from left). All lanes were loaded with 30 µg of protein.

The Tyrosine-phosphorylated Form of beta -Catenin Was Up-regulated in Non-tumor ApcMin/+ Tissue-- The signaling potential of beta -catenin at cell-cell adhesion sites is modulated by tyrosine phosphorylation, and distinct modification sites cause its dissociation from E-cadherin and alpha -catenin (50). Because we observed a reduced association of beta -catenin with E-cadherin in ApcMin/+ intestinal cells and the opposite effect in ApcMin/- adenomas, we predicted that the level of tyrosine-phosphorylated beta -catenin would be increased in the ApcMin/+ cells and decreased in the adenomas. To examine steady-state levels of tyrosine-phosphorylated beta -catenin in enterocytes, we performed IP and IB analyses using lysates of Apc+/+, ApcMin/+, and ApcMin/- adenoma cells prepared in the presence of ALLN. Cell lysates were immunoprecipitated with 4G10 anti-phosphotyrosine antibody, followed by IB analysis using anti-beta -catenin antibody. As shown in Fig. 6A, the relative level of tyrosine-phosphorylated beta -catenin was increased in ApcMin/+ enterocytes when compared with Apc+/+ intestine. Interestingly, two bands of ~92-95 kDa appeared in the lane containing the non-tumor ApcMin/+ cell lysate (arrow). Only the faster mobility band was evident in the lane containing the wild-type (Apc+/+) and ApcMin/- lysates. This result was confirmed by the reciprocal experiment in which the blot of samples precipitated with anti-beta -catenin antibody was probed with 4G10 (Fig. 6B). The intensity of the ~92-Da band in the lane containing the non-tumor ApcMin/+ cell lysate was roughly twice that of the corresponding band from the Apc+/+ lysate, and the expression of tyrosine-phosphorylated beta -catenin was again lower in the tumor cells. These data suggested that non-tumor ApcMin/+ enterocytes may contain beta -catenin that was phosphorylated at two different tyrosine residues, whereas Apc+/+ and ApcMin/- cells contain this protein modified at a single tyrosine.


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  		Fig. 6.   Expression of tyrosine-phosphorylated beta -catenin in Apc+/+, ApcMin/+, and ApcMin/- cells. Lysates from Apc+/+, non-tumor ApcMin/+, and ApcMin/- cells were prepared with ALLN (10 mM) and then immunoprecipitated with the anti-phosphotyrosine antibody, 4G10. IB was then performed using clone 14 anti-beta -catenin antibody (A). Reciprocal experiment in which IPs of lysates prepared in parallel used clone 14 anti-beta -catenin antibody, and the IB was performed with 4G10 (B). The Ig heavy chain (HC) bands serve as loading controls. IPs used 300 µg of protein each.

Altered Associations of beta -Catenin with RPTPs in ApcMin/+ and ApcMin/- Cells-- Tyrosine phosphorylation of adhesion proteins is controlled by the coordinated activities of kinases and phosphatases. We therefore determined whether the association of beta -catenin with RPTPs could be demonstrated in enterocytes and whether differences in the degree of this association would match the differences in the tyrosine-phosphorylated beta -catenin observed in these cells. IP/IB analyses were performed using antibodies specific for RPTPbeta /zeta , RPTPgamma , and PTP LAR. In the case of PTP LAR, no association with beta -catenin was detected in the enterocytes, although expression of this phosphatase was observed by IB of total cell lysates from all three cell types (data not shown). An association of beta -catenin with RPTPbeta /zeta and RPTPgamma in Apc+/+, ApcMin/+, and ApcMin/- cells was observed as shown in Fig. 7. IB analyses of the overall expression of RPTPbeta /zeta and RPTPgamma are presented in Fig. 7, A and C, respectively. The ectodomains of these receptor proteins are processed in a manner that leaves the phosphatase-containing cytoplasmic portion anchored in the membrane and the soluble extracellular portion non-covalently attached (51). Arrows indicate the extracellular soluble fragment (top) and the intracellular phosphatase fragment (bottom). The processed forms of these RPTPs are also apparent in positive control cell lysates (Jurkat and A431). In all three tissues, the expression of these phosphatases appears invariant and thus is unaffected by the Apc genotype. Consistent with the increased tyrosine phosphorylation of beta -catenin in non-tumor ApcMin/+ enterocytes, however, IP/IB analyses showed a reduced association of beta -catenin with RPTPbeta /zeta (Fig. 7B). The same results were obtained with a second anti-RPTPbeta /zeta antibody, MAB5210 (data not shown). In addition, consistent with the reduced tyrosine phosphorylation of beta -catenin in ApcMin/- adenoma cells, associations of this protein with both RPTPbeta /zeta and RPTPgamma were detected (Fig. 7, B and D). No association of these phosphatases with E-cadherin could be demonstrated in these tissues when blots of Fig. 7, B and D, were stripped and re-probed using clone 36 antibody (data not shown).


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  		Fig. 7.   Comparisons of the expression and associations with beta -catenin of RPTPbeta /zeta and -gamma in Apc+/+, ApcMin/+, and ApcMin/- enterocyte lysates. IB using clone 12 anti-RPTPbeta /zeta and 50 µg of protein of the three cell samples (A, left). A similar analysis of RPTPgamma using goat antibody M-18 (C, right). The bottom portions of these blots were separately probed for beta -actin as loading controls. IP of 500 µg of protein from each cell lysate using clone 12 anti-RPTPbeta /zeta (B, left) or M-18 anti-RPTPgamma antibody (D, right). Ig heavy chain (HC) bands serve as loading controls. The processed extracellular (120 kDa) and phosphatase (~75 kDa) fragments of these RPTPs are indicated by arrows. These specific bands were not detected when these blots were stripped and re-probed with mouse Ig as a negative control (not shown). The processed RPTP forms are also present in the positive control Jurkat (A) and A431 (C) lysates. Although the full-length proteins were present in the control lysates, very little of these forms was detected in the mouse intestinal cells (data not shown). The data of A and B were exactly reproduced using MA5210, another antibody for RPTPbeta /zeta , but is not shown.

In epithelial cells, EGFR can tyrosine-phosphorylate beta -catenin (33), and we therefore explored the possibility that beta -catenin is phosphorylated by EGFR in our specimens. An IP of the lysates was performed using antibody against EGFR (clone 13), followed by IB with beta -catenin antibody. This experiment showed minimal or no association between beta -catenin and EGFR (data not shown). Stripping and reprobing the IB with the anti-EGFR antibody confirmed that the 180-kDa EGFR protein was precipitated. The expression of EGFR in the Apc+/+, ApcMin/+, and ApcMin/- cells was relatively low and invariant among the samples (data not shown). In addition, Src kinase activity was assessed by IP of the cell lysates using antibodies against c-Src (SRC2) and phospho-Src (Tyr-416) (1:200 antibody dilution). By this method, no differences in Src expression or phosphorylation were observed between the Apc+/+, ApcMin/+, and ApcMin/- cells (data not shown). Thus, we have no evidence that the increased expression of tyrosine-phosphorylated beta -catenin in the ApcMin/+ enterocytes involved up-regulation of these kinases.

Association between beta - and alpha -Catenin Was Altered during Apc-associated Tumor Formation-- alpha -Catenin is an actin-bundling protein that links the transmembrane cadherin to the actin cytoskeleton indirectly via association with beta - or gamma -catenin at the AJ (33, 52, 53). The interaction between alpha -catenin and beta -catenin in the various cell types was therefore examined. In the slowly migrating ApcMin/+ enterocytes, increased expression of alpha -catenin was observed (Fig. 8A), although its association with beta -catenin was not significantly different than that of wild-type enterocytes (Fig. 8B). The overall expression of alpha -catenin was up-regulated in the ApcMin/- adenoma cells (Fig. 8A), a result consistent with the increased expression of E-cadherin in the same samples (Fig. 2A). The association of alpha -catenin with beta -catenin was also markedly increased in ApcMin/- adenoma cells (Fig. 8B). The increased binding between these two catenins and E-cadherin in adenoma cells is consistent with the complex being associated with the actin cytoskeleton and therefore detergent-insoluble (Fig. 3).


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  		Fig. 8.   IB analyses of the expression and association with beta -catenin of alpha -catenin in Apc+/+, ApcMin/+, and ApcMin/- enterocyte lysates. IB of the intestinal cell lysates (50 µg of protein) in which the top portion was probed with clone alpha CAT-7A4 antibody against alpha -catenin, and the bottom portion of the membrane was probed with beta -actin as a loading control (A). B, IP of lysates (500 µg of protein) using alpha CAT-7A4 followed by IB using clone 14 anti-beta -catenin antibody. Ig heavy chain (HC) bands serve as internal loading controls.

P120ctn, a cadherin-associated SFK, binds directly to E-cadherin at the AJ (reviewed in Ref. 54). P120ctn is phosphorylated in response to ligand stimulation of receptor tyrosine kinases (55) and may regulate cadherin-mediated adhesion. To determine whether p120ctn was affected by the Min mutation and as a further test of the specificity of the results presented in Figs. 2-8, we examined the expression and phosphorylation of p120ctn in Apc+/+, ApcMin/+, and ApcMin/- adenoma cells. As shown in Fig. 9, both the overall expression of p120ctn and its tyrosine phosphorylation status were unchanged throughout these tumor progression stages. As a control, the anti-phosphotyrosine IBs were stripped and re-probed with anti-p120ctn antibody. These re-probed blots showed that intact p120ctn proteins were present in all of the samples (data not shown).


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  		Fig. 9.   Comparisons of the expression and tyrosine phosphorylation of p120ctn in Apc+/+, ApcMin/+, and ApcMin/- enterocyte lysates. IB of the intestinal cell lysates (50 µg of protein) in which the top portion was probed with clone clone 98 antibody against p120ctn, and the bottom portion of of the membrane was probed with beta -actin as a loading control (A). B, IP of lysates (500 µg of protein) using 4G10 anti-phosphotyrosine antibody followed by IB using clone 98. Ig heavy chain (HC) bands serve as internal loading controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the epithelium, the signaling pathways controlling cell migration are integrated with those regulating cell proliferation and survival. These processes are altered during tumorigenesis, resulting in a cell that escapes normal growth controls. In the majority of human colon carcinomas and in the germ line of patients with familial adenomatous polyposis, early tumor formation is associated with APC protein truncation (56, 57). A truncated form of Apc protein together with the full-length product of a wild-type Apc allele is found in the histologically normal small intestine of the Min/+ mouse, and Apc truncation with loss of heterozygosity at the second Apc allele is characteristic of adenomas from Min/+ mice (48, 49). The phenotype of this animal, therefore, illustrates two stages of tumor progression, i.e. ApcMin/+ enterocytes and ApcMin/- adenoma cells. The earliest tumor-associated changes, found in ApcMin/+ cells, are alterations of adhesion. Although normal-appearing by histology, these enterocytes migrate slowly (40), have increased residence time in the intestine (40), and demonstrate decreased AJ structural integrity. It is not until the second Apc allele is lost that the cells acquire a hyperproliferative phenotype, as evidenced by up-regulation of cyclin D1 expression in adenomas from Min/+ mice (58, 59). In ApcMin/- adenoma cells, we found that the association of both alpha - and beta -catenin with E-cadherin was increased. This state of augmented AJ structure occurred in association with a presumed increase in beta -catenin/Tcf4-mediated gene expression caused by loss of wild-type Apc at the adenoma stage. Similar conditions were found in intestinal epithelial cells expressing a mutant form of beta -catenin that was protected from degradation because it lacked the serine-threonine phosphorylation sites (60). When this condition was examined in vivo, enterocytes with increased beta -catenin/Tcf-mediated gene transcription also demonstrated increased E-cadherin expression and a 4-fold elevation in proliferation within the murine crypts (39).

The tyrosine phosphorylation of catenins regulates their interaction with adhesion complexes and consequently modulates the activity of the AJ. We found that increased tyrosine phosphorylation of beta -catenin was present in ApcMin/+ enterocytes and occurred together with reduced beta -catenin-E-cadherin binding and reduced enterocyte migration (40). In multiple experimental systems, tyrosine phosphorylation of beta -catenin was associated with decreased cadherin-dependent adhesion (14, 33, 35). Reaction of recombinant c-Src in vitro with beta -catenin produced tyrosine phosphorylation of two residues, Tyr-86, located near the N terminus, and Tyr-654, found in the final armadillo repeat (31). In addition, modification of beta -catenin at Tyr-654 prevented the interaction of beta -catenin with E-cadherin in Caco-2 colon cancer cells (31). It remains to be determined whether the increased phosphorylation of ApcMin/+ enterocytes occurred at one or both of these sites. Our data shows that decreased beta -catenin-E-cadherin association occurred in ApcMin/+ enterocytes together with the appearance of full-length E-cadherin in the cytoplasm. Taken together, these results suggest that, in slowly migrating ApcMin/+ enterocytes, decreased beta -catenin-E-cadherin binding leads to increased E-cadherin internalization and may therefore alter the cycling of E-cadherin from membrane to cytoplasm as adhesion contacts form and re-form.

Insight into the mechanisms of early Apc-associated intestinal tumorigenesis can be obtained by comparing the differences in AJ structure between ApcMin/+ enterocytes and ApcMin/- adenoma cells. In the ApcMin/- tumor cells, in addition to decreased tyrosine-phosphorylated beta -catenin and increased beta -catenin-E-cadherin binding, we also observed increased association between beta -catenin and alpha -catenin. alpha -Catenin is an actin bundling protein that joins the cadherin-associated adhesion complexes to the cytoskeleton (33, 53). We found that E-cadherin, beta -catenin, and alpha -catenin were assembled at the membrane of ApcMin/- adenoma cells in a manner that suggests tight adhesion of the tumor cells, a condition associated with reduced turnover of these proteins (61, 62). alpha -Catenin binds to the N-terminal domain of beta -catenin (63), whereas E-cadherin binds to the C terminus (28, 37). In view of the reduced binding between E-cadherin and beta -catenin in ApcMin/+ enterocytes, it is reasonable to suggest that differential tyrosine phosphorylation of beta -catenin separately affects the interactions of these proteins. By this model, the lack of augmented alpha -catenin/beta -catenin association in ApcMin/+ enterocytes is consistent with the increased tyrosine phosphorylation of beta -catenin observed in these cells (Fig. 6).

Tyrosine phosphorylation of beta -catenin is modulated by several factors, including SFKs, receptor tyrosine kinases, and specific transmembrane and non-receptor tyrosine phosphatases. We have not yet identified an Src kinase specifically activated in ApcMin/+ enterocytes, although likely candidates include c-Src, c-Yes, Fyn, and Fer (29, 30, 64). EGFR can also tyrosine-phosphorylate beta -catenin (35), as activation of this growth factor receptor produced beta -catenin tyrosine phosphorylation and dissociation of the actin cytoskeleton from the AJ in breast cancer cells (33). In breast epithelial cells, signaling via EGFR also induced tyrosine phosphorylation of p120ctn, a condition not seen in ApcMin/+ or ApcMin/- cells (Fig. 5). Our data suggest that neither EGFR nor c-Src association was responsible for the increased tyrosine-phosphorylated beta -catenin in ApcMin/+ enterocytes.

Through the modification of AJ-associated proteins, phosphatases may regulate adhesion-mediated signaling. AJ functions are altered by c-Src, a signal transduction protein that is activated by tyrosine phosphatases (reviewed in Ref. 65). Phosphatase activity may control adhesion protein cell surface distribution, including protein movement into and out of lipid rafts (51). Although the targets of many phosphatases are as yet uncharacterized, gene knockout studies indicate that they have very distinct biological effects that are likely to be tissue-specific (66-71). The association of phosphatases with the AJ has not been described previously. In addition, the appropriate interaction of beta -catenin with tyrosine phosphatases in post-mitotic cells whose differentiated function requires them to be mobile is not known. A number of different phosphatases interact with beta -catenin, including cytosolic PTPs such as PTPalpha (72), hPTPkappa (73), and PTP LAR (50), and the receptor protein-tyrosine phosphatase, RPTPbeta /zeta (34, 74-76). RPTPbeta /zeta was first characterized in neural tissue, where it regulates cell migration, adhesion, and neurite outgrowth (50). In unstimulated cells, RPTPbeta /zeta is intrinsically active and controls the tyrosine phosphorylation status of beta -catenin (34). In our enterocyte assays, even through we do not demonstrate phosphatase activity directly, the decreased association of RPTPbeta /zeta with beta -catenin observed in ApcMin/+ enterocytes is consistent with increased levels of tyrosine-phosphorylated beta -catenin and decreased cell migration. The expression of RPTPbeta /zeta was reported previously (77) to be restricted to the nervous system, whereas RPTPgamma is ubiquitously expressed. Under the assay conditions reported here, IP studies using antibody specific for RPTPgamma showed significantly increased association of this phosphatase with beta -catenin in the ApcMin/- adenoma cells (Fig. 7D). This association may account for the diminished level of tyrosine-phosphorylated beta -catenin observed in these tumors.

The data presented here do not define the exact link between APC truncation and modulation of AJ structure. APC and E-cadherin do not directly interact, although both of these proteins serve as scaffolds for catenin binding (78). APC binds to the core region of beta -catenin, which is composed of 12 armadillo repeats, an amino acid sequence motif that is common to catenins and APC (78, 79). The Min mutation at codon 850 produces a 95-kDa protein truncated at the C terminus. This truncated protein retains the homodimerization domain and armadillo repeats of APC but lacks both the constitutive and kinase-regulated beta -catenin binding regions (17, 80). In ApcMin/+ tissues, however, a dimer containing both full-length and truncated APC may bind beta -catenin, possibly creating conditions where its tyrosine phosphorylation is facilitated. This argument is supported by the lack of a second tyrosine phosphorylation site in the ApcMin/- tumor cells, as these cells retain the truncated APC but have lost the full-length protein (49). Alternatively, the Min truncation may prohibit the assembly of a functional degradation complex, causing an accumulation of the beta -catenin-Tyr(P)-654 species. Finally, PTPs bind to beta -catenin at the armadillo repeats (50), and it is possible that dephosphorylation is inhibited when truncated APC is associated with beta -catenin-Tyr(P)-654.

These experiments show successive changes in E-cadherin-beta -catenin association and the relative levels of tyrosine-phosphorylated beta -catenin, as enterocytes progress from Apc+/+ to ApcMin/+ to ApcMin/-, and illustrate several important concepts governing tumor development in the setting of Apc loss. First, they provide in vivo data showing that progressive alterations in AJ structure occur during adenoma formation. These changes suggest that there is an initial reduction of cell-cell adhesion in ApcMin/+ enterocytes, followed by an eventual increase in AJ formation in the ApcMin/- adenoma cells. These experiments also show that the tumor-promoting effect of the Min mutation involves a defect in cadherin-mediated adhesion, indicating a dominant negative effect of the truncated APC protein produced from the Min allele.

    ACKNOWLEDGEMENTS

We thank Dr. Anthony M. C. Brown for helpful discussions and Dr. Haiyan Liu for assistance in manuscript preparation.

    FOOTNOTES

* This work was supported by NCI Grant IR29CA74162 from the National Institutes of Health (to M. M. B.), the Irving Weinstein Foundation (to A. M. C.), and the Irving S. Paley Gastrointestinal Tumor Bank (to J. D. M.).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.

|| To whom correspondence should be addressed: Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-8910; Fax: 617-682-6177; E-mail: mbertagnolli@partners.org.

Published, JBC Papers in Press, August 1, 2001, DOI 10.1074/jbc.M103450200

    ABBREVIATIONS

The abbreviations used are: APC, adenomatous polyposis coli; AJ, adherens junction; Min/+, C57BL/6J-Min/+; IB, immunoblot; IP, immunoprecipitation; RPTP, receptor protein-tyrosine phosphatase; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; SFKs, c-Src family kinases; ECM, extracellular matrix; ALLN, N-acetyl-Leu-Leu-norleucinal.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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