The Role of Calpain in the Proteolytic Cleavage of E-cadherin in Prostate and Mammary Epithelial Cells*

Jonathan Rios-DoriaDagger §, Kathleen C. DayDagger , Rainer Kuefer||, Michael G. RashidDagger , Arul M. ChinnaiyanDagger **, Mark A. RubinDagger **, and Mark L. DayDagger §DaggerDagger

From the Dagger  Department of Urology and the § Program in Cellular and Molecular Biology, University of Michigan, the || Department of Urology, University of Ulm, Prittwitz-Strasse 43, 89075 Ulm, Germany, and the ** Department of Pathology, and the  University of Michigan Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, August 27, 2002, and in revised form, October 7, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The E-cadherin protein mediates Ca2+-dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad100) in prostate and mammary epithelial cells. E-cad100 was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad100 induction by ionomycin. Immunoblotting for spectrin and µ-calpain confirmed calpain activation in response to ionomycin treatment. Both the µ- and m-isoforms of calpain efficiently generated E-cad100 in vitro. The E-cad100 fragment was unable to bind to beta -catenin, gamma -catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the beta - and gamma -catenin binding motifs of E-cadherin. Because E-cadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad100 accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which E-cadherin is functionally inactivated through calpain-mediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cadherin family of transmembrane glycoproteins mediates Ca2+-dependent intercellular adhesion in a homophilic manner (1). The classical cadherins, including N-, P-, and E-cadherin are well-conserved and are structurally similar. The cadherin structure consists of an extracellular domain with five tandem repeats, a transmembrane domain, and a cytoplasmic domain that interacts with the catenin family binding proteins (2). The interepithelial binding of E-cadherin, which mediates lateral cell-cell adhesion in secretory tissues such as the prostate and mammary gland (3), results in the formation of desmosomes and adherens junctions that are required for tissue morphogenesis and maintenance of the differentiated phenotype (1). The integrity of E-cadherin binding is dependent on its interaction with the catenin binding proteins in the intracellular domain. E-cadherin binds to beta -, gamma -, and alpha -catenin (4), which physically associate with actin filaments and the actin cytoskeleton (5-7). Inactivation of E-cadherin through gene mutation, transcriptional inactivation, or promoter methylation has been demonstrated in many adenocarcinomas (8-13). However, these mechanisms are not likely to account for all mechanisms by which E-cadherin becomes inactivated in cancer.

One mechanism of E-cadherin inactivation involves the proteolytic ablation of the extracellular domain (14). The generation of a soluble 80-kDa fragment that was shed into the medium of cultured cancer cells in response to calcium influx (15, 16) resulted from cleavage by matrix metalloproteases (17, 18). It was also demonstrated that metalloproteases mediated the cleavage of the extracellular domain of VE-cadherin that was shed into cultures of human endothelial cells (19). Additionally, caspase-3 has recently been shown to cleave E-cadherin during the execution phase of apoptosis (20). Taken together, these studies have demonstrated that specific proteases can target and inactivate E-cadherin; however, the biological ramifications of these proteolytic mechanisms remain unknown.

We have previously identified a novel 97-kDa truncated E-cadherin species that may be important in localized prostate tumorigenesis (21). We have since identified a novel E-cad100 species that is generated by a calcium-dependent mechanism and may also be important in prostate tumor progression. Due to the apparent requirement of calcium in the generation of E-cad100, the calpain family of cysteine proteases emerged as a likely candidate mediating this cleavage event. There are two main isoforms of calpain that are ubiquitously expressed in tissue and are designated µ- and m-calpain based on the concentration of calcium required for their respective function in vitro (22-25). Because physiological concentrations of calcium are in the range of 100-300 nM, it is hypothesized that other mechanisms exist to lower the activation threshold of calcium required for calpain activation in vivo (22, 24). Calpains are heterodimeric proteins consisting of a large catalytic 80-kDa subunit, and a smaller 29-kDa regulatory subunit. Calpain is involved in diverse physiologic roles including cell-cycle regulation (26, 27), signal transduction (28), and apoptosis (29-31). Calpain proteolysis occurs in a limited fashion, producing distinct peptide fragments without further degradation (32). Secondary structure is likely to affect substrate specificity, since many proteins contain consensus calpain sequences but are not susceptible to cleavage (32). Proteins that have been identified as substrates of calpain include the cytoskeletal proteins spectrin and members of the integrin family (33, 34).

In adenocarcinoma, the disruption of cellular adhesion appears to rise, in part, through alterations of the E-cadherin cell-adhersion system (35). However, proteolytic mechanisms of E-cadherin inactivation in cancer have not been thoroughly investigated. In this study, we investigated whether E-cadherin is a substrate for calpain, and whether calpain-dependent proteolysis of E-cadherin was associated with prostate cancer progression.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue Culture Cell Lines, Chemicals, and Transfections-- The cell lines LNCaP, MCF-7, and SKBR3 were propagated in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Cells were incubated at 37 °C and subcultured weekly. The engineered stable cell lines were maintained by adding 0.8 µg/ml puromycin or 1 mg/ml G418 to the culture media. TPA1 (Alexis Biochemicals) was dissolved in 100% ethanol, aliquoted, and used at a final concentration of 10 nM. Ionomycin, calpain inhibitor I, calpain inhibitor II, calpain inhibitor III, and calpeptin were all obtained from Calbiochem. Cycloheximide was obtained from Sigma and used at a final concentration of 50 µM. Cells were treated with cycloheximide 30 min prior to TPA treatment. SK-Ecad was generated by transfection of a plasmid that contained wild-type human E-cadherin in the pLK-pac vector with a 2×-Myc epitope added to the last codon (gift from Dr. Margaret Wheelock) and was selected with puromycin. Transfection was done with Lipofectin (Invitrogen) as instructed. E-cadherin mutants were generated from wild-type human E-cadherin cDNA cloned into the pcDNA3 vector that contained a 2×-Myc epitope added to the last codon. Transfections with pcDNA3-based vectors were done with the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) and selected with G418.

Western Analysis and Immunoprecipitations-- Protein lysates were prepared from tissue and cultured cells in the following buffer: 50 mM Tris, pH 7.5, 120 mM NaCl, 0.5% Nonidet p-40, 40 µM PMSF (phenylmethylsulfonyl fluoride), 50 µg/ml leupeptin, 50 µg/ml aprotinin, 200 µM orthovanadate, 1 mM EGTA. Tissue was homogenized prior to lysing. Cells were allowed to lyse 1 h on ice, centrifuged, supernatants extracted, and quantitated using a Bradford assay. Lysates were separated by 6% Novex gels and analyzed using chemiluminescence (Amersham Biosciences) as described previously (36). Membranes were stripped with the following stripping solution: 62.5 mM Tris pH 6.8, 2% SDS, 100 mM beta -mercaptoethanol. Following ECL and washing, the membrane was incubated with the stripping solution for 30 min at 55 °C, with gentle shaking every 5 min. Densitometry analysis was performed as previously described (21).

For immunoprecipitations, cell lysates were prepared and quantitated as described above. The lysates were pre-cleared for 30 min at 4 °C with the appropriate Sepharose beads (Zymed Laboratories) conjugated with protein G or A prior to immunoprecipitation reactions. All beads were diluted and mixed for 1 h at room temperature with an equal volume of Tris-buffered saline (50 mM Tris, pH 7.5, 120 mM NaCl) + 2.5% milk prior to use. Following pre-clearing, lysates were aliquoted into microcentrifuge tubes, where an equal volume of lysate was used for each immunoprecipitation reaction. The volumes of each reaction were equalized to 500 µl with lysis buffer, 5 µg of primary antibody was added, and the reaction was carried out at 4 °C rotating end-over-end. The following day, the appropriate Sepharose beads were added and the reaction was allowed to continue to rotate end-over-end at 4 °C for 90 min. The beads were then pelleted and washed three times with lysis buffer (+ protease inhibitors). Reducing sample buffer was added to each pellet, boiled for 5 min, centrifuged, and the supernatants loaded onto 6% NOVEX Tris-Gly (Invitrogen) gels for Western analysis.

Ionomycin and Calpain Inhibitor Experiments-- 2.0 × 105 LNCaP or MCF-7 cells were plated in 6-well plates. 72 h later, cells were pre-treated with 20 µM of the calpain inhibitors in fresh serum-containing media for 15 min. Cells were then treated with 10 µM ionomycin for 20 min. Cells were harvested, lysed, quantitated, and 30 µg of protein per sample were loaded onto 6% NOVEX gels for Western analysis.

Antibodies-- Antibodies used for the detection of E-cadherin were HECD-1 (Zymed Laboratories), E-9 (16), 4A2 (37), C20820 (Transduction Laboratories), E2 (38), and SC-1499 (Santa Cruz Biotechnology). Antibodies used for co-immunoprecipitation experiments were p120 (Transduction Laboratories), beta -catenin (Zymed Laboratories, CAT-5H10), and gamma -catenin (Santa Cruz Biotechnology). µ-Calpain antibody was purchased from Calbiochem and alpha -spectrin was purchased from Chemicon. The normal goat IgG antibody was obtained from Santa Cruz Biotechnology. The appropriate horseradish peroxidase-conjugated anti-mouse, anti-rat, or anti-goat secondary antibodies were obtained from Amresco and Jackson ImmunoResearch Laboratories, Inc.

In Vitro Calpain and Caspase-3 Assay-- E-cadherin was immunoprecipitated (as described above) from LNCaP cell lysates with the C20820 antibody. After the third wash with lysis buffer (+ protease inhibitors), the supernatants were removed, and the reactions placed on ice. The Sepharose beads were then re-suspended in an appropriate volume of lysis buffer and divided equally into five Eppendorf tubes for five separate reactions. They were then centrifuged at 6,000 rpm for 3 min at 4 °C, the supernatants removed, and tubes placed on ice. For the calpain assay, the following proteolysis buffer was used at 25 µl per reaction: 100 mM HEPES, 10 mM dithiothreitol, 10% glycerol (v/v), 1 mM CaCl2. A separate buffer that contained 10 mM EGTA was also used. Reactions were started by adding 0.4 units of µ-calpain, 0.95 units of m-calpain, or 50 units of caspase-3 (all from Calbiochem) and were carried out at room temperature. For the caspase-3 assay, the following proteolysis buffer was used at 25 µl per reaction: 100 mM HEPES, 10 mM dithiothreitol, 10% glycerol (v/v), 1 mM EGTA. Reducing sample buffer was added to each tube to stop the reaction at each time point. The reactions were boiled for 5 min and the supernatants were loaded onto 6% NOVEX Tris/Gly (Invitrogen) gels for Western analysis.

Mutagenesis-- E-cadherin deletion mutants were generated using the Quikchange kit (Stratagene). The parental plasmid was wild-type full-length human E-cadherin cloned into the pcDNA3 vector (Invitrogen) with a 2× c-Myc tag cloned in-frame at the C terminus. The primers used were as follows: 791Delta 5, forward: 5'-GACGTTGCACCAACCCGGTATCTTCC-3', reverse: 5'-GGAAGATACCGGGTTGGTGCAACGTC-3'; 795Delta 5, forward: 5'-CTCATGAGTGTCCGCCCTGCCAATC- 3', reverse: 5'-GATTGGCAGGGCGGACACTCATGAG-3'; 782Delta 6, forward: 5'-GACGCTCGGCCTGAAGCACCAACCCTCATG-3', reverse: 5'-CATGAGGGTTGGTGCTTCAGGCCGAGCGTC-3'. All mutants were verified by DNA sequencing at the University of Michigan sequencing Core facility.

cDNA Microarray Analysis-- Analysis of samples were performed as previously described (39). In brief, cDNA generated from prostate samples (i.e. prostate cancer) and benign prostate tissue (reference) samples were labeled with distinguishable fluorescent dyes and hybridized to the cDNA microarray (10,000 clones). The cDNA microarray was analyzed using a scanner, and fluorescence ratios were determined for each gene. Color intensities were converted into ratios of gene expression. These ratios are imported into a data base for further analysis. To test for significant differences in the mean cDNA expression between all tissue types, we performed a one-way ANOVA test. To determine differences between all pairs (e.g. localized prostate cancer versus benign), we performed a post-hoc analysis using the Scheffé method. The mean expression scores for all examined cases were presented in a graphical format using error bars with 95% confidence intervals. The results were considered to be significant when p < 0.05.

Tissue Procurement-- Patients with localized prostate cancer provided written informed consent in an Institutional Review Board (IRB)- approved study at the University of Michigan to have their prostate tissue evaluated in experimental studies. These fresh radical prostatectomy specimens were obtained through the University of Michigan prostate cancer SPORE tissue bank and were frozen in liquid nitrogen within 30 min after surgical excision. Histological confirmation of both tumor and normal regions of each prostate gland were confirmed by a genitourinary pathologist at the University of Michigan, and the corresponding tumor or normal region was excised before processing for protein analysis (21).

A Rapid Autopsy Protocol has been previously described (40). Permission for an autopsy was granted from patients with hormone-refractory prostate cancer to participate in an IRB-approved tissue donor program at the University of Michigan. Metastases from prostate cancer were immediately frozen in liquid nitrogen. The metastases were confirmed as prostate cancer in origin by a genitourinary pathologist at the University of Michigan.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ionomycin Induces a Calpain-dependent Cleavage of E-cadherin to 100 kDa-- Previous studies from our laboratory have shown that PKC activation induces a signal transduction pathway that leads to the truncation of E-cadherin in prostate and mammary epithelial cells (41, 42). Activation of PKC has a number of physiological ramifications in the cell, including cell proliferation and differentiation, changes in gene expression, and transmembrane ion transport (43). We hypothesized that E-cadherin cleavage may be due to the influx of calcium ions initiated by PKC activation. To specifically address the role of calcium in the cleavage of E-cadherin, we treated LNCaP cells with the calcium ionophore, ionomycin, and observed the rapid accumulation of a novel 100-kDa fragment of E-cadherin that was designated E-cad100 (Fig. 1A, top panel). Ionomycin did not result in the accumulation of the previously described E-cadherin species, E-cad97. Due to the apparent role of calcium in E-cadherin cleavage, we speculated that the calcium-dependent protease calpain may mediate the cleavage of E-cadherin. E-cadherin cleavage and the accumulation of E-cad100 was dramatically inhibited in cells pretreated with the calpain inhibitors calpain inhibitor I, calpain inhibitor II or calpeptin (Fig. 1A, top panel). These inhibitors had no effect on the generation of the 97-kDa species ruling out the possibility that calpain generates both fragments. E-cadherin cleavage to 100 kDa was also prevented by calpain inhibitor I following TPA treatment (data not shown).


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Fig. 1.   Calpain inhibitors prevent induction of E-cad100, alpha -spectrin cleavage, and µ-calpain activation. LNCaP cells (A) or MCF-7 cells (B) were pretreated with 20 µM calpain inhibitors for 15 min, then treated with 10 µM ionomycin for 20 min. The inhibitors used were calpain inhibitor I, calpain inhibitor II, calpain inhibitor III, or calpeptin. 30 µg of protein extracts were resolved on a 6% SDS-PAGE gel. Each of the membranes were immunoblotted with HECD-1 antibody (top panels), stripped and re-probed with an alpha -spectrin (middle panels), and a µ-calpain antibody (bottom panels).

To help confirm that ionomycin induced a calpain-mediated cleavage of E-cadherin, we re-probed the membranes with an alpha -spectrin antibody. alpha -Spectrin is a well documented calpain substrate with a molecular mass of 280 kDa that is cleaved by calpain to produce a 145/150-kDa doublet (33). The resulting alpha -spectrin immunoblot revealed that spectrin cleavage was induced by ionomycin, and that this induction was effectively inhibited by calpain inhibitors (Fig. 1A, middle panel). In the inactive state, calpain exists as pro-enzyme heterodimer of 80-29 kDa, and following calcium binding undergoes autolysis to an active 78-18 kDa heterodimer (32). Again, using the same membrane, we assayed for the autolysis of the large subunit of calpain by immunoblot (Fig. 1A, bottom panel). Although faint, immunoblot analysis revealed a clear conversion of the 80-kDa large subunit to the 78-kDa active monomer following the addition of ionomycin. Pretreatment of LNCaP cells with calpain inhibitor I, calpain inhibitor II, and calpeptin prevented this autolysis.

A similar experiment was performed using MCF-7 cells (Fig. 1B). MCF-7 cells were pretreated with calpain inhibitor III and calpeptin prior to ionomycin treatment. Fig. 1B shows that these inhibitors effectively blocked E-cadherin cleavage by ionomycin. Re-probing the membrane for alpha -spectrin shows that calpain inhibitor III and calpeptin blocked calpain-mediated spectrin cleavage (Fig 1B, middle panel). Although partial, re-probing with µ-calpain antibody showed some inhibition of autolysis of the µ-calpain large subunit (Fig. 1B, bottom panel). We observed that calpain inhibitor 3 slightly reduced the amount of calpain; this may be an indirect effect of the inhibitor on calpain stability. Taken together, these data show that ionomycin treatment of LNCaP and MCF-7 cells induces a calpain-dependent truncation of E-cadherin to the 100-kDa form that was significantly inhibited by calpain inhibitors.

Calpain Cleaves E-cadherin in Vitro-- To determine if purified calpain could generate E-cad100 in vitro, we incubated E-cadherin immunopurified from untreated LNCaP cells with either µ- or m-calpain. In the presence of both isozymes, full-length E-cadherin was rapidly cleaved to the 100-kDa fragment, and this truncation could be completely inhibited by addition of the calcium chelator EGTA (Fig. 2, A and B). In the absence of calpain, the 100-kDa product was not detected. Over the course of 20 min, some general degradation of E-cadherin was observed, which was unrelated to the effects of calpain cleavage of E-cadherin. These results provide compelling evidence that both µ- and m-calpain can specifically cleave mature E-cadherin to the 100-kDa fragment in vitro.


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Fig. 2.   Calpain cleaves E-cadherin in vitro. E-cadherin, immunoprecipitated from LNCaP cells was pretreated with EGTA prior to the addition of µ-calpain (A) or m-calpain (B) for the indicated times. All cleavage reactions were resolved by 6% SDS-PAGE and immunoblotted with HECD-1 antibody.

Examination of the cytoplasmic domain of E-cadherin revealed a potential caspase-3 cleavage site within residues 747DTRD. Caspase-3 is a cysteine protease that is involved in proteolytic breakdown during the execution of apoptosis and has been implicated in the cleavage of E-cadherin (20). We wanted to determine if E-cad100 was generated by caspase-3. A similar in vitro assay was performed using purified caspase-3, which failed to demonstrate cleavage of E-cadherin to E-cad100 (data not shown).

PKC Activation Induces E-cad100-- We utilized the E-cad-/- mammary epithelial cell line SKBR3 (44) to exogenously express E-cadherin. We selected stable clones that expressed moderate to high levels of the 2×-Myc-tagged E-cadherin protein, which migrates at a slightly higher molecular weight of 130 kDa (37). Activation of PKC in these E-cad-expressing SKBR3 cells (termed SK-Ecad) revealed the rapid accumulation of E-cad100 over a course of 12 h (Fig. 3). PKC activation by TPA treatment did not induce expression of any endogenous E-cadherin protein in untransfected SKBR3 cells. We also noticed a dramatic increase in the expression levels of E-cadherin in the transfectants following TPA-treatment; however, the mechanism through which this up-regulation occurs is currently unclear, as the E-cadherin cDNA is driven by a cytomegalovirus promoter and does not contain any phorbol-responsive elements. To address whether increased expression of E-cadherin was required for cleavage, LNCaP cells were pretreated with cycloheximide prior to TPA treatment. Fig. 4 demonstrates that blocking E-cadherin synthesis with cycloheximide did not prevent cleavage. Thus, protein synthesis is not required for calpain-dependent cleavage of E-cadherin. In order to determine what proportion of E-cadherin is cleaved in cell culture, we performed densitometry on multiple E-cadherin Western blots in which E-cad100 has been induced with ionomycin or TPA. On average, the amount of total cellular E-cadherin cleaved was ~30% (data not shown).


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Fig. 3.   Expression and truncation of exogenous E-cadherin in SKBR3 cells. A, Protein extracts from SKBR3 parental cells (lanes 1-2) and SK-Ecad cells (lanes 3-6), treated with TPA at the indicated times, were immunoblotted with the HECD-1 antibody.


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Fig. 4.   E-cadherin synthesis is not required for cleavage. LNCaP cells were pretreated with 50 µM cycloheximide 30 min prior to TPA treatment. 50 µg of protein extracts from cells that were harvested at the indicated times were resolved by 6% SDS-PAGE and immunoblotted with HECD-1 antibody.

Epitope and Functional Mapping of E-cad100-- TPA-treated SK-Ecad cells were utilized to map the cleavage site on E-cadherin. The specific antibody epitopes that were used are shown in Fig. 5. TPA-treated SK-Ecad cell lysates were immunoblotted with the different antibodies that recognized epitopes in either the extracellular or cytoplasmic domains of E-cadherin. E-cad100 was recognized by the N-terminal antibody E-9, as well as by the cytoplasmic antibody 4A2, which has been mapped to residues 757-778 and by another cytoplasmic antibody C20820, which has been mapped to residues 773-791 (45). The antibody SC-1499 (46) that maps to the C terminus of E-cadherin failed to recognize E-cad100 (Fig. 5). Although three antibodies had variable sensitivities for E-cad100, the immunoblot data indicated that the cleavage site was in the cytoplasmic domain of E-cadherin located between the C20820 and SC-1499 epitopes.


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Fig. 5.   Epitope mapping of E-cad100. A schematic of full-length E-cadherin and a table of the antibodies and epitopes to which they are mapped are shown. TPA-treated SK-Ecad protein extracts were resolved by SDS-PAGE and immunoblotted with E-9 (site A), 4A2 (site B), C20820 (site C), and SC-1499 (site D) antibodies. Lysates were resolved by 6% SDS-PAGE.

To further verify the antibody mapping and to functionally map the truncation, we employed co-immunoprecipitations utilizing the catenin binding proteins to determine if the beta -catenin, gamma -catenin, and p120 binding domains are present in E-cad100. The beta -catenin and gamma -catenin binding domains have been mapped to residues 815-839 of the cytoplasmic domain of E-cadherin (47). The p120 binding site has been mapped to residues 758-773 (48). We predicted that p120 would retain adhesiveness to E-cad100, while beta -catenin and gamma -catenin would not. Co-immunoprecipitations from TPA-treated LNCaP lysates revealed that only full-length E-cadherin is bound to these proteins (Fig. 6). As a positive control, E-cadherin was also immunoprecipitated using a cytoplasmic antibody that recognizes E-cad100. The co-immunoprecipitation data demonstrate that E-cad100 has lost the beta - and gamma -catenin binding domains.


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Fig. 6.   E-cad100 does not associate with the catenin complex. TPA-treated LNCaP protein extracts were immunoprecipitated with E-cadherin antibody 4A2, beta -catenin, p120, and gamma -catenin antibodies. A control immunoprecipitation was also performed using a normal goat IgG antibody and an immunoprecipation using protein A Sepharose beads alone. Immunoprecipitations were resolved by 6% SDS-PAGE and immunoblotted with HECD-1 antibody.

Mutation of the E-cadherin Cytoplasmic Domain Inhibits Truncation of E-cadherin in Vivo-- Based on the data from the immunoblot and co-immunoprecipitation experiments, the calpain cleavage site appeared to reside between the C20820 antibody epitope and the beta -catenin binding domain. Examination of the amino acid sequence of E-cadherin between residues 773 and 815 revealed three potential calpain cleavage sites (Fig. 7A). To identify the calpain cleavage site within the E-cadherin cytoplasmic domain in vivo, we generated three E-cadherin deletion mutants targeted within the proposed region in the E-cadherin cytoplasmic domain (Fig. 7A). In a pcDNA3 vector harboring the wild-type human E-cadherin cDNA, we engineered in-frame deletion mutants of 5-6 amino acids spanning the three potential calpain cleavage sites and transfected the constructs into SKBR3 cells. Stable pools were selected, and PKC was activated by TPA over a 24-h time course. Two of the E-cadherin deletion mutants, SK-791Delta 5 and SK-795Delta 5 were truncated in an identical fashion as the wild type control, while deletion mutant SK-782Delta 6 exhibited complete inhibition of truncation (Fig. 7B). These data indicate that amino acid residues 782-787 are essential for calpain cleavage as SK-782Delta 6 failed to be truncated in vivo.


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Fig. 7.   E-cadherin mutagenesis inhibits truncation in vivo. A, E-cadherin amino acid sequence, from 771-810, is shown with the targeted mutation sites. B, the corresponding mutant constructs containing in-frame internal deletions were stably transfected into SKBR3 cells and protein extracts harvested at the indicated times following TPA treatment. Lysates from SK-791Delta 5 cells (lanes 1-3), SK-795Delta 5 cells (lanes 4-6), and SK-782Delta 6 (lanes 7-9) were resolved by 6% SDS-PAGE and immunoblotted with HECD-1 antibody. SKBR3-Ecad wild type cells are also shown (lane 10).

Calpain Is Up-regulated in Localized and Metastatic Prostate Tumors-- Both epigenetic and genetic mechanisms of E-cadherin inactivation have been demonstrated in adenocarcinoma (49). We explored the possibility that calpain activity and expression may be associated with prostate tumorigenesis, and that E-cadherin cleavage may represent a novel mechanism of E-cadherin inactivation in cancer. We hypothesized that the loss of the catenin binding domain ablated the adhesive function of E-cadherin, and that this loss of function might support the malignant transformation of prostate epithelium. Using a cDNA microarray (39), benign prostate (n = 26), localized prostate cancer (n = 49), and metastatic prostate cancer (n = 19) demonstrated mean cDNA expression for m-calpain of 1.0 (95% confidence interval (CI) 0.9-1.1), 1.2 (95% CI 1.1-1.2), and 1.4 (95% CI 1.3-1.4), respectively (one-way ANOVA, p < 0.001) (Fig. 8). Pair-wise comparisons demonstrated significant differences in cDNA expression levels between clinically localized prostate cancer and metastatic prostate cancer with respect to benign prostate tissue (post-hoc analysis using Scheffé method, p < 0.01). Immunoblot analysis of E-cad100 expression in matched normal and tumor prostate specimens revealed the appearance of E-cad100 in the tumor aspect of the prostate gland compared with the normal (Fig. 9A). Of 16 normal/tumor-matched pairs examined, 6 showed an increase in E-cad100 in the tumor aspect of the prostate gland compared with normal; two representative matched pairs are shown in Fig. 9. The E-cad100 species was also observed in prostate cancer metastases (Fig. 9B). Multiple metastatic samples were harvested from the liver, diaphragm, soft tissue, peritoneum, adrenal gland, and paratracheal lymph node. The samples were analyzed by Western blot for E-cadherin, and each of the metastases demonstrated the presence of E-cad100 (Fig. 9B). Of over 40 prostate cancer metastases examined, virtually 100% contained the E-cad100 species (data not shown). The abundance at which E-cad100 was detected in localized and metastatic prostate cancer compared with normal prostate tissue correlated with the increase in m-calpain expression observed in the cDNA microarray. These data demonstrate that increased expression of calpain in prostate tumors is associated with the generation of the 100-kDa fragment in localized and metastatic disease.


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Fig. 8.   m-Calpain is up-regulated in localized and metastatic prostate tumors. cDNA microarray analysis was performed using 26 benign prostate, 49 localized prostate cancer, and 19 metastatic prostate cancer samples. m-Calpain transcript levels were found to be significantly up-regulated in localized prostate and metastatic cancer samples compared with benign prostate.


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Fig. 9.   E-cad100 is detected in localized and metastatic prostate cancer. A, protein extracts (30 µg) of prostate tissue from two different patients were analyzed for E-cadherin expression. Extracts from both the normal and tumor aspect of the prostate gland were run on a 6% SDS-PAGE gel and immunoblotted with HECD-1 antibody. B, protein extracts were prepared from metastatic lesions removed from the following sites: 1, liver; 2, diaphragm; 3, soft tissue; 4, peritoneum; 5, adrenal gland; 6, paratracheal lymph node. 30 µg of protein were resolved on a 6% SDS-PAGE gel and immunoblotted with HECD-1 antibody.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Results from the current study demonstrate that calpain induces a specific, inactivating proteolytic cleavage within the cytoplasmic domain of E-cadherin in prostate and mammary epithelial cells. Initial experiments indicated that cleavage of E-cadherin to the 100-kDa fragment was a calcium-dependent mechanism that implicated a number of calcium-regulated proteases. Pretreatment of LNCaP and MCF-7 cells with calpain inhibitors blocked the generation of ionomycin-induced E-cad100, strongly implicating calpain in this process.

In vitro experiments also supported the hypothesis that E-cadherin is a substrate for calpain. Both µ- and m-calpain effectively cleaved full-length E-cadherin in vitro to 100 kDa. We proceeded to characterize this truncation using the mammary epithelial cell line SKBR3, which does not express E-cadherin protein (44). We showed that the truncation of exogenous E-cadherin to 100-kDa was rapidly induced by PKC activation in SKBR3 cells transfected with wild-type E-cadherin. We found that this truncation occurred in the cytoplasmic domain of E-cadherin, and that this truncated species of E-cadherin is unable to bind to beta -catenin, gamma -catenin, and p120, which are essential for the adhesive and cell signaling functions of E-cadherin (50). In our study E-cad100 does not associate with p120; however, an association would be predicted from previous studies that have mapped the p120 binding site on E-cadherin (48). One explanation is that cleavage of E-cadherin changes the protein conformation near the juxtamembrane domain, resulting in the loss of the p120 binding site.

An examination of the amino acid sequence of the cytoplasmic domain of E-cadherin revealed potential calpain cleavage sites that were targeted for mutation. The E-cadherin 782Delta 6 mutant failed to generate E-cad100 when transfected into cells, while other constructs encoding adjacent mutant sites were truncated in the same fashion as wild-type E-cadherin. While the calpain cleavage site may reside within residues 782-787 in the E-cadherin cytoplasmic domain, it is possible that the 782Delta 6 mutation has merely disrupted secondary structure or a calpain binding site that is required for cleavage. Based on our mutagenic analysis, we hypothesize that the residues 782-787 are either critical for calpain binding or represent the actual cleavage site.

Calpain has been implicated in the cleavage of other membrane-proteins involved adhesion, most notably members of the integrin family (34). However, the precise function of calpain in cellular adhesion remains to be elucidated. Caspases are a well-studied family of cysteine proteases that act as the executers of apoptosis (51). Caspase-3 has been shown to cleave E-cadherin during apoptosis (20). However, we found that caspase-3, was not responsible for the specific cleavage of E-cadherin to generate the 100-kDa species (data not shown).

In a variety of adenocarcinomas, aberrations in E-cadherin expression have been noted in malignant transformation of epithelium, metastatic progression, and decreased patient survival (9, 52-54). The development of metastatic prostate cancer is a multistep process in which the disruption of cellular adhesion promotes both local invasion and distant dissemination. In prostate cancer the loss of E-cadherin function does not appear to result from allelic loss or gene mutation to any great extent but rather to other mechanisms, such as decreased transcription due to the aberrant methylation of its promoter (13). Although important in the etiology of some cancers, genetic alteration and promoter methylation do not account for all of the physiologic mechanisms by which E-cadherin function is abrogated during tumorigenesis.

The first reports of E-cadherin proteolysis emerged nearly two decades ago when it was discovered that a novel, 80-kDa fragment of E-cadherin was released into the culture media of mouse and human mammary tumor cells (16). This proteolytic fragment, which was derived from the amino-terminal, extracellular domain of E-cadherin was generated by a number of stress stimuli, including serum deprivation and high calcium loading and was particularly intriguing due to its ability to disrupt the lateral adhesion of mammary epithelium. These findings were also of interest due to the fact this fragment was elevated in the serum of patients suffering from a variety of adenocarcinomas (55). This laboratory has been involved in the characterization of proteolytic mechanisms that target and inactivate E-cadherin during prostate and mammary tumorigenesis and had previously demonstrated the generation of a novel, membrane bound fragment of E-cadherin that migrated at 97-kDa (E-cad97) in prostate and mammary epithelial cells (41). This fragment resulted from the truncation of E-cadherin in the cytosolic domain and had lost its ability to bind beta -catenin thus abolishing the adherent function of the molecule. In a later study, we observed that the accumulation of E-cad97 was significantly associated with localized prostate cancer as compared with adjacent normal tissue derived from the same prostate specimen (21). We have since observed the presence of E-cad100, that while not as prominent as E-cad97 in localized prostate cancer, appears to be present in all metastatic prostate tumors examined. We have also found that calpain is up-regulated in localized and metastatic prostate tumors and this is the first report associating calpain with prostate tumorigenesis. Based on these observations, we hypothesized that specific proteolytic cleavage events, such as that mediated by calpain, target and inactivate E-cadherin. Such a loss of E-cadherin function may result in the net reduction of interepithelial adhesion and promote the malignant transformation of prostate epithelial cells.

    ACKNOWLEDGEMENTS

We thank K. Wang for expert advice on calpain and critical reading of the article, M. Wheelock and K. Johnson for providing the wild-type E-cadherin construct and for helpful discussions, and Ken Pienta for assistance with the Rapid Autopsy Protocol. We also thank M. McCabe and J. Davis for critical reading of the article, and the University of Michigan DNA Sequencing Core for the sequencing of the E-cadherin mutants.

    FOOTNOTES

* This study was supported by Grant RO1 DK56137 from the National Institutes of Health.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.

Dagger Dagger To whom correspondence should be addressed: Box 0944, Room 6219 CCGC, 1500 East Medical Center Dr., Ann Arbor, MI 48109-0944. Tel.: 734-763-9968; Fax: 734-647-9271; E-mail: mday@umich.edu.

Published, JBC Papers in Press, October 19, 2002, DOI 10.1074/jbc.M208772200

    ABBREVIATIONS

The abbreviations used are: TPA, phorbol 12-myristate 13-acetate; SK-Ecad, SKBR3-wild type E-cadherin-transfected cells; 782Delta 6, E-cadherin with a 6-amino acid deletion; 791Delta 5, E-cadherin with a 5-amino acid deletion; 795Delta 5, E-cadherin with a 5-amino acid deletion; E-cad100, Mr = 100,000 E-cadherin; E-cad97, Mr = 97,000 E-cadherin; ANOVA, analysis of variance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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