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Biochemical processing of E-cadherin under cellular stress
BBRC Biochemical and Biophysical Research Communications 307 (2003) 215–223 www.elsevier.com/locate/ybbrc
Biochemical processing of E-cadherin under cellular stressq Steven H. Kellera,* and Sanjay K. Nigamb a
Department of Medicine, University of California, San Diego, 210A Dickinson Street, San Diego, CA 92103-8382, USA b Department of Medicine and Pediatrics, University of California, San Diego, La Jolla, CA 92093-0693, USA Received 2 June 2003
Abstract The proteolytic cleavage pathways of E-cadherin endogenously expressed in MDCK (Madin–Darby canine kidney) cells were characterized in cells treated with antimycin A and deoxyglucose to examine transmembrane protein processing under cellular stress. E-cadherin is a type I transmembrane protein which operates as the cell adhesion molecule component of the adherens junction, a complex of proteins involved in epithelial tissue development and integrity. We now demonstrate that treatment of MDCK cells with antimycin A and deoxyglucose activates caspase mediated pathways that cleave E-cadherin. E-cadherin is cleaved into two major fragments, with the sizes predicted by the location of a caspase-3 cleavage consensus sequence. Cleavage of E-cadherin and deposition of the C-terminal fragment into the cytoplasm are inhibited by the caspase inhibitor DEVD-CHO. Thus, a major mechanism for E-cadherin cleavage and dissolution of the adherens junction under antimycin/deoxyglucose treatment is caspase mediated, initiated by activation of an apoptosis pathway. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Caspase; MDCK cells; Antimycin; Deoxyglucose; Cellular stress; Ischemia; Adherens junction; E-cadherin; Protein degradation; Apoptosis
To study the biochemical processing of transmembrane proteins in epithelial cells subjected to cellular stress, we treated MDCK cells with antimycin A and deoxyglucose and followed the fate of endogenously expressed E-cadherin. Treatment of cells with antimycin A and deoxyglucose has been employed to model ischemia in cell culture due to ATP depletion and glucose deprivation [1–3]. Antimycin A inhibits oxidative phosphorylation and activates apoptosis pathways signaled through mitochondrial membrane depolarization [4–6]. Antimycin A binds to and inactivates Bcl-2, which normally inhibits mitochondrial membrane depolarization during the initiation of apoptosis [5,7,8]. Deoxyglucose activates endoplasmic reticulum stress pathways q Abbreviations: MDCK, Madin–Darby canine kidney; Ant/DG, antimycin/deoxyglucose; PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene-bis(oxyethylenenitrilo)]tetraacetic acid; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; DXXD, aspartate–X–X–aspartate, where X is any amino acid. * Corresponding author. Fax: 1-619-543-3432. E-mail address: [email protected] (S.H. Keller).
[9]. Although the biochemical and cellular consequences of staurosporine treatment are well characterized [10– 13], the biochemical fate of proteins in antimycin/ deoxyglucose-treated cells has not been studied extensively. E-cadherin is a single-pass transmembrane protein that operates as the cell adhesion molecule component of the adherens junction [14–16]. E-cadherin is assembled at the cytoplasmic tail with b- or c-catenin, which in turn are joined with a-catenin that bridges the extracellular domain of E-cadherin to the actin cytoskeleton [14]. It has been hypothesized that degradation of E-cadherin is a central lesion in creation of the ischemic epithelial phenotype, which includes a loss of epithelial polarity and disruption of tight junctions [17]. We now demonstrate that treatment of MDCK cells with antimycin A and deoxyglucose results in the release of cytochrome c into the cytoplasm, indicating activation of apoptosis. E-cadherin is cleaved into two major fragments by this treatment. One fragment encompasses the extracellular domain, as elucidated by a specific monoclonal antibody [15]. The other fragment is
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01143-4
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recognized by a monoclonal antibody directed to an epitope in the cytoplasmic domain. The size of both fragments together is equivalent to the mass of intact Ecadherin. A caspase-3 consensus site is located in the cytoplasmic domain in human E-cadherin (747-DTRD750; GenBank Accession No. P12830) at a location consistent with the size of the two cleaved fragments. The cytoplasmic fragment of E-cadherin in antimycin/deoxyglucose treated cells collects intracellularly into a diffuse pattern overlain with punctate structures. Incubating cells with the membrane permeable caspase inhibitor DEVD-CHO, but not the serine protease inhibitor, PMSF, significantly diminished the cleavage of E-cadherin, and preserves its cell membrane localization. These observations strongly suggest that caspase activation contributes to the cleavage and redistribution of E-cadherin fragments. Under severe respiratory stress and apoptosis, transmembrane proteins appear to be subjected to caspase mediated cleavage that operates independently of the proteasomal and lysosomal degradation characterized previously for transmembrane protein processing [18–20].
Materials and methods Cells, reagents, antibodies, and protease inhibitors. MDCK type I cells, a canine polarized epithelial cell line, and MCF-7 cells, an epithelial breast cancer line deficient in the caspase-3 activation pathway [21] purchased from ATCC were employed in this study. MDCK cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% FBS and antibiotics. MCF-7 cells were grown in minimal essential medium with 10% FBS and antibiotics. Antibodies to a cytoplasmic epitope of E-cadherin (Clone 36) and to b-catenin (Clone 14) were purchased from Transduction Laboratories, San Diego, CA. Antibody to cytochrome c was purchased from Santa Cruz Biotechnologies (Clone H104). Cell permeable caspase inhibitors (caspase 6: Z-VEID-FMK; caspase 8: ITED-CHO; and caspase 3: DEVD-CHO) were purchased from Calbiochem (San Diego, CA). Antimycin and deoxyglucose were acquired from Sigma (St. Louis, MO). The protease inhibitor cocktail added to cell lysates was purchased from Sigma and contained AEBSF, E-64, bestatin, leupeptin, aprotinin, and EDTA. PMSF was also added to the cell lysates. Antimycin and deoxyglucose treatment. Confluent plates of cells were incubated in PBS (supplemented with 1.5 mM CaCl2 and 2 mM MgCl2 ) in the presence of 10 mM antimycin A and 2 mM deoxy-D glucose. Control cells were incubated in the PBS solution supplemented with CaCl2 and MgCl2 , without addition of antimycin or deoxyglucose. Cells were incubated in these solutions at 32 °C in a 5% CO2 incubator for the specified time intervals and then lysed in the RIPA buffer described below, or processed for immunofluorescent microscopy. Caspase inhibitor treatment. Confluent monolayers of cells were preincubated in 17 lM caspase inhibitor solubilized in DMSO, and then subjected to antimycin/deoxyglucose treatment for 3.5 h also in the presence of 17 lM caspase inhibitor. For control, an equivalent volume of DMSO was added to the cells untreated with caspase inhibitors. Immunofluorescent microscopy. Cells grown on coverslips were fixed in 4% formaldehyde in PBS, and permeabilized in 0.1% Triton, 1% BSA, 1% fish gelatin, and 1% BSA. Primary and fluorophore-conjugated secondary antibodies (or rhodamine–phalloidin for actin stain-
ing) were added to this solution diluted by one-half in PBS to stain cells. Fluoromount G was employed for preservation. Images were taken with a confocal laser scanning microscope. Immunoprecipitation and Western blots. Cells were lysed in a RIPA buffer (0.2% SDS, 0.4% deoxycholate, 1% NP-40, 130 mM NaCl, 1 mM EDTA, and 50 mM Tris, pH 8.0) with protease inhibitors on ice. Solutions were clarified and aliquots of supernatant were added to Laemmli sample buffer and boiled, or subjected to immunoprecipitation. For immunoprecipitations, antibody was added to clarified lysate, followed by the addition of protein G immunoprecipitation beads (Ultalink Protein G; Pierce, IL). Laemmli sample buffer was added to the beads and boiled to elute bound proteins. As much as 4–20% gradient gels purchased from Novex (San Diego, CA) were employed to resolve proteins, which were transferred to nitrocellulose for 1 h at 100 V. Western blots were developed with chemiluminescence techniques (Pierce Supersignal, Rockford, IL). Cell fractionation. To isolate membrane and cytoplasmic fractions by ultracentrifugation, cells were sonicated briefly in the buffer 0.33 M sucrose, 0.5 M EGTA, 2 mM EDTA, 1 mM Na3 VO4 , 10 mM NaF, and 20 mM Tris, pH 7.4, supplemented with protease inhibitors. The homogenate was placed in an ultracentrifuge tube and fractionated at 100,000g for 1 h. Supernatant was collected and the pellet was briefly rinsed in the above buffer. Laemmli sample buffer was added to the cell pellet, which was vortexed and solubilized. Aliquots of the supernatant were also solubilized in Laemmli sample buffer. Samples were resolved in gels and Western blots, which were developed with antibody to the C-terminus of E-cadherin. Tunnel assay. The “Apoalert” tunnel assay kit (Clontech, Palo Alto, CA) employing DAB stain was used according to the manufacturer’s instructions to detect nicked DNA. A dark brown deposit in the nucleus displays nicked DNA.
Results and discussion Cytochrome c release into the cytoplasm Mitochondrial depolarization, permeability barrier disruption, and cytochrome c release into the cytoplasm are upstream events in the caspase-3 activation pathway [22]. Released cytochrome c distributes into a diffuse intracellular pattern which is distinct from the punctate appearance of cytochrome c located in intact mitochondria [22,23]. MDCK cells were treated with antimycin/deoxyglucose for 2 h and cytochrome c was detected with an anti-cytochrome c antibody in paraformaldehyde fixed and permeabilized cells (Fig. 1). A confocal microscope was employed for imaging: two representative images are displayed for untreated (Fig. 1A) and treated cells (Fig. 1B) in Fig. 1. Well-resolved punctate structures in perinuclear locations were observed for the untreated cells, revealing intact mitochondria (Fig. 1A). Activation of apoptosis initiated in mitochondria is thus minimal in the untreated cells. In contrast, a significant fraction (approximately 48%) of antimycin/deoxyglucose treated cells exhibited a diffusely appearing cytochrome c stain, suggesting that cytochrome c was released into the cytoplasm (Fig. 1B). Diffusely appearing cytochrome c stain implicates apoptosis pathway and caspase-3 activation [22,24,25]. These data therefore raised the
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Fig. 1. Antimycin/deoxyglucose (Ant/DG) treatment of MDCK cells for 2 h results in cytochrome c release into the cytoplasm. MDCK cells were untreated (A; control) or treated (B) with Ant/DG for 2 h, fixed, permeabilized, and processed for immunofluorescent microscopy using an anti-cytochrome c antibody. Images were taken with a confocal microscope. Two representative images for control and Ant/DG treated are displayed. A significant fraction of cells (48%) treated with Ant/DG display diffusely appearing cytochrome c stain, implicating release into the cytoplasm.
Fig. 2. Antimycin/deoxyglucose (Ant/DG) treatment of MDCK cells removes E-cadherin from the cell surface and elevates an intracellular pool. MDCK cells were treated or untreated with Ant/DG for 3.5 h and processed for immunofluorescent microscopy by paraformaldehyde fixing and permeabilization. The top two images (A) were developed with a monoclonal antibody recognizing an extracellular epitope. The bottom two images (B) represent detection with a monoclonal antibody that recognizes the C-terminal domain of Ecadherin.
possibility that antimycin/deoxyglucose treatment of MCDK cells activates an apoptosis pathway mediated through cytochrome c release, with expected caspase-3 activation.
E-cadherin was largely removed from the cell surface (Fig. 2A; Ant/DG+). The intracellular stain with the antibody to the N-terminus epitope likely also reflected E-cadherin in the biosynthetic or endocytosis pathway compartments in the antimycin/deoxyglucose treated cells (Fig. 2A; Ant/DG+). A relatively faint stain was also observed on the cell periphery using the antibody directed to the C-terminal epitope of E-cadherin in cells treated with antimycin/deoxyglucose (Fig. 2B; Ant/ DG+). An intense intracellular stain was observed in antimycin/deoxyglucose treated cells stained with the antibody directed to the c-terminus epitope (Fig. 2B; Ant/ DG+). The intracellular stain appeared diffuse in most cells, a pattern consistent with deposition into the cytoplasm. The E-cadherin stain was brightest intracellularly when focal planes perpendicular to the microscope slide were examined in series. Immunofluorescent images of antimycin/deoxyglucose treated cells using a non-specific mouse IgG and the same anti-mouse FITC conjugated secondary antibody did not reveal resolvable patterns (data not shown), indicating that the immunofluorescent images reflected specific detection of Ecadherin. The distinctly appearing cellular distributions of Nand C-terminal peptides of E-cadherin in antimycin/ deoxyglucose treated cells suggested that cleavage resulted in removal of the N-terminal-extracellular fragment from the cell surface and release of a C-terminal fragment into the cytoplasm. In support of this interpretation, cell surface E-cadherin stains were faint when
Removal of the E-cadherin N-terminal fragment and release of the C-terminal fragment into the cytoplasm MDCK cells were treated or untreated with antimycin/deoxyglucose (Ant/DG) for 3 h and then prepared for immunofluorescent microscopy using the anti-Ecadherin antibodies that recognize the N- or C-terminus epitopes. Cells were fixed in paraformaldehyde and permeabilized prior to antibody application: images were taken with a confocal microscope. Untreated cells revealed an intense E-cadherin stain at the cell periphery (Figs. 2A and B; Ant/DG)). The most intense E-cadherin stain corresponded to the cell periphery when the confocal fields were viewed perpendicular to the coverslip (Figs. 2A and B; Ant/DG)). This distribution pattern is consistent with E-cadherin located on the plasma membrane. A fainter appearing intracellular stain was also detected in cells, which likely reflected E-cadherin in the biosynthesis or endocytosis pathway compartments (Figs. 2A and B; Ant/DG)). Observing E-cadherin at the cell periphery has been reported in numerous studies and is consistent with its role as a cell adhesion molecule. In contrast, cells treated with antimycin/deoxyglucose revealed a relatively faint stain with the antibody to the N-terminus, suggesting that the N-terminal fragment of
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Fig. 3. Antimycin/deoxyglucose treatment of MDCK cells diminishes membrane embedded full-length E-cadherin and releases a cleaved fragment into the cytoplasm (arrow; lane 4). Cells were fractionated into membrane and cytoplasmic fractions by ultracentrifugation at 100,000g for 1 h. Supernatant and pellet fractions were resolved in gels and Western blots, and E-cadherin was detected with the monoclonal antibody that recognizes a cytoplasmic epitope. Lane 1: unfractionated-total (T) cell lysate control. Lane 2: unfractionated-total (T) cell lysate in cells treated with Ant/DG for 3.5 h. Lane 3: 100,000g pellet (P) fraction in cells treated with Ant/DG. Lane 4: 100,000g supernatant (S) fraction in the same cells as in Lane 3 (treated with Ant/DG).
No. P12830) was cut. Cellular fractionation also enables identifying whether an E-cadherin fragment is released into the cytoplasm. Ultracentrifugation at 100,000g for 1 h was employed to fractionate membrane and cytoplasmic fractions, and E-cadherin fragments were detected by Western blot (Fig. 3). Cells were treated, or untreated with antimycin/deoxyglucose (Ant/DG), and sonicated in the fractionation buffer described in Materials and methods. Aliquots of total cell lysates before fractionation were removed (T: total) and the remaining lysate was applied into an ultracentrifugation tube. Following 100,000g centrifugation for 1 h, pellet (P) and supernatant (S) were isolated and diluted in Laemmli sample buffer. Samples were resolved in gels and Western blots, which were developed with anti-E-cadherin antibody directed to the C-terminal epitope (Fig. 3). Western blots were also developed with secondary antibody alone to verify specific detection of E-cadherin fragments. Resulting Western blots revealed enrichment of an E-cadherin fragment in cytoplasmic fractions of antimycin/deoxyglucose treated cells (Fig. 3, compare lane 2 to lane 4; arrow). The size of this fragment was approximately 30 kDa, which, based on the resolving capacity of SDS–PAGE, is consistent with the location of caspase-3 consensus cleavage site 747-DTRD-750. Data from the cellular fractionation experiment thus support the interpretation that E-cadherin C-terminal fragment is released into the cytoplasm during antimycin/deoxyglucose treatment. Inhibition of E-cadherin cleavage with the caspase inhibitor DEVD-CHO
detected with antibodies to the N- and C-termini in antimycin/deoxyglucose treated cells (Figs. 2A and B; Ant/DG+). Cleavage and release of the C-terminal fragment into the cytoplasm are also consistent with the cytoplasmic location of caspases in the cell. As detailed below, ultracentrifuge fractionation of antimycin/deoxyglucose treated cells into membrane and cytoplasmic fractions also supports the interpretation of cytoplasmic fragment release into the cytoplasm (see Fig. 3). Membrane and cytoplasmic fractionation of cleaved Ecadherin We previously demonstrated cleavage of E-cadherin in antimycin/deoxyglucose treated cells and generation of an 80 kDa fragment by employing the monoclonal antibody to the N-terminus to develop Western blots [15]. However, Western blot data employing the monoclonal antibody to the C-terminus epitope were not studied. Elucidation of the size of the N-terminal and C-terminal products could provide supportive evidence that a caspase-3 cleavage site at a homologous position to the 747-DTRD-750 caspase consensus cleavage site in human E-cadherin (GenBank Accession
As noted above, a caspase-3 consensus site in the form of DXXD is present in the cytoplasmic tail of Ecadherin, suggesting the possibility for cleavage by this enzyme. To identify the enzyme(s) involved in the cleavage, MDCK cells were pre-treated with caspase inhibitors and subjected to antimycin/deoxyglucose incubation, also in the presence of caspase inhibitors. Confluent monolayers of MDCK cells were pretreated overnight (16 h) with the cell permeable caspase inhibitor DEVD-CHO (17 lM), followed by antimycin/deoxyglucose treatment for 3.5 h in the presence of the same caspase inhibitor. Cells were lysed on ice in RIPA buffer with protease inhibitors, solutions were clarified, and equivalent sample volumes were resolved in gels and Western blots, which were developed with antibody to the C-terminus of E-cadherin (Fig. 4). Cells subjected to 3.5 h of antimycin/deoxyglucose treatment exhibited a significantly diminished full length 120 kDa E-cadherin band (Fig. 4, lane 2), a finding consistent with the data displayed in Fig. 3. In contrast, the full length E-cadherin band was largely preserved in cells treated with the cell permeable caspase inhibitor DEVD-CHO (Fig. 4, lane 3). This finding indicates that
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Addition of DEVD-CHO to antimycin/deoxyglucose treated cells maintains the pool of E-cadherin associated with b-catenin
Fig. 4. Treatment of MDCK cells with the cell permeable caspase-3 inhibitor DEVD-CHO reduces E-cadherin cleavage during antimycin/ deoxyglucose treatment. Confluent monolayers of MDCK cells were subjected to Ant/DG treatment for 3.5 h in the absence of inhibitor ()) or pretreated with inhibitor DEVD-CHO for 16 h and then subjected to Ant/DG for 3.5 h in the presence of DEVD-CHO (+). Equivalent numbers of cell plates were lysed and clarified and supernatants were resolved in gels and Western blots. Antibody to the cytoplasmic epitope of E-cadherin was used for Western blot detection.
the caspase inhibitor DEVD-CHO inhibits the cleavage of E-cadherin during antimycin/deoxyglucose treatment. [Equivalent protein loads among the gel lanes were demonstrated by similarly dense background staining bands (Fig. 4).] DEVD-CHO is known to inhibit caspases 3, 6, 7, 8, and 10 that cleave at the consensus sequence DXXD (X is any amino acid) [26,27]. The caspase inhibition experiment thus implicates a candidate group of caspases involved in E-cadherin cleavage. Subsequent experiments that employed the cell permeable inhibitors specific to caspase 6 (Z-VEID-FMK) and caspase 8 (ITED-CHO), also at 17 lM with 16 h of pretreatment, did not reveal inhibition of E-cadherin cleavage during antimycin/deoxyglucose treatment (data not shown). These results therefore narrowed identification of the candidate protease to caspases 3, 7, and 10. Inhibitors specific to caspases 7 and 10 were not available commercially, which limited more specific identification of the protease involved in E-cadherin cleavage. Moreover, addition of the proteosomal or lysosomal inhibitors ALLN, ALLM, E64, calpeptin, chloroquine, primaquine, and BAPTA to antimycin/deoxyglucose treated cells did not inhibit cleavage, which suggested that proteasomal and lysosomal proteases were not involved in the cleavage [15]. Although the selection of protease inhibitors in our experiments was not exhaustive, substantial inhibition of cleavage by DVED-CHO implicates a prominent role for caspases in the degradation of E-cadherin during antimycin/deoxyglucose treatment.
The total cellular E-cadherin pool consists of (1) nascent-unassembled molecules undergoing synthesis and processing in the endoplasmic reticulum (ER); (2) Ecadherin assembled with b-catenin in the ER and upstream in the secretory pathway; and (3) E-cadherin assembled into the mature-functional adherens junction configuration located at the cell surface. (Other intracellular pools conceivably exist as well, such as a cytoplasmic pool in a degradative pathway.) In the mature adherens junction, E-cadherin is associated with b-catenin in the cytoplasmic tail, which in turn, is assembled with c or a-catenin, a complex that links the extracellular domain of E-cadherin to the cytoskeleton [28,29]. Co-immunoprecipitation experiments between Ecadherin and b-catenin were performed to examine whether addition of DEVD-CHO preserved the adherens junction during antimycin/deoxyglucose treatment. It should also be noted here that a-, b-, and c-catenins in MDCK cells did not degrade during the 3.5 h of antimycin/deoxyglucose treatment under the conditions employed in this study [15]. Cells were pretreated with DEVD-CHO for 16 h and subsequently exposed to antimycin/deoxyglucose in the presence of DEVD-CHO (Fig. 5, lane 3). For control,
Fig. 5. Treatment of MDCK cells with DEVD-CHO protects the Ecadherin pool assembled with b-catenin in antimycin/deoxyglucose (Ant/DG) treated cells. Cells were treated with Ant/DG in the absence ()) or presence (+) of inhibitor DEVD-CHO. Aliquots of cell lysates were subjected to immunoprecipitation with antibody to b-catenin. Western blots were developed with anti-E-cadherin antibody.
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other cells were not treated with antimycin/deoxyglucose (Fig. 5, lane 1) or treated with antimycin/deoxyglucose in the absence of DEVD-CHO (Fig. 5, lane 2). Equivalent numbers of confluent cell plates of control and treated samples were lysed and clarified following 3.5 h of treatment. Samples were subjected to immunoprecipitation employing anti-b-catenin antibody and subsequent Western blots were developed with anti-Ecadherin (Fig. 5). DEVD-CHO treatment largely maintained the Ecadherin assembled with b-catenin, as evidenced by the robust co-immunopreciptation between b-catenin and E-cadherin (Fig. 5, lane 3). In the absence of DEVDCHO, reduced co-immunoprecipitation was evident (Fig. 5, lane 2), presumably due to the cleavage of Ecadherin. These data therefore suggest that breakdown of the adherens junction during antimycin/deoxyglucose treatment is mediated primarily by one class of proteases (caspase). The cellular fate of E-cadherin during antimycin/deoxyglucose treatment in the presence of DEVD-CHO Confocal immunofluorescent microscopy was also employed to determine whether the addition of DEVDCHO maintains E-cadherin at the cell surface in antimycin/deoxyglucose treated cells. Consistent with the
data presented above in Fig. 2B, antimycin/deoxyglucose treatment largely removed E-cadherin from the cell membrane (Fig. 6, compare A to B). In contrast, DEVD-CHO treatment preserved E-cadherin at the cell periphery in the antimycin/deoxyglucose treated cells, although relatively minor degradation was apparent (Fig. 6, panel C). The immunofluorescent images are thus consistent with the Western blot data indicating that DEVD-CHO inhibited E-cadherin cleavage. In summary, DEVD-CHO treatment limits E-cadherin cleavage and disassembly of the adherens junction during antimycin/deoxyglucose treatment. To demonstrate the relative specificity of DEVDCHO to limiting E-cadherin redistribution in antimycin/ deoxyglucose treated cells, the arrangement of the actin cytoskeleton was also examined. Rhodamine–phalloidin staining of fixed and permeabilized cells was employed to detect the actin cytoskeleton. In the absence of antimycin/deoxyglucose treatment, the stress-fiber arrangement was clearly visible at the cell periphery (Fig. 7, panel A). However, the actin staining patterns were extensively altered by antimycin/deoxyglucose treatment, revealing punctate structures located in the cell interior (Fig. 7, panel B). Treatment of cells with DEVD-CHO did not protect the actin cytoskeleton, since the appearance of the actin stain was very similar to that detected in the cells untreated with DEVD-CHO
Fig. 6. Addition of the caspase inhibitor DEVD-CHO largely maintains E-cadherin at the cell surface in antimycin/deoxyglucose treated MDCK cells. Cells were untreated (control) or treated with Ant/DG in the absence or presence of DEVD-CHO for 3.5 h, fixed, permeabilized, and exposed to the anti-E-cadherin antibody that recognizes the C-terminus. A confocal microscope was employed for imaging.
Fig. 7. DEVD-CHO addition to antimycin/deoxyglucose treated MDCK cells does not inhibit actin rearrangement. Cells were co-treated with antimycin/deoxyglucose (Ant/DG) and DEVD-CHO as in Fig. 6, except staining was to actin using rhodamine–phalloidin.
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(Fig. 7, panel C). These observations therefore suggest that mechanisms other than caspase mediated (that is blockable by DEVD-CHO) are involved in actin cytoskeleton damage. It should be noted that antimycin/ deoxyglucose treatment depletes ATP levels and abrogates kinesin molecular motor functions required to maintain the actin cytoskeleton [30,31]. Degradation of E-cadherin is not evident in caspase-3 deficient MCF-7 cells MCF-7 is an epithelial breast cancer line deficient in the caspase-3 activation pathway [32]. Cells were subjected to antimycin/deoxyglucose treatment employing the protocol and time spans described above for MDCK cells. Following treatment, cells and media were collected, the cells were sedimented and lysed, and aliquots were resolved in gels and Western blots. E-cadherin degradation was minimal in MCF-7 cells following 3.0 h of antimycin/deoxyglucose treatment, as evidenced by preservation of the full length 120 kDa band and minimal detection of fragment (Fig. 8). These data therefore indicate that E-cadherin is preserved in antimycin/deoxyglucose treated cells that have an impaired caspase-3 activation pathway. Data derived from DEVD-CHO treated MDCK cells, and separately, from MCF-7 cells, implicate a caspase mediated pathway in E-cadherin cleavage during antimycin/deoxyglucose treatment.
Fig. 8. Full length E-cadherin is maintained in caspase-3 deficient MCF-7 cells during antimycin/deoxyglucose treatment. Cells were treated with antimycin/deoxyglucose at the specified time spans and solubilized, and E-cadherin was detected in Western blots with the anti-E-cadherin antibody to the cytoplasmic domain.
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Tunnel assay of MDCK cells treated with antimycin/ deoxyglucose The tunnel assay detects DNA fragmentation, a downstream event of apoptosis. MDCK cells were treated, or untreated with antimycin/deoxyglucose for 3.5 h and processed with the ApoAlert tunnel assay kit (Clonetech, CA) to detect fragmented DNA. The control cells grown for 3.5 h in PBS supplemented with 0.1 mM CaCl2 , 1 mM MgCl2 at 37 °C did not react with the tunnel assay reagents (Fig. 9, panel A). In contrast, cells treated with antimycin/deoxyglucose revealed positive tunnel stain in a small fraction of the cells (approximately 1%; Fig. 9, panel B). Although approximately 48% of the cells subjected to 2.0 h of antimycin/ deoxyglucose exhibited cytochrome c release into the cytoplasm, the smaller fraction of cells revealing DNA fragmentation by the tunnel assay indicated that 3.5 h of antimycin/deoxyglucose treatment was not a sufficient time span to reach a downstream event of the apoptosis pathway in the majority of cells. Thus, E-cadherin is
Fig. 9. Tunnel assay identified DNA fragmentation in a small fraction of antimycin/deoxyglucose treated MDCK cells. The DAB stained cells exhibit DNA fragmentation, as detected by the tunnel “Apoalert” kit (Clonetech, CA). (A) Control, cells grown for 3.5 h in PBS supplemented with 1 mM MgCl2 , 0.1 mM CaCl2 . (B) Cells treated with Ant/DG in PBS, 1 mM MgCl2 , and 0.1 mM CaCl2 for 3.5 h.
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cleaved by antimycin/deoxyglucose treatment at relatively upstream steps of the apoptosis pathway. Treatment of MDCK cells with antimycin/deoxyglucose has been employed widely to model ischemia in epithelial tissues [1–3]. The combination of antimycin A and 2-deoxy-D -glucose depletes cellular ATP by inhibition of both glycolysis and oxidative phosphorylation. Recent experimental evidence has also demonstrated that antimycin treatment generates pores in the mitochondrial outer membrane and disrupts the mitochondrial permeability barrier [5], which are early steps in the apoptosis pathway initiated in mitochondria. The combined cellular damage caused by antimycin/deoxyglucose treatment is therefore due to ATP depletion and the activation of mitochondrial apoptosis pathways. Ecadherin cleavage and the redistribution of fragments however appear to be a direct result of the apoptosis pathway activation. Following 2 h of antimycin/deoxyglucose treatment, nearly one-half of the cells displayed cytochrome c released into the cytoplasm, with the remaining cells showing evidence for localization in intact mitochondria (Fig. 1). Cytochrome c release into the cytoplasm is a hallmark for caspase-3 activation [33]. The 2 h time span for antimycin/deoxyglucose treatment was chosen instead of the 3.5 h interval employed elsewhere in this study to reveal the contrasts between damaged mitochondria and intact punctate appearing mitochondria on the same image field. It can be expected that proteins with the consensus sequence DXXD (X is any amino acid) located in cytoplasmic domains will become cleaved upon cytochrome c release [34–36]. Western blots of E-cadherin extracted from cells subjected to 2 h of antimycin/deoxyglucose treatment revealed partial cleavage of E-cadherin [15], which closely overlapped quantitatively with the fraction of cells exhibiting damaged mitochondria: approximately one-half of the cellular E-cadherin pool was cleaved [15] corresponding to approximately 48% of the cells exhibiting damaged mitochrondria (Fig. 1, panel B). The relationship between the fraction of cells exhibiting cleaved E-cadherin and damaged mitochondria further supports a significant role for a caspase mediated pathway in E-cadherin cleavage. Treatment of cells with the caspase inhibitor DEVDCHO largely inhibited the cleavage and preserved intact E-cadherin at the cell periphery. The cell permeable inhibitor DEVD-CHO blocks degradation at the consensus sequence DXXD, which is cleaved by caspases 3, 6, 7, 8, and 10 [26]. Treatment of cells with specific inhibitors to caspases 6 and 8 did not inhibit degradation, and inhibitors unique to caspases 7 and 10 were not available. Thus, the experimental analysis employing a battery of caspase inhibitors narrowed identification of the candidate protease that cleaves E-cadherin to caspase 3, 7 or 10. A study using staurosporin, an inducer of mi-
tochondrial mediated apoptosis, also demonstrated Ecadherin cleavage in MDCK cells that was partially blockable with a DEVD analogue [10]. Taken together, the experimental observations in our study are consistent with the interpretation that a caspase mediated pathway is involved in the dissolution of the adherens junction when the mitochondrial membrane permeability transition is disrupted by antimycin/deoxyglucose treatment. These data therefore strongly implicate a caspase mediated mechanism for the disassembly of the adherens junction upon activation of upstream events in the mitochondrial apoptosis pathway.
Acknowledgments This work was supported by an American Heart Association Grant to S.H.K. and NIH R01 to S.K.N. The imaging experiments utilized facilities of the National Center of Microscopy and Imaging Research, UCSD, which is supported by RR04050 to Mark Ellisman.
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Biochemical processing of E-cadherin under cellular stressq Steven H. Kellera,* and Sanjay K. Nigamb a
Department of Medicine, University of California, San Diego, 210A Dickinson Street, San Diego, CA 92103-8382, USA b Department of Medicine and Pediatrics, University of California, San Diego, La Jolla, CA 92093-0693, USA Received 2 June 2003
Abstract The proteolytic cleavage pathways of E-cadherin endogenously expressed in MDCK (Madin–Darby canine kidney) cells were characterized in cells treated with antimycin A and deoxyglucose to examine transmembrane protein processing under cellular stress. E-cadherin is a type I transmembrane protein which operates as the cell adhesion molecule component of the adherens junction, a complex of proteins involved in epithelial tissue development and integrity. We now demonstrate that treatment of MDCK cells with antimycin A and deoxyglucose activates caspase mediated pathways that cleave E-cadherin. E-cadherin is cleaved into two major fragments, with the sizes predicted by the location of a caspase-3 cleavage consensus sequence. Cleavage of E-cadherin and deposition of the C-terminal fragment into the cytoplasm are inhibited by the caspase inhibitor DEVD-CHO. Thus, a major mechanism for E-cadherin cleavage and dissolution of the adherens junction under antimycin/deoxyglucose treatment is caspase mediated, initiated by activation of an apoptosis pathway. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Caspase; MDCK cells; Antimycin; Deoxyglucose; Cellular stress; Ischemia; Adherens junction; E-cadherin; Protein degradation; Apoptosis
To study the biochemical processing of transmembrane proteins in epithelial cells subjected to cellular stress, we treated MDCK cells with antimycin A and deoxyglucose and followed the fate of endogenously expressed E-cadherin. Treatment of cells with antimycin A and deoxyglucose has been employed to model ischemia in cell culture due to ATP depletion and glucose deprivation [1–3]. Antimycin A inhibits oxidative phosphorylation and activates apoptosis pathways signaled through mitochondrial membrane depolarization [4–6]. Antimycin A binds to and inactivates Bcl-2, which normally inhibits mitochondrial membrane depolarization during the initiation of apoptosis [5,7,8]. Deoxyglucose activates endoplasmic reticulum stress pathways q Abbreviations: MDCK, Madin–Darby canine kidney; Ant/DG, antimycin/deoxyglucose; PMSF, phenylmethylsulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene-bis(oxyethylenenitrilo)]tetraacetic acid; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; DXXD, aspartate–X–X–aspartate, where X is any amino acid. * Corresponding author. Fax: 1-619-543-3432. E-mail address: [email protected] (S.H. Keller).
[9]. Although the biochemical and cellular consequences of staurosporine treatment are well characterized [10– 13], the biochemical fate of proteins in antimycin/ deoxyglucose-treated cells has not been studied extensively. E-cadherin is a single-pass transmembrane protein that operates as the cell adhesion molecule component of the adherens junction [14–16]. E-cadherin is assembled at the cytoplasmic tail with b- or c-catenin, which in turn are joined with a-catenin that bridges the extracellular domain of E-cadherin to the actin cytoskeleton [14]. It has been hypothesized that degradation of E-cadherin is a central lesion in creation of the ischemic epithelial phenotype, which includes a loss of epithelial polarity and disruption of tight junctions [17]. We now demonstrate that treatment of MDCK cells with antimycin A and deoxyglucose results in the release of cytochrome c into the cytoplasm, indicating activation of apoptosis. E-cadherin is cleaved into two major fragments by this treatment. One fragment encompasses the extracellular domain, as elucidated by a specific monoclonal antibody [15]. The other fragment is
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01143-4
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recognized by a monoclonal antibody directed to an epitope in the cytoplasmic domain. The size of both fragments together is equivalent to the mass of intact Ecadherin. A caspase-3 consensus site is located in the cytoplasmic domain in human E-cadherin (747-DTRD750; GenBank Accession No. P12830) at a location consistent with the size of the two cleaved fragments. The cytoplasmic fragment of E-cadherin in antimycin/deoxyglucose treated cells collects intracellularly into a diffuse pattern overlain with punctate structures. Incubating cells with the membrane permeable caspase inhibitor DEVD-CHO, but not the serine protease inhibitor, PMSF, significantly diminished the cleavage of E-cadherin, and preserves its cell membrane localization. These observations strongly suggest that caspase activation contributes to the cleavage and redistribution of E-cadherin fragments. Under severe respiratory stress and apoptosis, transmembrane proteins appear to be subjected to caspase mediated cleavage that operates independently of the proteasomal and lysosomal degradation characterized previously for transmembrane protein processing [18–20].
Materials and methods Cells, reagents, antibodies, and protease inhibitors. MDCK type I cells, a canine polarized epithelial cell line, and MCF-7 cells, an epithelial breast cancer line deficient in the caspase-3 activation pathway [21] purchased from ATCC were employed in this study. MDCK cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5% FBS and antibiotics. MCF-7 cells were grown in minimal essential medium with 10% FBS and antibiotics. Antibodies to a cytoplasmic epitope of E-cadherin (Clone 36) and to b-catenin (Clone 14) were purchased from Transduction Laboratories, San Diego, CA. Antibody to cytochrome c was purchased from Santa Cruz Biotechnologies (Clone H104). Cell permeable caspase inhibitors (caspase 6: Z-VEID-FMK; caspase 8: ITED-CHO; and caspase 3: DEVD-CHO) were purchased from Calbiochem (San Diego, CA). Antimycin and deoxyglucose were acquired from Sigma (St. Louis, MO). The protease inhibitor cocktail added to cell lysates was purchased from Sigma and contained AEBSF, E-64, bestatin, leupeptin, aprotinin, and EDTA. PMSF was also added to the cell lysates. Antimycin and deoxyglucose treatment. Confluent plates of cells were incubated in PBS (supplemented with 1.5 mM CaCl2 and 2 mM MgCl2 ) in the presence of 10 mM antimycin A and 2 mM deoxy-D glucose. Control cells were incubated in the PBS solution supplemented with CaCl2 and MgCl2 , without addition of antimycin or deoxyglucose. Cells were incubated in these solutions at 32 °C in a 5% CO2 incubator for the specified time intervals and then lysed in the RIPA buffer described below, or processed for immunofluorescent microscopy. Caspase inhibitor treatment. Confluent monolayers of cells were preincubated in 17 lM caspase inhibitor solubilized in DMSO, and then subjected to antimycin/deoxyglucose treatment for 3.5 h also in the presence of 17 lM caspase inhibitor. For control, an equivalent volume of DMSO was added to the cells untreated with caspase inhibitors. Immunofluorescent microscopy. Cells grown on coverslips were fixed in 4% formaldehyde in PBS, and permeabilized in 0.1% Triton, 1% BSA, 1% fish gelatin, and 1% BSA. Primary and fluorophore-conjugated secondary antibodies (or rhodamine–phalloidin for actin stain-
ing) were added to this solution diluted by one-half in PBS to stain cells. Fluoromount G was employed for preservation. Images were taken with a confocal laser scanning microscope. Immunoprecipitation and Western blots. Cells were lysed in a RIPA buffer (0.2% SDS, 0.4% deoxycholate, 1% NP-40, 130 mM NaCl, 1 mM EDTA, and 50 mM Tris, pH 8.0) with protease inhibitors on ice. Solutions were clarified and aliquots of supernatant were added to Laemmli sample buffer and boiled, or subjected to immunoprecipitation. For immunoprecipitations, antibody was added to clarified lysate, followed by the addition of protein G immunoprecipitation beads (Ultalink Protein G; Pierce, IL). Laemmli sample buffer was added to the beads and boiled to elute bound proteins. As much as 4–20% gradient gels purchased from Novex (San Diego, CA) were employed to resolve proteins, which were transferred to nitrocellulose for 1 h at 100 V. Western blots were developed with chemiluminescence techniques (Pierce Supersignal, Rockford, IL). Cell fractionation. To isolate membrane and cytoplasmic fractions by ultracentrifugation, cells were sonicated briefly in the buffer 0.33 M sucrose, 0.5 M EGTA, 2 mM EDTA, 1 mM Na3 VO4 , 10 mM NaF, and 20 mM Tris, pH 7.4, supplemented with protease inhibitors. The homogenate was placed in an ultracentrifuge tube and fractionated at 100,000g for 1 h. Supernatant was collected and the pellet was briefly rinsed in the above buffer. Laemmli sample buffer was added to the cell pellet, which was vortexed and solubilized. Aliquots of the supernatant were also solubilized in Laemmli sample buffer. Samples were resolved in gels and Western blots, which were developed with antibody to the C-terminus of E-cadherin. Tunnel assay. The “Apoalert” tunnel assay kit (Clontech, Palo Alto, CA) employing DAB stain was used according to the manufacturer’s instructions to detect nicked DNA. A dark brown deposit in the nucleus displays nicked DNA.
Results and discussion Cytochrome c release into the cytoplasm Mitochondrial depolarization, permeability barrier disruption, and cytochrome c release into the cytoplasm are upstream events in the caspase-3 activation pathway [22]. Released cytochrome c distributes into a diffuse intracellular pattern which is distinct from the punctate appearance of cytochrome c located in intact mitochondria [22,23]. MDCK cells were treated with antimycin/deoxyglucose for 2 h and cytochrome c was detected with an anti-cytochrome c antibody in paraformaldehyde fixed and permeabilized cells (Fig. 1). A confocal microscope was employed for imaging: two representative images are displayed for untreated (Fig. 1A) and treated cells (Fig. 1B) in Fig. 1. Well-resolved punctate structures in perinuclear locations were observed for the untreated cells, revealing intact mitochondria (Fig. 1A). Activation of apoptosis initiated in mitochondria is thus minimal in the untreated cells. In contrast, a significant fraction (approximately 48%) of antimycin/deoxyglucose treated cells exhibited a diffusely appearing cytochrome c stain, suggesting that cytochrome c was released into the cytoplasm (Fig. 1B). Diffusely appearing cytochrome c stain implicates apoptosis pathway and caspase-3 activation [22,24,25]. These data therefore raised the
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Fig. 1. Antimycin/deoxyglucose (Ant/DG) treatment of MDCK cells for 2 h results in cytochrome c release into the cytoplasm. MDCK cells were untreated (A; control) or treated (B) with Ant/DG for 2 h, fixed, permeabilized, and processed for immunofluorescent microscopy using an anti-cytochrome c antibody. Images were taken with a confocal microscope. Two representative images for control and Ant/DG treated are displayed. A significant fraction of cells (48%) treated with Ant/DG display diffusely appearing cytochrome c stain, implicating release into the cytoplasm.
Fig. 2. Antimycin/deoxyglucose (Ant/DG) treatment of MDCK cells removes E-cadherin from the cell surface and elevates an intracellular pool. MDCK cells were treated or untreated with Ant/DG for 3.5 h and processed for immunofluorescent microscopy by paraformaldehyde fixing and permeabilization. The top two images (A) were developed with a monoclonal antibody recognizing an extracellular epitope. The bottom two images (B) represent detection with a monoclonal antibody that recognizes the C-terminal domain of Ecadherin.
possibility that antimycin/deoxyglucose treatment of MCDK cells activates an apoptosis pathway mediated through cytochrome c release, with expected caspase-3 activation.
E-cadherin was largely removed from the cell surface (Fig. 2A; Ant/DG+). The intracellular stain with the antibody to the N-terminus epitope likely also reflected E-cadherin in the biosynthetic or endocytosis pathway compartments in the antimycin/deoxyglucose treated cells (Fig. 2A; Ant/DG+). A relatively faint stain was also observed on the cell periphery using the antibody directed to the C-terminal epitope of E-cadherin in cells treated with antimycin/deoxyglucose (Fig. 2B; Ant/ DG+). An intense intracellular stain was observed in antimycin/deoxyglucose treated cells stained with the antibody directed to the c-terminus epitope (Fig. 2B; Ant/ DG+). The intracellular stain appeared diffuse in most cells, a pattern consistent with deposition into the cytoplasm. The E-cadherin stain was brightest intracellularly when focal planes perpendicular to the microscope slide were examined in series. Immunofluorescent images of antimycin/deoxyglucose treated cells using a non-specific mouse IgG and the same anti-mouse FITC conjugated secondary antibody did not reveal resolvable patterns (data not shown), indicating that the immunofluorescent images reflected specific detection of Ecadherin. The distinctly appearing cellular distributions of Nand C-terminal peptides of E-cadherin in antimycin/ deoxyglucose treated cells suggested that cleavage resulted in removal of the N-terminal-extracellular fragment from the cell surface and release of a C-terminal fragment into the cytoplasm. In support of this interpretation, cell surface E-cadherin stains were faint when
Removal of the E-cadherin N-terminal fragment and release of the C-terminal fragment into the cytoplasm MDCK cells were treated or untreated with antimycin/deoxyglucose (Ant/DG) for 3 h and then prepared for immunofluorescent microscopy using the anti-Ecadherin antibodies that recognize the N- or C-terminus epitopes. Cells were fixed in paraformaldehyde and permeabilized prior to antibody application: images were taken with a confocal microscope. Untreated cells revealed an intense E-cadherin stain at the cell periphery (Figs. 2A and B; Ant/DG)). The most intense E-cadherin stain corresponded to the cell periphery when the confocal fields were viewed perpendicular to the coverslip (Figs. 2A and B; Ant/DG)). This distribution pattern is consistent with E-cadherin located on the plasma membrane. A fainter appearing intracellular stain was also detected in cells, which likely reflected E-cadherin in the biosynthesis or endocytosis pathway compartments (Figs. 2A and B; Ant/DG)). Observing E-cadherin at the cell periphery has been reported in numerous studies and is consistent with its role as a cell adhesion molecule. In contrast, cells treated with antimycin/deoxyglucose revealed a relatively faint stain with the antibody to the N-terminus, suggesting that the N-terminal fragment of
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Fig. 3. Antimycin/deoxyglucose treatment of MDCK cells diminishes membrane embedded full-length E-cadherin and releases a cleaved fragment into the cytoplasm (arrow; lane 4). Cells were fractionated into membrane and cytoplasmic fractions by ultracentrifugation at 100,000g for 1 h. Supernatant and pellet fractions were resolved in gels and Western blots, and E-cadherin was detected with the monoclonal antibody that recognizes a cytoplasmic epitope. Lane 1: unfractionated-total (T) cell lysate control. Lane 2: unfractionated-total (T) cell lysate in cells treated with Ant/DG for 3.5 h. Lane 3: 100,000g pellet (P) fraction in cells treated with Ant/DG. Lane 4: 100,000g supernatant (S) fraction in the same cells as in Lane 3 (treated with Ant/DG).
No. P12830) was cut. Cellular fractionation also enables identifying whether an E-cadherin fragment is released into the cytoplasm. Ultracentrifugation at 100,000g for 1 h was employed to fractionate membrane and cytoplasmic fractions, and E-cadherin fragments were detected by Western blot (Fig. 3). Cells were treated, or untreated with antimycin/deoxyglucose (Ant/DG), and sonicated in the fractionation buffer described in Materials and methods. Aliquots of total cell lysates before fractionation were removed (T: total) and the remaining lysate was applied into an ultracentrifugation tube. Following 100,000g centrifugation for 1 h, pellet (P) and supernatant (S) were isolated and diluted in Laemmli sample buffer. Samples were resolved in gels and Western blots, which were developed with anti-E-cadherin antibody directed to the C-terminal epitope (Fig. 3). Western blots were also developed with secondary antibody alone to verify specific detection of E-cadherin fragments. Resulting Western blots revealed enrichment of an E-cadherin fragment in cytoplasmic fractions of antimycin/deoxyglucose treated cells (Fig. 3, compare lane 2 to lane 4; arrow). The size of this fragment was approximately 30 kDa, which, based on the resolving capacity of SDS–PAGE, is consistent with the location of caspase-3 consensus cleavage site 747-DTRD-750. Data from the cellular fractionation experiment thus support the interpretation that E-cadherin C-terminal fragment is released into the cytoplasm during antimycin/deoxyglucose treatment. Inhibition of E-cadherin cleavage with the caspase inhibitor DEVD-CHO
detected with antibodies to the N- and C-termini in antimycin/deoxyglucose treated cells (Figs. 2A and B; Ant/DG+). Cleavage and release of the C-terminal fragment into the cytoplasm are also consistent with the cytoplasmic location of caspases in the cell. As detailed below, ultracentrifuge fractionation of antimycin/deoxyglucose treated cells into membrane and cytoplasmic fractions also supports the interpretation of cytoplasmic fragment release into the cytoplasm (see Fig. 3). Membrane and cytoplasmic fractionation of cleaved Ecadherin We previously demonstrated cleavage of E-cadherin in antimycin/deoxyglucose treated cells and generation of an 80 kDa fragment by employing the monoclonal antibody to the N-terminus to develop Western blots [15]. However, Western blot data employing the monoclonal antibody to the C-terminus epitope were not studied. Elucidation of the size of the N-terminal and C-terminal products could provide supportive evidence that a caspase-3 cleavage site at a homologous position to the 747-DTRD-750 caspase consensus cleavage site in human E-cadherin (GenBank Accession
As noted above, a caspase-3 consensus site in the form of DXXD is present in the cytoplasmic tail of Ecadherin, suggesting the possibility for cleavage by this enzyme. To identify the enzyme(s) involved in the cleavage, MDCK cells were pre-treated with caspase inhibitors and subjected to antimycin/deoxyglucose incubation, also in the presence of caspase inhibitors. Confluent monolayers of MDCK cells were pretreated overnight (16 h) with the cell permeable caspase inhibitor DEVD-CHO (17 lM), followed by antimycin/deoxyglucose treatment for 3.5 h in the presence of the same caspase inhibitor. Cells were lysed on ice in RIPA buffer with protease inhibitors, solutions were clarified, and equivalent sample volumes were resolved in gels and Western blots, which were developed with antibody to the C-terminus of E-cadherin (Fig. 4). Cells subjected to 3.5 h of antimycin/deoxyglucose treatment exhibited a significantly diminished full length 120 kDa E-cadherin band (Fig. 4, lane 2), a finding consistent with the data displayed in Fig. 3. In contrast, the full length E-cadherin band was largely preserved in cells treated with the cell permeable caspase inhibitor DEVD-CHO (Fig. 4, lane 3). This finding indicates that
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Addition of DEVD-CHO to antimycin/deoxyglucose treated cells maintains the pool of E-cadherin associated with b-catenin
Fig. 4. Treatment of MDCK cells with the cell permeable caspase-3 inhibitor DEVD-CHO reduces E-cadherin cleavage during antimycin/ deoxyglucose treatment. Confluent monolayers of MDCK cells were subjected to Ant/DG treatment for 3.5 h in the absence of inhibitor ()) or pretreated with inhibitor DEVD-CHO for 16 h and then subjected to Ant/DG for 3.5 h in the presence of DEVD-CHO (+). Equivalent numbers of cell plates were lysed and clarified and supernatants were resolved in gels and Western blots. Antibody to the cytoplasmic epitope of E-cadherin was used for Western blot detection.
the caspase inhibitor DEVD-CHO inhibits the cleavage of E-cadherin during antimycin/deoxyglucose treatment. [Equivalent protein loads among the gel lanes were demonstrated by similarly dense background staining bands (Fig. 4).] DEVD-CHO is known to inhibit caspases 3, 6, 7, 8, and 10 that cleave at the consensus sequence DXXD (X is any amino acid) [26,27]. The caspase inhibition experiment thus implicates a candidate group of caspases involved in E-cadherin cleavage. Subsequent experiments that employed the cell permeable inhibitors specific to caspase 6 (Z-VEID-FMK) and caspase 8 (ITED-CHO), also at 17 lM with 16 h of pretreatment, did not reveal inhibition of E-cadherin cleavage during antimycin/deoxyglucose treatment (data not shown). These results therefore narrowed identification of the candidate protease to caspases 3, 7, and 10. Inhibitors specific to caspases 7 and 10 were not available commercially, which limited more specific identification of the protease involved in E-cadherin cleavage. Moreover, addition of the proteosomal or lysosomal inhibitors ALLN, ALLM, E64, calpeptin, chloroquine, primaquine, and BAPTA to antimycin/deoxyglucose treated cells did not inhibit cleavage, which suggested that proteasomal and lysosomal proteases were not involved in the cleavage [15]. Although the selection of protease inhibitors in our experiments was not exhaustive, substantial inhibition of cleavage by DVED-CHO implicates a prominent role for caspases in the degradation of E-cadherin during antimycin/deoxyglucose treatment.
The total cellular E-cadherin pool consists of (1) nascent-unassembled molecules undergoing synthesis and processing in the endoplasmic reticulum (ER); (2) Ecadherin assembled with b-catenin in the ER and upstream in the secretory pathway; and (3) E-cadherin assembled into the mature-functional adherens junction configuration located at the cell surface. (Other intracellular pools conceivably exist as well, such as a cytoplasmic pool in a degradative pathway.) In the mature adherens junction, E-cadherin is associated with b-catenin in the cytoplasmic tail, which in turn, is assembled with c or a-catenin, a complex that links the extracellular domain of E-cadherin to the cytoskeleton [28,29]. Co-immunoprecipitation experiments between Ecadherin and b-catenin were performed to examine whether addition of DEVD-CHO preserved the adherens junction during antimycin/deoxyglucose treatment. It should also be noted here that a-, b-, and c-catenins in MDCK cells did not degrade during the 3.5 h of antimycin/deoxyglucose treatment under the conditions employed in this study [15]. Cells were pretreated with DEVD-CHO for 16 h and subsequently exposed to antimycin/deoxyglucose in the presence of DEVD-CHO (Fig. 5, lane 3). For control,
Fig. 5. Treatment of MDCK cells with DEVD-CHO protects the Ecadherin pool assembled with b-catenin in antimycin/deoxyglucose (Ant/DG) treated cells. Cells were treated with Ant/DG in the absence ()) or presence (+) of inhibitor DEVD-CHO. Aliquots of cell lysates were subjected to immunoprecipitation with antibody to b-catenin. Western blots were developed with anti-E-cadherin antibody.
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other cells were not treated with antimycin/deoxyglucose (Fig. 5, lane 1) or treated with antimycin/deoxyglucose in the absence of DEVD-CHO (Fig. 5, lane 2). Equivalent numbers of confluent cell plates of control and treated samples were lysed and clarified following 3.5 h of treatment. Samples were subjected to immunoprecipitation employing anti-b-catenin antibody and subsequent Western blots were developed with anti-Ecadherin (Fig. 5). DEVD-CHO treatment largely maintained the Ecadherin assembled with b-catenin, as evidenced by the robust co-immunopreciptation between b-catenin and E-cadherin (Fig. 5, lane 3). In the absence of DEVDCHO, reduced co-immunoprecipitation was evident (Fig. 5, lane 2), presumably due to the cleavage of Ecadherin. These data therefore suggest that breakdown of the adherens junction during antimycin/deoxyglucose treatment is mediated primarily by one class of proteases (caspase). The cellular fate of E-cadherin during antimycin/deoxyglucose treatment in the presence of DEVD-CHO Confocal immunofluorescent microscopy was also employed to determine whether the addition of DEVDCHO maintains E-cadherin at the cell surface in antimycin/deoxyglucose treated cells. Consistent with the
data presented above in Fig. 2B, antimycin/deoxyglucose treatment largely removed E-cadherin from the cell membrane (Fig. 6, compare A to B). In contrast, DEVD-CHO treatment preserved E-cadherin at the cell periphery in the antimycin/deoxyglucose treated cells, although relatively minor degradation was apparent (Fig. 6, panel C). The immunofluorescent images are thus consistent with the Western blot data indicating that DEVD-CHO inhibited E-cadherin cleavage. In summary, DEVD-CHO treatment limits E-cadherin cleavage and disassembly of the adherens junction during antimycin/deoxyglucose treatment. To demonstrate the relative specificity of DEVDCHO to limiting E-cadherin redistribution in antimycin/ deoxyglucose treated cells, the arrangement of the actin cytoskeleton was also examined. Rhodamine–phalloidin staining of fixed and permeabilized cells was employed to detect the actin cytoskeleton. In the absence of antimycin/deoxyglucose treatment, the stress-fiber arrangement was clearly visible at the cell periphery (Fig. 7, panel A). However, the actin staining patterns were extensively altered by antimycin/deoxyglucose treatment, revealing punctate structures located in the cell interior (Fig. 7, panel B). Treatment of cells with DEVD-CHO did not protect the actin cytoskeleton, since the appearance of the actin stain was very similar to that detected in the cells untreated with DEVD-CHO
Fig. 6. Addition of the caspase inhibitor DEVD-CHO largely maintains E-cadherin at the cell surface in antimycin/deoxyglucose treated MDCK cells. Cells were untreated (control) or treated with Ant/DG in the absence or presence of DEVD-CHO for 3.5 h, fixed, permeabilized, and exposed to the anti-E-cadherin antibody that recognizes the C-terminus. A confocal microscope was employed for imaging.
Fig. 7. DEVD-CHO addition to antimycin/deoxyglucose treated MDCK cells does not inhibit actin rearrangement. Cells were co-treated with antimycin/deoxyglucose (Ant/DG) and DEVD-CHO as in Fig. 6, except staining was to actin using rhodamine–phalloidin.
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(Fig. 7, panel C). These observations therefore suggest that mechanisms other than caspase mediated (that is blockable by DEVD-CHO) are involved in actin cytoskeleton damage. It should be noted that antimycin/ deoxyglucose treatment depletes ATP levels and abrogates kinesin molecular motor functions required to maintain the actin cytoskeleton [30,31]. Degradation of E-cadherin is not evident in caspase-3 deficient MCF-7 cells MCF-7 is an epithelial breast cancer line deficient in the caspase-3 activation pathway [32]. Cells were subjected to antimycin/deoxyglucose treatment employing the protocol and time spans described above for MDCK cells. Following treatment, cells and media were collected, the cells were sedimented and lysed, and aliquots were resolved in gels and Western blots. E-cadherin degradation was minimal in MCF-7 cells following 3.0 h of antimycin/deoxyglucose treatment, as evidenced by preservation of the full length 120 kDa band and minimal detection of fragment (Fig. 8). These data therefore indicate that E-cadherin is preserved in antimycin/deoxyglucose treated cells that have an impaired caspase-3 activation pathway. Data derived from DEVD-CHO treated MDCK cells, and separately, from MCF-7 cells, implicate a caspase mediated pathway in E-cadherin cleavage during antimycin/deoxyglucose treatment.
Fig. 8. Full length E-cadherin is maintained in caspase-3 deficient MCF-7 cells during antimycin/deoxyglucose treatment. Cells were treated with antimycin/deoxyglucose at the specified time spans and solubilized, and E-cadherin was detected in Western blots with the anti-E-cadherin antibody to the cytoplasmic domain.
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Tunnel assay of MDCK cells treated with antimycin/ deoxyglucose The tunnel assay detects DNA fragmentation, a downstream event of apoptosis. MDCK cells were treated, or untreated with antimycin/deoxyglucose for 3.5 h and processed with the ApoAlert tunnel assay kit (Clonetech, CA) to detect fragmented DNA. The control cells grown for 3.5 h in PBS supplemented with 0.1 mM CaCl2 , 1 mM MgCl2 at 37 °C did not react with the tunnel assay reagents (Fig. 9, panel A). In contrast, cells treated with antimycin/deoxyglucose revealed positive tunnel stain in a small fraction of the cells (approximately 1%; Fig. 9, panel B). Although approximately 48% of the cells subjected to 2.0 h of antimycin/ deoxyglucose exhibited cytochrome c release into the cytoplasm, the smaller fraction of cells revealing DNA fragmentation by the tunnel assay indicated that 3.5 h of antimycin/deoxyglucose treatment was not a sufficient time span to reach a downstream event of the apoptosis pathway in the majority of cells. Thus, E-cadherin is
Fig. 9. Tunnel assay identified DNA fragmentation in a small fraction of antimycin/deoxyglucose treated MDCK cells. The DAB stained cells exhibit DNA fragmentation, as detected by the tunnel “Apoalert” kit (Clonetech, CA). (A) Control, cells grown for 3.5 h in PBS supplemented with 1 mM MgCl2 , 0.1 mM CaCl2 . (B) Cells treated with Ant/DG in PBS, 1 mM MgCl2 , and 0.1 mM CaCl2 for 3.5 h.
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cleaved by antimycin/deoxyglucose treatment at relatively upstream steps of the apoptosis pathway. Treatment of MDCK cells with antimycin/deoxyglucose has been employed widely to model ischemia in epithelial tissues [1–3]. The combination of antimycin A and 2-deoxy-D -glucose depletes cellular ATP by inhibition of both glycolysis and oxidative phosphorylation. Recent experimental evidence has also demonstrated that antimycin treatment generates pores in the mitochondrial outer membrane and disrupts the mitochondrial permeability barrier [5], which are early steps in the apoptosis pathway initiated in mitochondria. The combined cellular damage caused by antimycin/deoxyglucose treatment is therefore due to ATP depletion and the activation of mitochondrial apoptosis pathways. Ecadherin cleavage and the redistribution of fragments however appear to be a direct result of the apoptosis pathway activation. Following 2 h of antimycin/deoxyglucose treatment, nearly one-half of the cells displayed cytochrome c released into the cytoplasm, with the remaining cells showing evidence for localization in intact mitochondria (Fig. 1). Cytochrome c release into the cytoplasm is a hallmark for caspase-3 activation [33]. The 2 h time span for antimycin/deoxyglucose treatment was chosen instead of the 3.5 h interval employed elsewhere in this study to reveal the contrasts between damaged mitochondria and intact punctate appearing mitochondria on the same image field. It can be expected that proteins with the consensus sequence DXXD (X is any amino acid) located in cytoplasmic domains will become cleaved upon cytochrome c release [34–36]. Western blots of E-cadherin extracted from cells subjected to 2 h of antimycin/deoxyglucose treatment revealed partial cleavage of E-cadherin [15], which closely overlapped quantitatively with the fraction of cells exhibiting damaged mitochondria: approximately one-half of the cellular E-cadherin pool was cleaved [15] corresponding to approximately 48% of the cells exhibiting damaged mitochrondria (Fig. 1, panel B). The relationship between the fraction of cells exhibiting cleaved E-cadherin and damaged mitochondria further supports a significant role for a caspase mediated pathway in E-cadherin cleavage. Treatment of cells with the caspase inhibitor DEVDCHO largely inhibited the cleavage and preserved intact E-cadherin at the cell periphery. The cell permeable inhibitor DEVD-CHO blocks degradation at the consensus sequence DXXD, which is cleaved by caspases 3, 6, 7, 8, and 10 [26]. Treatment of cells with specific inhibitors to caspases 6 and 8 did not inhibit degradation, and inhibitors unique to caspases 7 and 10 were not available. Thus, the experimental analysis employing a battery of caspase inhibitors narrowed identification of the candidate protease that cleaves E-cadherin to caspase 3, 7 or 10. A study using staurosporin, an inducer of mi-
tochondrial mediated apoptosis, also demonstrated Ecadherin cleavage in MDCK cells that was partially blockable with a DEVD analogue [10]. Taken together, the experimental observations in our study are consistent with the interpretation that a caspase mediated pathway is involved in the dissolution of the adherens junction when the mitochondrial membrane permeability transition is disrupted by antimycin/deoxyglucose treatment. These data therefore strongly implicate a caspase mediated mechanism for the disassembly of the adherens junction upon activation of upstream events in the mitochondrial apoptosis pathway.
Acknowledgments This work was supported by an American Heart Association Grant to S.H.K. and NIH R01 to S.K.N. The imaging experiments utilized facilities of the National Center of Microscopy and Imaging Research, UCSD, which is supported by RR04050 to Mark Ellisman.
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