From the Departments of Medical Oncology and
Tumorimmunology and
Protein Chemistry, Max Delbrück Center
of Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin
and the ¶ University Hospital Benjamin Franklin, Institute of
Clinical Chemistry and Pathobiochemistry, Hindenburgdamm 30,
D-12200 Berlin, Germany
Received for publication, July 11, 2000, and in revised form, October 26, 2000
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ABSTRACT |
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Apoptotic cell death induces dramatic molecular
changes in cells, becoming apparent on the structural level as membrane
blebbing, condensation of the cytoplasm and nucleus, and loss of
cell-cell contacts. The activation of caspases is one of the
fundamental steps during programmed cell death. Here we report a
detailed analysis of the fate of the
Ca2+-dependent cell adhesion molecule
E-cadherin in apoptotic epithelial cells and show that during apoptosis
fragments of E-cadherin with apparent molecular masses of 24, 29, and 84 kDa are generated by two distinct proteolytic activities. In
addition to a caspase-3-mediated cleavage releasing the cytoplasmic
domain of E-cadherin, a metalloproteinase sheds the extracellular
domain from the cell surface during apoptosis. Immunofluorescence
analysis confirmed that concomitant with the disappearance of
E-cadherin staining at the cell surface, the E-cadherin cytoplasmic
domain accumulates in the cytosol. In the presence of inhibitors of
caspase-3 and/or metalloproteinases, cleavage of E-cadherin was almost
completely blocked. The simultaneous cleavage of the intracellular and
extracellular domains of E-cadherin may provide a highly efficient
mechanism to disrupt cadherin-mediated cell-cell contacts in apoptotic
cells, a prerequisite for cell rounding and exit from the epithelium.
The crucial role of apoptosis during development and for tissue
homeostasis of multicellular organisms is well established (1).
Malfunctions of the death program and its control mechanisms often
result in prenatal death during development and contribute to immune
and neuronal diseases or cancer in the adult organism (2-4). The
central mechanism of this cell death machinery is a proteolytic cascade
mediated by the caspase family of cysteine proteinases (5, 6), which
specifically cleave their substrates after aspartate residues. Caspases
are synthesized as inactive proenzymes. After initiation of the
apoptotic program these proenzymes are processed by two proteolytic
events, generating a large subunit and a small subunit that form a
heterodimer. The association of two heterodimers results in the
formation of the active enzyme containing two catalytic sites (7,
8). Depending on their position in the proteolytic cascade,
caspase family members are divided into upstream initiator caspases and
downstream effector caspases.
The activation of this caspase cascade leads to the specific cleavage
of substrate proteins and finally results in the morphological changes
becoming apparent in apoptotic cells. The identification of an
increasing number of caspase substrates has revealed different classes
of cellular proteins that are cleaved during the effector phase of
apoptosis. A set of substrate proteins is represented by proteins that
protect living cells from apoptosis, e.g. ICAD/DFF45 (9, 10)
and Bcl-2 (11, 12). Nuclear envelope proteins (e.g. lamin A
and B (13-15) and LAP2 and Nup153 (16)), and cytoskeletal proteins and
regulators (e.g. fodrin (17), keratins (18, 19), gelsolin (20), Gas2 (21), and FAK (22-24)) represent targets of
caspases, resulting in disruption of the cyto-architecture of cells.
Furthermore, inactivation of proteins involved in cell cycle regulation
(e.g. pRb (25), Mdm2 (26), p21 (27)) and DNA repair
(DNA-PKCS) and of the splicing machinery (U1-70K) is assumed to support the disruption of structural and signaling networks
in an apoptotic cell (28).
Morphological changes observed during apoptosis in part result from
effects on cell-cell contacts. The cadherin-catenin adhesion complex
represents one of the major adhesive systems in multiple epithelial
tissues. Cadherins comprise a large family of
Ca2+-dependent, homophilic cell-cell adhesion
molecules essential for morphogenic movements and tissue formation
during development and for maintenance of tissue integrity in the adult
organism (29-31). Mutations in E-cadherin were identified in a number
of carcinomas and highly invasive tumor-derived cell lines (32). For
their functional integrity, cadherin extracellular domains have to form
lateral dimers, and the cytoplasmic domain has to be connected to the
actin cytoskeleton by the catenins (33). Within the cadherin-catenin
complex, Here we present a detailed analysis of the apoptotic fate of E-cadherin
in epithelial cells. Induction of apoptosis resulted in the formation
of three detectable E-cadherin cleavage products with apparent
molecular masses of 24, 29, and 84 kDa, respectively, that were
generated by two distinct proteolytic activities. One of these
activities could be blocked by the caspase-3 inhibitor Z-DEVD-FMK,1 whereas the
other proteolytic event, shedding the E-cadherin extracellular domain
into the cell culture medium, was mediated by a metalloprotease.
According to these data both proteases are required for the efficient
cleavage of E-cadherin during apoptosis.
Cell Culture--
MDCK (Madin-Darby canine kidney) cells were
cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) with 10% (v/v) fetal calf serum in the presence of
100 units/ml penicillin and 100 µg/ml streptomycin (Life
Technologies, Inc.) at 5% CO2. The human breast epithelial
cell line H184A1 was grown in Dulbecco's modified Eagle's
medium/Ham's F12 (1:1) (Biochrom, Berlin, Germany) supplemented
with 5% (v/v) fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml transferrin (Sigma), 10 µg/ml insulin (Biochrom), and 1.8 µg/ml hydrocortisone (Biochrom). The human breast carcinoma cells MCF-7.3.28 (transfected with caspase-3) and MCF-7/Vector (17) (kindly provided by Dr. A. Porter) were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.8 mg/ml G418 (Life Technologies, Inc.).
Reagents and Antibodies--
The monoclonal antibody directed
against the E-cadherin cytoplasmic domain (clone 36) was purchased from
Transduction Laboratories (Lexington, KY); antibody HECD-1 against the
human E-cadherin extracellular domain was obtained from R & D Systems
(Wiesbaden, Germany); and DECMA-1 was kindly provided by Dr.
Rolf Kemler (Max Planck Institute of Immunobiology, Freiburg, Germany).
Horseradi peroxidase-labeled anti-mouse and anti-rabbit
antibodies were purchased from Dianova (Hamburg, Germany). Alexa
FluorTM 488 goat anti-mouse IgG and Alexa
FluorTM 594 phalloidin were obtained from Molecular Probes
(MoBiTec, Gottingen, Germany). Caspase-3 inhibitor Z-DEVD-FMK
and MMP inhibitor I were purchased from Calbiochem; TAPI
(N Induction of Apoptosis and Preparation of Cell
Lysates--
Apoptosis was induced in confluent monolayers of cells
cultivated in 6-well dishes by addition of 1 µM
staurosporine in Me2SO or 2 µg/ml camptothecin.
Adherent cells were washed twice with PBS, incubated with ice-cold
lysis buffer (10 mM imidazole (pH 6.8), 0.1 M
KCl, 0.3 M sucrose, 2 mM MgCl2, 10 mM EGTA, 1 mM NaF, 1 mM
MbO42 Western Blot Analysis--
For Western blot analysis of cell
lysates, 50 µg of total protein in 4× SDS loading buffer was
separated on 14% ProSieve SDS-polyacrylamide gels (FMC BioProducts
obtained from Biozym, Hess. Oldendorf, Germany) and transferred
onto polyvinylidene difluoride membranes (ImmobilonTM-P,
Millipore). Membranes were blocked with TST buffer (10 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.1% (v/v)
Tween 20) for 1 h at room temperature and incubated with first
antibody at a concentration of 1 µg/ml in TST for 1 h. After
three washes, membranes were incubated with horseradish
peroxidase-conjugated second antibody diluted 1:10,000 in TST. After
washing, chemoluminescence detection was performed by exposure of
Lumi-Light Western blotting substrate (Roche Molecular
Biochemicals)-treated membranes to Biomax MR films (Kodak, Rochester,
NY). For quantification of the chemoluminescence signals, membranes
were scanned with a FujiFilm LAS-1000 system and analyzed with the
Image Gauge version 3.2 software. Molecular weights of fragments
were determined using the BenchMarkTM Prestained Protein
Ladder (Life Technologies, Inc.).
Immunoprecipitations--
To detect the 84-kDa E-cadherin
fragment released from the cell surface, cell culture supernatants were
collected at different time points after induction of apoptosis. After
centrifugation for 10 min at 20,800 × g, 1 ml of the
supernatant was pre-cleared by incubation with 30 µl of protein
A-Sepharose for 30 min at 4 °C under constant agitation. Protein A
beads were removed by centrifugation at 20,800 × g for
10 min at 4 °C. For immunoprecipitation, 0.5 µg of HECD-1 antibody
or 5 µg of DECMA-1 antibody were added. After 30 min at 4 °C, 40 µl of a 1:1 slurry of protein A-Sepharose beads were added and
incubated for 1 h as described above. Protein A beads were
sedimented by centrifugation (1 min, 2700 × g,
4 °C), washed five times with wash buffer (50 mM NaCl,
300 mM sucrose, 10 mM imidazole (pH 6.8), 3 mM MgCl2, 0.5% (v/v) Triton X-100), and
resuspended in 20 µl of 2× SDS sample buffer. After boiling, proteins were separated by 7.5% SDS-polyacrylamide gel
electrophoresis, and Western blotting was performed as described above.
Immunofluorescence--
Cells were grown on glass coverslips.
3 h or 6 h after induction of apoptosis, cells were briefly
washed with PBS and fixed in ice-cold methanol for 10 min.
Subsequently, cells were washed in PBS, and after blocking with 0.1%
(v/v) goat serum in PBS for 30 min at room temperature, cells were
incubated with anti-E-cadherin antibodies for 30 min at room
temperature (0.5 µg/ml for anti-E-cadherin clone 36 and 2 µg/ml for
anti-HECD-1). After three washes in PBS, cells were incubated with
Alexa FluorTM 488 goat anti-mouse IgG for another 30 min
and washed again before mounting in elvanol. For double staining, cells
were washed twice with prewarmed PBS, fixed in 3% paraformaldehyde for
20 min, and incubated with blocking buffer (PBS + 25 mM
glycine) for 5 min. After two additional washes with PBS, cells were
permeabilized with 0.1% Triton X-100 in PBS for 3 min and subsequently
blocked with 0.1% (v/v) goat serum in PBS for 30 min at room
temperature. E-cadherin staining was performed as described above, and
subsequent phalloidin staining was performed with Alexa
FluorTM 594 phalloidin according to the manufacturer's
instructions. Analysis and photography were performed on a Zeiss LSM510
confocal microscope with × 63 magnification at excitation
wavelengths of 543 and 488 nm. Details on the microscopy setup are
available on request.
In Vitro Caspase Cleavage--
GST·ECT was expressed in
Escherichia coli as described (41). 5 µg of recombinant
protein were digested with 50 ng of recombinant caspase-3, -6, or -7 in
50 mM HEPES (pH 7.4), 0.1% (w/v) CHAPS, 5 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 µM leupeptin, 200 µg/ml aprotinin at 37 °C for 2-4
h. After separation by SDS-polyacrylamide gel electrophoresis, cleavage
products were transferred to a polyvinylidene difluoride membrane.
Coomassie-stained protein bands were excised, destained in 60%
acetonitrile, and analyzed by Edman sequencing on a Procise sequencer
(Applied Biosystems).
E-cadherin Cleavage during Apoptosis of Epithelial Cells--
To
investigate the fate of E-cadherin during programmed cell death,
lysates of MDCK cells were examined by Western analysis upon induction
of apoptosis by staurosporine treatment. Changes in morphology,
fragmentation of the nucleus, and detachment from the substrate
indicated that MDCK cells responded to the apoptotic stimulus. Nearly
all full-length E-cadherin was cleaved 24 h after addition of
staurosporine (Fig. 1A). As
determined by quantitative chemoluminescence imaging, 50% of
E-cadherin was proteolytically processed during the first 11-13 h of
staurosporine treatment (Fig. 1B). Distinct cleavage
products of E-cadherin with apparent molecular masses of about 24 kDa
and 29 kDa, designated fragment 1 and fragment 2, respectively, were
detectable by 3 h after induction of apoptosis with an antibody
(clone C36) directed against the cytoplasmic domain of E-cadherin. A
35-kDa polypeptide band reacting with the antibody was also present in
nonapoptotic cells, indicating that this band is not related to the
apoptotic cleavage of E-cadherin. Furthermore, by comparing
Ltk
To find out whether fragments 1 and 2 were generated by caspase
cleavage, the membrane-permeable irreversible caspase inhibitor Z-DEVD-FMK was added to the cultured cells prior to staurosporine treatment, and cell lysates were analyzed for E-cadherin 6 h
later. In the presence of Z-DEVD-FMK, formation of fragment 1 was
completely blocked, whereas formation of fragment 2 was not affected
(Fig. 2). This observation explains why,
despite the presence of caspase-inhibitor, full-length E-cadherin still
was proteolytically processed (data not shown) and suggests that
E-cadherin is targeted by a distinct second cleavage event.
In Vitro Cleavage of the E-cadherin Cytoplasmic Domain with
Recombinant Caspase-3, -6, and -7--
Because fragment 1 was detected
with an antibody directed against the cytoplasmic domain of E-cadherin,
and generation of fragment 1 was blocked by Z-DEVD-FMK, it was expected
that this fragment resulted from a caspase-mediated intracellular
cleavage event. To identify the cleavage site and the caspase(s)
responsible for this cleavage, recombinant mouse E-cadherin cytoplasmic
tail expressed as a GST fusion protein (GST·ECT) was digested
in vitro with recombinant caspase-3, -6, and -7, respectively. GST·ECT was efficiently digested by caspase-3,
resulting in the formation of 30- and 24-kDa cleavage products (Fig.
3A). Caspase-7 generated fragments of identical molecular mass, however, with a markedly reduced
efficiency and thus were barely detectable. Caspase-6 treatment did not
generate detectable cleavage products at all. Treatment with the
caspase inhibitor Z-DEVD-FMK blocked the generation of the 24-kDa
fragment (Fig. 3A). Edman degradation revealed that the
30-kDa fragment represented the N terminus of the GST moiety. The amino
acid sequence obtained from the 24-kDa fragment precisely defined the
caspase-3 cleavage site C-terminal to Asp752 in an
Asp-Asp-Asp-Thr-Arg-Asp752-Asn-Val-Tyr-Tyr
motif. This site is highly conserved in different species, located next
to the transmembrane domain, and represents the only caspase-3
consensus sequence in the cytoplasmic tail (Fig. 3C). The
24-kDa in vitro cleavage product and fragment 1 produced
in vivo in apoptotic cells comigrated on SDS-polyacrylamide gels, indicating that both fragments are identical (Fig.
3B). The discrepancy in the apparent (24 kDa) and calculated
molecular masses (15 kDa) for this E-cadherin cytoplasmic domain
fragment might be explained by an unusual migration behavior of the
cytoplasmic domain of E-cadherin during SDS-polyacrylamide gel
electrophoresis or because of migration differences of molecular mass
standards.
Further evidence that caspase-3 is the major caspase responsible for
cleavage of the E-cadherin cytoplasmic tail in apoptotic cells was
given by analyses of MCF-7 cells that have been previously shown to be
deficient in caspase-3 (17). Induction of apoptosis in these cells did
not result in the generation of detectable amounts of fragments 1 and
2, respectively. In contrast, in MCF-7 cells that have been stably
transfected with caspase-3, both fragments were generated (Fig.
4).
Shedding of the E-cadherin Extracellular Fragment from the Cell
Surface during Apoptosis--
The inhibitor studies and the mapping of
the caspase-3 cleavage site to Asp752 proximal to the
transmembrane segment of E-cadherin suggested that fragment 2 was
generated by an extracellular cleavage event. This, however, should
result in an extracellular domain fragment that is released from the
cell surface into the culture medium. Indeed, in immunoprecipitation
experiments with antibodies directed against the extracellular domain
of E-cadherin, increasing amounts of an 84-kDa polypeptide (fragment 3)
were precipitated from cell culture supernatants of apoptotic cells
with time after staurosporine addition (Fig.
5).
Next we wanted to characterize the enzyme(s) responsible for the
formation of the 84-kDa extracellular fragment of E-cadherin. There is
clear evidence now that shedding of the extracellular domain of a
number of cell surface proteins is a regulated process that can be
blocked by metalloproteinase inhibitors (42). To test whether the
formation of the 84-kDa extracellular fragment of E-cadherin is
mediated by this type of shedding protease, inhibitor studies were
performed. In the presence of the matrix metalloproteinase inhibitor I,
no significant inhibitory effect was detected (data not shown). In
contrast, TAPI, a metalloproteinase inhibitor that was shown to block
tumor necrosis factor- Localization of E-cadherin in Apoptotic Cells--
Caspase-3
cleavage of E-cadherin cytoplasmic domains proximal to the
transmembrane region was expected to release fragment 1 into the
cytosol. To analyze this, E-cadherin and E-cadherin fragments were
localized in apoptotic cells by confocal immunofluorescence microscopy.
Staining of E-cadherin at the plasma membrane of MDCK cells with
anti-E-cadherin C36 antibody directed against the cytoplasmic domain of
E-cadherin was reduced and became diffuse after onset of apoptosis.
Cells started to shrink, and E-cadherin cell surface staining was
finally undetectable in cells that had lost their cell-cell contacts.
Concomitant with cell rounding and disintegration of cell nuclei,
E-cadherin staining became detectable in the cytoplasm, confirming the
release of the E-cadherin fragment 1 into the cytosol (Fig.
7A, a-c). Cells
treated with Z-DEVD-FMK retained staining of E-cadherin at the plasma
membrane, and in consequence cytoplasmic E-cadherin staining was
reduced (Fig. 7A, d). Moreover, in the presence
of the caspase inhibitor, STS-treated cells exhibited significantly
less rounding, and morphological integrity was less affected.
Confocal immunofluorescence analysis of H184A1 cells with antibody
HECD-1 directed against the extracellular domain also showed a
reduction of E-cadherin staining at the cell membrane. TAPI treatment
inhibited this decrease in E-cadherin staining, showing that by
blocking the extracellular cleavage event, fragment 4 containing the
transmembrane domain remained attached in the cell membrane.
Furthermore, cell morphology appeared to be less affected in these
cells. E-cadherin staining in cells treated with both TAPI and
Z-DEVD-FMK was highly similar to that of nonapoptotic cells;
however, cells appeared flattened and the cell surface was more
ruffled (Fig. 7B). To visualize the reorganization of the
actin cytoskeleton during apoptosis, we performed costaining with Alexa
FluorTM 594-phalloidin. In untreated cells strong staining
at sites of cell-cell contact and beyond the cell surface was
detectable. STS treatment resulted in a nearly complete disruption of
actin filaments within 3-6 h. Residual staining could often be
detected in irregularly shaped structures especially prominent in
rounded cells, similar to those shown by Brancolini et al.
(35) (data not shown). TAPI treatment did not block actin fiber
destruction. In the presence of both inhibitors cell size remained
comparable with nonapoptotic control cells, and phalloidin staining was
detectable at sites of cell-cell contacts. This emphasizes the
importance of the cadherin-catenin system for the establishment and
maintenance of the submembranous actin filament system and suggests
that destruction of cadherins in this way promotes drastic cell shape
changes during apoptosis.
Specific cell-cell and cell-matrix contacts regulate cell growth
in epithelial cells, and disruption of these contacts induces apoptotic
cell death (44, 45). In the course of apoptosis the activation of
caspases is the central step driving cells into the execution phase of
programmed cell death. Among the substrates of the effector caspases, a
number of proteins involved in formation and/or regulation of
cell-matrix and cell-cell contacts were identified, including FAK
(22-24), PAK2 (46), fodrin (17), Here we show that during apoptosis, in addition to Because fragment 1 still contains the binding sites for The generation of the 29- and 84-kDa fragments could not be blocked by
caspase inhibitors but was efficiently blocked by TAPI, an inhibitor
originally used to study tumor necrosis factor- Consistent with our observations, it was recently reported that
during apoptosis of endothelial cells, the extracellular domain of the endothelium-specific VE-cadherin is shed from the cell surface by a TAPI-inhibited activity (39). However, for VE-cadherin no
caspase-mediated cleavage of the cytoplasmic tail was shown. This may
be due to the antibody used in this study, which was directed against
the VE-cadherin extracellular domain. VE-cadherin fragments A and B
described in this study might correspond to E-cadherin fragments 3 and
4 described in our study. Therefore, it is also likely that the
VE-cadherin cytoplasmic tail is a caspase target in endothelial cells.
In another recent report, E-cadherin and P-cadherin were shown to be
subjected to cleavage during early stages of apoptosis (38).
E-cadherin cleavage generated a 48-kDa cleavage product, and formation
of this fragment was reported to be blocked by the caspase inhibitor
Z-VAD-FMK. This cleavage event was assigned to residue
Asp479 in the extracellular domain of E-cadherin. However,
this is contradictory to the localization of caspases in the cytoplasm.
The 104-kDa P-cadherin fragment might correspond to fragment 4 described in our study. Furthermore, in this report shedding was not
analyzed for both cadherins. These discrepancies might be explained by the different cell lines used in both studies. Nevertheless, it would
be interesting to analyze the supernatant of these cells for cadherin fragments.
During recent years it became evident that membrane protein secretases
(often named sheddases) play an important regulatory role in the
activation/inactivation of transmembrane proteins as seen for tumor
necrosis factor- The following mechanisms can be considered. 1) the sheddase
cytoplasmic tail itself might represent a substrate for caspases, and
cleavage might induce a conformational change that subsequently allows
extracellular cleavage of E-cadherin. 2) the shedding protease might be
activated as a secondary target during programmed cell death. 3)
cleavage of Caspase-3 activity was shown not to be essential for the induction of
apoptosis in MCF-7 cells by staurosporine (17). With respect to the
present data, we assume that caspase-3 is instead required for
efficient E-cadherin cytoplasmic domain cleavage during apoptosis.
There is accumulating evidence now that cadherins contribute to cell
survival and that the cytoplasmic tail of cadherins has an
antiapoptotic function (40, 66, 67). For VE-cadherin it was shown that
cytoplasmic domain-deleted molecules are not able to respond to the
survival activity of VEGF-A that requires the formation of a
VE-cadherin, In summary our data demonstrate that during apoptosis of epithelial
cells cadherin-mediated adhesion is efficiently disrupted by two
distinct, presumably cooperative, mechanisms (Fig.
8). A caspase-3-mediated cleavage event
near the transmembrane domain releases the cytoplasmic tail and thus
results in a disconnection from the actin cytoskeleton. Cadherin
molecules that have lost their contact to the actin microfilament
system no longer show full adhesiveness (68, 69). The remaining
E-cadherin fragment including the extracellular and transmembrane
domain, however, still exhibits basic and weak adhesiveness (70, 71).
Thus, the shedding of the E-cadherin extracellular domain mediated by the metalloproteinase eliminates this residual adhesive activity and
results in a complete disruption of cadherin-mediated cell-cell adhesion. The regulated cleavage of E-cadherin is likely to represent an important process during the extrusion of apoptotic cells from epithelial cell layers, e.g. during release of enterocytes
at the tips of intestinal villi or during prostate and mammary gland involution.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin or plakoglobin is directly associated with the
cytoplasmic domain of cadherins, and
-catenin establishes the
connection to the actin microfilament system (34). Recently
-catenin
and
-catenin/plakoglobin were reported to be proteolytically
targeted by caspases during the apoptotic effector phase, whereas
-catenin was not affected (35-37). Recent reports showed that
cadherins are also targeted during apoptosis (38-40); however,
major questions about the molecular mechanisms involved in E-cadherin
cleavage remained unanswered.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
{
,L[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl} L-3-(2'naphthyl)-alanyl-L-alanine, 2-aminoethyl
amide) was kindly provided by Dr. Roy Black (Immunex, Seattle, WA).
Staurosporine, camptothecin, ALLN
(N-acetyl-Leu-Leu-NorLeu-CHO), and ionomycin were
obtained from Sigma, and CompleteTM-EDTA protease inhibitor
mix was from Roche Molecular Biochemicals.
, 1 mM NaV03,
0.2% (v/v) Triton X-100, and CompleteTM-EDTA protease
inhibitor mixture) for 10 min at 4 °C, and scraped off from the
culture dish. Floating cells were collected by gentle centrifugation at
310 × g for 10 min and pooled with adherent cells in
lysis buffer. After centrifugation (10 min, 4 °C, 20,800 × g), the supernatant was used for Western blotting. Total
protein concentration of the cell lysates was determined with the BCA protein assay system (Pierce). For inhibitor studies, cells were preincubated for 30 min with 50 µM Z-DEVD-FMK and/or 50 µM TAPI before addition of staurosporine.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells and E-cadherin-transfected Ltk
cells, it could be shown that this band reflects nonspecific cross-reactivity of the antibody with an unknown polypeptide. The
35-kDa band was detected in Ltk
and
E-cadherin-transfected Ltk
cells, whereas a
120-kDa cadherin band was only present in E-cadherin-transfected Ltk
cells (data not shown). The accumulation of fragments
1 and 2 peaked at about 6 h and subsequently declined over the
next 6 h, indicating that these fragments were further degraded
(Fig. 1, A and B). This might explain why the
intensity of the signals for the cleavage products is considerably
weaker than the signal for full-length cadherin at all time points
following induction of apoptosis. In the presence of ALLN, fragment 1 was stabilized, suggesting that further degradation of fragment 1 might
be a proteasome-mediated process (Fig. 1C). In contrast, the
amount of fragment 2 was not affected by ALLN, suggesting that this
fragment might be an intermediate product. Similar results were
obtained after induction of apoptosis with camptothecin, independent of
whether MDCK or the human mammary epithelial cell line H184A1 was used
(Fig. 1D), confirming that the described fragmentation of
E-cadherin is not cell-line specific.
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Fig. 1.
E-cadherin is proteolytically cleaved during
apoptosis of epithelial cells. A, cell lysates from
MDCK cells were analyzed by Western blotting with anti-E-cadherin C36
antibody at different times after induction of apoptosis with
staurosporine. Fragments 1 and 2 represent specific cleavage products.
The band marked with an asterisk represents an unidentified
and unspecific band. B, cleavage of full-length E-cadherin
and generation of fragments 1 and 2 as quantified by chemoluminescence
imaging on a FujiFilm LAS-1000 system. Data present mean values of
three independent experiments including the representative Western blot
shown in A. The signal intensity for full-length E-cadherin
at t = 0 h and for fragments 1 and 2 at t = 6 h was set to 100%. C, ALLN treatment inhibits
further degradation of fragment 1. D, identical apoptotic
cleavage patterns of E-cadherin were obtained in H184A1 and MDCK cells
independent of the inducing agent camptothecin (Camp) or
STS. In C and D cells were analyzed 6 h
after induction of apoptosis. MW, molecular weight.
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Fig. 2.
Formation of fragment 1 is inhibited by a
caspase inhibitor. In apoptotic MDCK and H184A1 cells generation
of E-cadherin fragment 1 was blocked in the presence of the caspase
inhibitor Z-DEVD-FMK (DEVD), whereas formation of fragment 2 was not affected. C, Me2SO control.
MW, molecular weight.
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Fig. 3.
Recombinant E-cadherin cytoplasmic domain is
efficiently cleaved by caspase-3. A, the E-cadherin
cytoplasmic tail expressed as a glutathione S-transferase
fusion protein (GST·ECT) was subjected to cleavage by recombinant
caspase-3, -6, and -7 and analyzed on a Coomassie-stained
SDS-polyacrylamide gel. Caspase-3 cleavage was blocked by Z-DEVD-FMK
(DEVD). B, buffer control; C3,
caspase-3; C6, caspase-6; C7, caspase-7;
MW, molecular weight. B, alignment of E-cadherin
fragment 1 generated by in vivo or in
vitro cleavage. C, schematic representation of the
highly conserved caspase-3 cleavage sites in human, mouse, and rat
E-cadherin cytoplasmic domains.
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Fig. 4.
Caspase-3-deficient MCF-7 cells do not form
fragment 1. Total cell lysates from mock-transfected MCF-7 cells
were analyzed for E-cadherin on Western blots with anti-E-cadherin C36
antibody and compared with caspase-3-retransfected cells. C,
control; Vec, vector-transfected; Casp3,
caspase-3-transfected MCF-7 cells; MW, molecular
weight.
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Fig. 5.
The E-cadherin extracellular domain is shed
from the cell surface during apoptosis. The E-cadherin
extracellular domain (fragment 3) released from H184A1 cells was
immunoprecipitated from the cell culture supernatant with anti-HECD-1
antibody at different time points after induction of apoptosis and
analyzed on Western blots with anti-HECD-1 antibody. The strong signal
at a molecular mass of 50 kDa represents the heavy chain of the
precipitating antibody. MW, molecular weight.
convertase (43), markedly inhibited formation
of E-cadherin fragment 3 in a concentration-dependent manner (Fig. 6A). Consistent
with this observation, generation of fragment 2 was also reduced,
whereas formation of fragment 1 was unaffected (Fig. 6B,
lane 3). In the presence of both TAPI and caspase inhibitor
Z-DEVD-FMK, formation of both fragments was significantly inhibited
(Fig. 6B, lane 4). Moreover, in cells treated
with both inhibitors the amount of full-length E-cadherin was nearly
comparable with nonapoptotic control cells (Fig. 6B, lane 4). In consequence, TAPI treatment should result in the
formation of a new cleavage product that contains the E-cadherin
transmembrane domain and thus remains attached to the membrane. Indeed,
with an antibody directed against the extracellular domain of
E-cadherin, we could identify a fragment of about 88 kDa (fragment 4)
in Western blots from lysates of TAPI-treated apoptotic H184A1 cells
(Fig. 6C, lane 1). The 88-kDa fragment was
undetectable in apoptotic cells not treated with TAPI (Fig.
6C, lane 3). In MDCK cells a TAPI-dependent reduction of fragment 3 (data not shown) and
generation of fragment 4 were even more prominent (Fig. 6D,
lane 3).
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Fig. 6.
TAPI inhibits shedding of the E-cadherin
extracellular domain (fragment 3) during STS-induced apoptosis.
A, increasing concentrations of TAPI were added to H184A1
cells prior to induction of apoptosis by STS. After 6 h of STS
treatment fragment 3 was immunoprecipitated from the cell culture
supernatants and analyzed on Western blots with anti-HECD-1 antibody.
Lane 1, control; lane 2, STS; lanes
3-6, STS + 6.25, 12.5, 25, or 50 µM TAPI,
respectively. B, lysates of cells treated with TAPI or TAPI + Z-DEVD-FMK were analyzed by Western blotting with anti-E-cadherin
antibody C36. Lane 1, Me2SO control; lane
2, STS; lane 3, STS + TAPI; lane 4, STS + TAPI + Z-DEVD-FMK. *, not identified. C, H184A1 cells were
treated as described in B, and cell lysates were analyzed on
Western blots with anti-HECD-1 antibody. In the presence of TAPI, a new
fragment (fragment 4) with a slightly higher molecular mass of 88 kDa
was generated (lane 1) compared with fragment 3 (lane
2) immunoprecipitated from the cell culture supernatant. In
control cell lysates, fragment 4 was not present (lane 3).
D, fragment 4 generated in TAPI-treated MDCK cells was
detected with anti-DECMA-1 antibody. Lanes 1 and
2, 0 and 6 h after induction of apoptosis; lane
3, 6 h after induction of apoptosis in the presence of TAPI.
MW, molecular weight.
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Fig. 7.
Confocal immunofluorescence microscopy of
apoptotic cells. A, MDCK cells were analyzed for
E-cadherin by immunofluorescence staining with anti-E-cadherin C36
antibody at different time points after induction of apoptosis with
STS. a, 0 h; b, 3 h; c,
6 h; d, 6 h in the presence of Z-DEVD-FMK. The
inset in c shows completely rounded cells
with diffuse E-cadherin staining in the cytoplasm (arrows).
B, H184A1 cells analyzed by indirect immunofluorescence
double-staining with anti-HECD-1 antibody for E-cadherin
(green) and Alexa FluorTM 594-phalloidin
(red) for F-actin. a, d, g,
and j, E-cadherin; b, e, h,
and k, phalloidin; c, f, i,
and l, merged images of E-cadherin and phalloidin stainings.
a-c, 0 h; d-f, 6 h STS;
g-i, 6 h STS + TAPI; j-l, 6 h STS + TAPI + Z-DEVD-FMK.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin (35-37, 47, 48), and
plakoglobin (36, 47).
-catenin and
plakoglobin, E-cadherin is efficiently cleaved in epithelial cells.
Three fragments with apparent molecular masses of 24 kDa (fragment 1),
29 kDa (fragment 2), and 84 kDa (fragment 3) were generated after
induction of apoptosis by staurosporine or camptothecin. These cleavage
products resulted from two distinct proteolytic activities. From
inhibitor studies and in vitro cleavage reactions we assume
that caspase-3 is predominantly responsible for the generation of the
24-kDa fragment. Caspase-7 was also able to generate fragment 1, however, with a much lower efficiency, whereas caspase-6, another
effector caspase, did not cleave recombinant GST·ECT. Mapping of the
caspase-3 cleavage site C-terminal to Asp752 showed that
fragment 1 represents nearly the entire cytoplasmic domain of
E-cadherin. These observations were confirmed by indirect immunofluorescence microscopy, revealing that the E-cadherin
intracellular domain was lost at the plasma membrane and accumulated in
the cytoplasm after induction of apoptosis. Phalloidin staining
revealed disintegration of the actin microfilament system within 3 h after induction of apoptosis. Disruption of actin fibers could not be prevented by the addition of Z-DEVD-FMK or TAPI alone. In the presence
of both inhibitors strong phalloidin staining at cell membranes
suggests that the cadherin-based adhesion system is retained and allows
formation of a subcortical actin filament network, whereas in the
presence of either one of the inhibitors, affected clustering of
cadherins or absence of the cytoplasmic domains appears to impair
linkage of cadherin molecules to the actin cytoskeleton.
-catenin and
p120, the question arises whether this fragment has a physiological
role during apoptosis, especially in respect to the signaling function
of
-catenin in association with lymphocyte enhancer factor-1/T cell
factor transcription factors (49, 50) where
-catenin provides
transcriptional activation domains (51-53). Previous studies have
shown that ectopic expression of the E-cadherin cytoplasmic domain can
block the lymphocyte enhancer factor-1/T cell
factor
-catenin-mediated transactivation process (54, 55),
indicating that generation of fragment 1 might be a mechanism to block
the signaling function of
-catenin. This assumption is in line with
the finding that
-catenin is also fragmented during apoptosis
(35-37) and that the resulting apoptotic
-catenin fragments exhibit
reduced transactivation potential (48). In this context it is
interesting to note that recently the adenomatous polyposis coli tumor
suppressor protein APC involved in the regulation of the
proteasome-mediated degradation of
-catenin was reported to be
another target of caspase-3 during apoptosis (56). All these
data indicate that the Wnt-signaling pathway is affected during
apoptosis, although the molecular consequences remain to be unraveled.
-converting enzyme
(43, 57), showing that these two fragments are generated by a
metalloproteinase. This observation and the finding that the soluble
84-kDa fragment 3 was released to the cell culture medium demonstrated
that the cleavage site generating the 84- and 29-kDa fragments is
localized in the extracellular domain of E-cadherin. Tumor necrosis
factor-
-converting enzyme is a member of the growing ADAM (a
disintegrin and metalloproteinase) family of metalloproteinases (58,
59). Because TAPI also blocks other metalloproteinases, it is
intriguing to figure out whether an ADAM family member or even tumor
necrosis factor-
-converting enzyme itself might shed the 84-kDa
fragment into the cell culture supernatant. Unfortunately, up to now we
have not been able to precisely determine the cleavage site in the
extracellular domain of E-cadherin. When we compared the mobility of
immunoprecipitated E-cadherin fragments released to the cell culture
supernatant after induction of apoptosis or by ionomycin-induced
Ca2+ influx, both fragments perfectly aligned on Western
blots (data not shown). This suggests that the metalloproteinase(s)
activated during apoptosis have the same substrate specificity reported for the metalloproteinase(s) induced by Ca2+ influx, which
was mapped C-terminal to amino acids Leu581 and
Ser582 in human E-cadherin (59).
converting enzyme, transforming growth
factor
secretase, Notch-activating enzyme Kuzbanian, or FasL
secretase (42). In this respect, soluble forms of the N-cadherin
extracellular domain were reported to be generated during embryonic
retinal histogenesis by proteolysis, representing a novel functional
form of N-cadherin involved in retinal development (60). Soluble
E-cadherin fragments were detected in the urine, in the blister fluid
of cutaneous diseases, and in the circulation of cancer patients
(61-63). Interestingly, expression of a stromelysin transgene in a
mammary epithelial cell line led to an epithelial-mesenchymal transition phenotype concomitant with a disappearance of E-cadherin and
shedding of minor amounts of E-cadherin to the cell culture supernatant, suggesting that either stromelysin itself or a
stromelysin-activated secondary product might be involved in this
process (64). At present the mechanisms generating these fragments are
unknown, and it is an open question how these sheddase activities are regulated.
-catenin and plakoglobin normally bound to the
cytoplasmic domain might be a prerequisite for the release of a
sterical block, subsequently allowing metalloproteinase access to its
substrate, or might induce a change in the E-cadherin conformation that
leads to the exposure of the metalloproteinase target site. Results
obtained in kinetic experiments with caspase-negative MCF-7 cells
indicate that their metalloproteinase activity is markedly reduced but
not completely absent, because we could detect minor amounts of soluble
fragment 3 in the cell culture supernatant (data not shown).
Retransfection of caspase-3 coincided with a higher metalloproteinase
activity in the transfected cells. This observation suggests that
caspase-3-mediated cleavage of
-catenin or plakoglobin might enhance
metalloproteinase activity on E-cadherin. A similar observation has
been reported for L-selectin shedding by a
metalloproteinase, where removal of calmodulin from the cytoplasmic tail of L-selectin allows effective L-selectin
shedding (65).
-catenin, phosphatidylinositol 3-kinase, VEGFR-2
complex resulting in an increase of the antiapoptotic mediator Bcl-2
(67). Consistent with these results, it was reported that disruption of
E-cadherin function by anti-E-cadherin antibodies leads to the
down-regulation of Bcl-2 (66). The rapid cleavage of cadherins and
-catenin observed during apoptosis thus might represent a mechanism
that ensures that the antiapoptotic function of cadherins is eliminated
after the death program is started.
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Fig. 8.
Model of the apoptotic E-cadherin
cleavage. During apoptosis full-length E-cadherin is cleaved by
caspase-3 and a metalloproteinase, resulting in three detectable
products, a cytoplasmic fragment (fragment 1), an extracellular
fragment (fragment 3), and the transmembrane domain fused to the
cytoplasmic domain (fragment 2) (A). The cytoplasmic
cleavage event is predominantly mediated by caspase-3 and was mapped
C-terminal to Asp752 close to the transmembrane domain.
Inhibition of caspase-3 activity with Z-DEVD-FMK results in the
formation of fragments 2 and 3 (B). The extracellular
cleavage is mediated by a metalloprotease activity that releases the
E-cadherin extracellular domain (fragment 3) to the cell culture
supernatant. This shedding is blocked by TAPI, resulting in the
formation of fragments 1 and 4 (C). PM,
plasma membrane.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Roy Black for the gift of the metalloproteinase inhibitor TAPI, Dr. Rolf Kemler for the DECMA-1 antibody, Dr. A. Porter for providing caspase-transfected MCF-7 cells, Anja Fromm for introduction to the FujiFilm LAS-1000 system, Ina Krukenberg and Barbara Kosel for technical assistance, and Dr. Claudia Fieger for discussion. We are especially grateful to Dr. Torsten Schöneberg for his kind help in confocal immunofluorescence microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 366/B3) (to K. B.), the Volkswagen Stiftung (to R. T. and O. H.), and the Sonnenfeld Stiftung.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.
§ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Institut für Klinische Chemie und Pathobiochemie, Universitätsklinikum Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.: 49-30-8445-2525; Fax: 49-30-8445-4152; E-mail: Huber@ukbf.fu-berlin.de.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M006102200
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ABBREVIATIONS |
---|
The abbreviations used are:
Z-DEVD-FMK, Z-Asp(methoxy)-Glu(methoxy)-Val-Asp(methoxy)fluoromethylketone;
MDCK, Madin-Darby canine kidney;
TAPI, N{
,L[2(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl}
L-3-(2'naphthyl)-alanyl-L-alanine, 2-aminoethyl
amide;
ALLN, N-acetyl-Leu-Leu-NorLeu-CHO;
PBS, phosphate-buffered saline;
GST, glutathione S-transferase;
ECT, E-cadherin cytoplasmic tail;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
STS, staurosporine;
VE, vascular endothelial.
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