From the ¶ Department of Pharmacology, College of Medicine,
University of Illinois, Chicago, Illinois 60612, the
Department of Vascular Biology, The Scripps Research
Institute, La Jolla, California 92037, and the
Laboratory for
Proteolytic Neuroscience, RIKEN Brain Science Institute,
Saitama 351-0198, Japan
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ABSTRACT |
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In this study, we report that the cytoplasmic
domain of the integrin 3 subunit is a target for
limited proteolysis during apoptosis of human umbilical vein
endothelial cells. Calpain inhibitors inhibited the cleavage of the
3 cytoplasmic domain, indicating that calpain is
required. Calpain-mediated proteolysis of fodrin was also detected,
indicating that calpain is activated during endothelial cell apoptosis.
A phosphatase inhibitor, sodium orthovanadate, inhibited endothelial
cell apoptosis and cleavage
3, suggesting that protein
dephosphorylation preceded integrin cleavage in the apoptosis signaling
pathway.
3 cleavage was observed in cells that were
viable, suggesting that it is an early event and not the consequence of
post-death proteolysis. The extent of
3 cleavage correlated with a loss in the capacity of cells to reattach to matrix
proteins. Loss of reattachment capacity during apoptosis was
significantly retarded by a calpain inhibitor. As the
3
cytoplasmic domain is required for integrin signaling and interaction
with the cytoskeleton, our results suggest that cleavage in the
3 cytoplasmic domain by calpain or a calpain-like
protease negatively regulates integrin-mediated adhesion,
signaling, and cytoskeleton association.
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INTRODUCTION |
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Programmed cell death or apoptosis is a cell suicide pathway involved in a variety of physiological and pathological events such as tissue morphogenesis, development, cancer, and neurodegenerative disorders (reviewed in Refs. 1 and 2). The mechanism of programmed cell death involves the activation of various intracellular proteases (reviewed in Ref. 3). The caspase family of proteases appears to play a key role in the signaling pathway (3, 4). In addition to caspases, the Ca2+-dependent neutral protease, calpain, was also found to be activated during apoptosis in T cells and to cleave fodrin during apoptosis (3, 5-8).
Although the role of calpain during apoptosis is not clear, its
substrates include several important intracellular signaling and
cytoskeletal proteins, particularly those associated with integrin-focal adhesion complexes, such as focal adhesion kinase (pp125fak) (9), pp60src (10, 11), fodrin (12), filamin
and talin (13). In addition, we have recently found that calpain
cleaves the cytoplasmic domain of the integrin 3
subunit, and that calpain cleavage sites flank two NXXY
motifs in the
3 cytoplasmic domain required for integrin function (14).
Integrins are a family of cell adhesion receptors, each of which is a
heterodimer complex of two transmembrane subunits, and
(reviewed in Ref. 15). Integrins bind to extracellular matrix adhesion
proteins such as vitronectin, fibronectin, collagen, and laminin.
Ligand binding to integrins transduces signals across the plasma
membrane, resulting in activation of protein kinases, influx of
calcium, elevation of intracellular pH, and hydrolysis of membrane
phospholipids and reorganization of the cytoskeleton (reviewed in Ref.
16). Integrin-mediated signals are required for the survival of
anchorage-dependent cells including endothelial and
epithelial cells (17-21). Integrins interact with cytoskeletal proteins at focal adhesion sites and are critical for cytoskeletal organization and morphological characteristics of
anchorage-dependent cells (22). Cytoskeleton reorganization
and morphological changes such as membrane blebbing occur during
apoptosis (3).
To determine if and how integrins are regulated during apoptosis, we
investigated the possible relationship between intracellular proteases
and integrins. We report here that the cytoplasmic domain of the
integrin 3 subunit is cleaved during endothelial cell apoptosis and that calpain is likely to be the protease involved. Since
the integrin
-subunit cytoplasmic domain plays an important role in
integrin-mediated cell adhesion and signaling (23-34), its proteolysis
may block integrin-mediated signals and disrupt cytoskeleton
organization during apoptosis.
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EXPERIMENTAL PROCEDURES |
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Materials--
Rabbit anti-peptide antibody,
anti-3C, is directed against the C-terminal 20 amino
acid residues of the
3 cytoplasmic domain. Rabbit
antibody anti-
3 was produced using purified
3 subunit as an immunogen and is directed against the
extracellular domain. These antibodies have been described previously
(14). Calpain inhibitors E64 and calpain inhibitor I were purchased
from Boehringer Mannheim. The cell-permeable calpain inhibitor E64d was
purchased from Sigma, and the antibody specific for calpain-cleaved
fodrin was described previously (12).
Cell Culture-- Human umbilical vein endothelial cells (HUVECs)1 were grown and maintained in endothelial cell growth medium (EGM, Clonetics) supplemented with an additional 8% fetal bovine serum (Life Technologies, Inc.). Cells were used between passages 2 and 10.
HUVECs were serum-starved by incubation in serum-free medium containing M199 (Life Technologies, Inc.), 0.1% bovine serum albumin (nuclease- and protease-free; Calbiochem), and insulin, selenium, and transferrin (GMS-G; Life Technologies, Inc.). For incubation in suspension culture, cells were detached with 0.01 M Na2HPO4, 0.15 M NaCl, 2.5 mM EDTA, pH 7.4 (PBS/EDTA) and resuspended in EGM containing 0.5% methylcellulose (Sigma). Cells were plated on tissue culture dishes coated with 2% agarose (Sigma) in M199 medium to prevent attachment (17). For reattachment experiments, cells incubated in suspension for 12 h were isolated by centrifugation, washed with PBS/EDTA for 5 min at 37 °C and then re-plated on tissue culture dishes in EGM. Cells were allowed to adhere and spread for 30 min. Adherent cells were lysed directly on the dish or were detached by PBS/EDTA and quantitated by cell counts prior to lysis. Unattached cells were collected by centrifugation. Both adherent and unattached cells were solubilized in buffer as described below and analyzed by immunoblotting.Immunoblotting--
Cells were isolated and washed twice with
PBS/EDTA; for serum-starved cells, adherent cells were detached by
incubation with PBS/EDTA and then recombined with the floating cells
prior to lysis. Cells were lysed by incubation in 1% Triton X-100, 100 mM Tris, pH 7.4, 150 mM NaCl, 10 mM
EDTA, 0.1 mM E64, and 1 mM phenylmethylsulfonyl
fluoride for 10 min on ice. Lysates were cleared by centrifugation at
14,000 × g for 5 min. Protein concentrations of cell
lysates were determined by BCA assay (Pierce). Equivalent amounts of
cell lysates were analyzed by SDS-polyacrylamide gel electrophoresis
followed by Western blotting either with antibody, anti-3C, against the C-terminal region of the
3 cytoplasmic domain or with the anti-
3
antibody. Reactions with antibodies were visualized using an enhanced
chemiluminescence kit (Amersham Pharmacia Biotech), and Kodak X-Omat AR
film. The immunoblots were scanned, and the uncalibrated optical
density of each band was quantitated using NIH Image.
Terminal Deoxynucleotidyltransferase-mediated Fluorescein-dUTP Nick End Labeling (TUNEL)-- Cells in suspension were isolated and incubated in PBS/EDTA for 5 min at 37 °C prior to fixation in 2% formaldehyde (methanol-free; Polysciences)/PBS for 15 min at room temperature. Following fixation, cells were washed twice with PBS and then extracted in 0.1% Triton X-100, 0.1% trisodium citrate for 2 min on ice. Cells were washed once with PBS and then incubated in 0.1% bovine serum albumin/PBS for 10 min at room temperature. Cells were labeled by incubation with terminal deoxynucleotidyltransferase and fluorescein-dUTP (Boehringer Mannheim) for 1 h at 37 °C. Labeled cells were washed with PBS and analyzed by flow cytometry (FACScan).
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RESULTS |
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Loss of the Cytoplasmic Domain of the Integrin 3
subunit during Suspension-induced Apoptosis--
To investigate the
possibility that proteolysis may regulate integrin function during
apoptosis, HUVECs were induced to undergo apoptosis by incubation in
suspension. We and others have shown previously that endothelial cells
undergo apoptosis when cultured in suspension which is characterized by
DNA fragmentation and apoptotic morphology (17, 18). In agreement with
these previous results, cells in suspension showed characteristic
apoptotic morphology (Fig. 1), and DNA
fragmentation (Fig. 2). Cells in
suspension began to undergo apoptosis after 6 h of incubation, as
demonstrated by labeling of fragmented DNA (Fig. 2A). The
percentage of apoptotic cells increased with increasing time in
suspension (Fig. 2A). Cells incubated in suspension for
various lengths of time were solubilized in the presence of protease
inhibitors. Equal amounts of lysates were then immunoblotted with
anti-
3 antibodies. Two different antibodies were used;
the antibody anti-
3 recognizes epitopes in the
extracellular domain, while the antibody anti-
3C recognizes epitopes located in the C-terminal 20 residues of the cytoplasmic domain (14). Expression of the extracellular domain epitopes is essentially constant over the time course
(anti-
3, Fig. 2, B and C).
However, expression of the cytoplasmic domain epitopes significantly
decreased with increasing lengths of time in suspension
(anti-
3C, Fig. 2, B and C). These
results indicate that the C-terminal domain is lost from a population
of
3 subunits as a result of suspension culture. The
time course of this loss of immunoreactivity with
anti-
3C paralleled the time course of DNA fragmentation
(Fig. 2, compare A and B), suggesting that this effect may be linked to the process of apoptosis.
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Cleavage of the Endothelial 3 Cytoplasmic Domain by
Calpain--
We previously showed that calpain cleaves the cytoplasmic
domain of the integrin
3 subunit during platelet
aggregation (14). Since there have been reports that calpain is
activated during apoptosis of T cells (5-7), we investigated whether
calpain may be responsible for the loss in endothelial
3
cytoplasmic domain immunoreactivity. HUVECs were incubated in
suspension for various lengths of time in the absence or presence of
the membrane-permeable calpain inhibitor E64d. 20 µM E64d
was used since a higher concentration of E64d (100 µM)
was toxic to cells. Cell lysates were then analyzed for reactivity with
anti-
3C by Western blot. As shown in Fig. 3, E64d significantly inhibited the loss
of the
3 cytoplasmic domain immunoreactivity during
apoptosis, indicating that calpain or a calpain-like protease is
responsible for the cleavage of the
3 cytoplasmic
domain. Similar to E64d, another membrane permeable calpain inhibitor,
calpain inhibitor I, also inhibited cleavage of the
3
cytoplasmic domain (not shown).
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Calpain Cleavage of Fodrin during Endothelial Cell Apoptosis-- To verify whether calpain is activated during apoptosis of endothelial cells, we examined whether fodrin, a known calpain substrate (8, 12), is cleaved during suspension culture-induced apoptosis. Lysates from suspension-cultured endothelial cells were immunoblotted with an antibody specifically recognizing a calpain cleavage site (GMMPR) at the N terminus of the 150-kDa calpain-generated fragment of fodrin (12). This antibody recognizes its epitope only when fodrin is cleaved by calpain at this specific site (12). No calpain-generated fragments of fodrin were detected in control endothelial cell lysates. After suspension culture, however, generation of the 150-kDa fodrin fragment was shown by reactivity with the calpain cleavage-specific antibody (Fig. 4). Generation of the 150-kDa fodrin fragment was maximal after 12 h; however, longer incubation in suspension resulted in a decrease in the fodrin fragment reactive with the antibody, suggesting further degradation of fodrin. In the presence of the calpain inhibitor, E64d (20 µM), there was a significant decrease in the generation of the 150-kDa fodrin fragment after 12 h of incubation and the peak of 150-kDa fragment generation was retarded to the 24-h time point. This result indicates that calpain is activated and cleaves fodrin during endothelial cell apoptosis, and, consistent with the effects of E64d on integrin cleavage (Fig. 3), suggests that intracellular calpain-like activity was only partially inhibited by E64d.
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Cleavage of 3 Cytoplasmic Domain Occurs after
Initiation of Apoptosis Signaling--
To determine whether the
cleavage of
3 cytoplasmic domain was a consequence of
apoptosis signaling, we tested the effect of a tyrosine phosphatase
inhibitor, Na3VO4. We have shown previously that Na3VO4 blocks apoptosis of
suspension-cultured HUVECs (17). Cells were incubated in suspension for
12 h in the absence or presence of Na3VO4.
In agreement with previous results, Na3VO4 prevented apoptosis of suspension cultured endothelial cells as indicated by TUNEL assay (Fig.
5A). When these
suspension-cultured cells were solubilized and immunoblotted with
anti-
3 antibodies, we found that the
3
cytoplasmic domain was protected from cleavage in the presence of
Na3VO4 (Fig. 5B). This effect was
dose-dependent (data not shown) and correlated with the
ability of Na3VO4 to block apoptosis (Fig.
5A). Thus, suspension culture per se is not responsible for
the cleavage of
3 cytoplasmic domain. Rather, cleavage
of
3 cytoplasmic domain occurs downstream of protein dephosphorylation in the apoptosis signaling pathway.
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Cleavage of Integrin 3 Cytoplasmic Domain during
Apoptosis Induced by Serum Withdrawal--
To investigate whether
cleavage of the integrin cytoplasmic domain is unique in
suspension-culture induced apoptosis, endothelial cells were induced to
undergo apoptosis by serum withdrawal. As shown in Fig.
6, cells in serum-free medium became
apoptotic, but at a much slower rate compared with that induced by
suspension culture. Fig. 6 (D and E) shows a
time-dependent reduction in the reaction of
anti-
3C with the integrin from endothelial cells cultured under serum-free conditions, indicating that cleavage of the
integrin cytoplasmic domain occurred during apoptosis induced by serum
withdrawal. Thus, cleavage of the integrin
3 cytoplasmic domain is not unique for suspension-induced apoptosis, but is likely to
be in the convergent signaling pathway induced by different apoptotic
stimuli.
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Cleavage of the 3 Cytoplasmic Domain Is an Early
Event, but Not a Consequence of Cell Death--
We examined whether
3 modification occurs as a step in the apoptosis
signaling pathway or is a result of post-death proteolysis. To address
this issue, we induced endothelial cell apoptosis by incubating cells
in suspension culture for 12 h, then separated the late-phase
apoptotic cells from cells in earlier phases of the apoptosis pathway.
Cells were separated by their ability to re-adhere to serum-coated
dishes (vitronectin is known to be the integrin ligand responsible for
cell adhesion to serum-coated plates; Ref. 35). We found that
nonadherent cells displayed the characteristic apoptotic morphology
(not shown, cf. Fig. 1) while reattached cells appeared
normal (Fig. 7). Lysates generated from
both re-attached cells and nonadherent cells were then analyzed for
immunoreactivity with the anti-
3C antibody. Equivalent
amounts of lysates from adherent endothelial cells were also examined as controls. In comparison with control cells, suspension-cultured cells showed significantly less (about 60%) reactivity with the anti-
3C antibody even though these cells reattached to
the matrix (Fig. 7). This indicates that cleavage of the
3 cytoplasmic domain was initiated before cells lost the
capacity to reattach. As dead cells do not adhere, this result suggests
that integrin cleavage is not the result of post-death proteolysis.
Furthermore, the reattached apoptotic cells appeared to have a normal
morphology (Fig. 7, A and B), which also suggests
that cleavage of
3 is an early event and may be
associated with the early phases of apoptosis signaling. Integrin
3 from non-adherent cells showed further reduction in
immunoreactivity with anti-
3C compared with the cells
reattached to serum-coated plates (Fig. 7, C and
D). Similar results were obtained when cells were replated
on collagen or fibronectin (Fig. 7E). Thus, it appears that
the majority of integrin
3 subunits are cleaved in late
phase apoptotic cells that have lost their capacity to reattach and
spread onto matrix proteins.
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Inhibition of the Loss of Cell Re-attachment Capacity by Calpain Inhibitor E64d-- The above study suggests a correlation between the loss of cell reattachment capacity and cleavage of the integrin cytoplasmic domain by calpain. To investigate whether calpain is indeed involved in regulation of cell adhesion during apoptosis, we examined the effects of E64d on cell readhesion. HUVECs were incubated in suspension for increasing lengths of time in the absence or presence of E64d, and then allowed to reattach to tissue culture plates. The percentage of reattached cells at each time point was then determined. Although the percentage of cell reattachment at each time point varied from experiment to experiment, we did observe a significant rescue of cell reattachment in the presence of E64d in each of the these paired experiments at the 6- and 12-h points (Fig. 8). The paired Student's t test of data from three experiments indicated a significant rescue of cell reattachment by E64d (p < 0.01). However, no significant inhibition by E64d was observed after 24 h of incubation in suspension (Fig. 8). Thus, our results suggest that calpain or a calpain-like protease is indeed involved in the loss of cell reattachment capacity during early phases of apoptosis. Incomplete inhibition of reattachment by E64d is consistent with a partial inhibition of calpain or calpain-like proteolytic activity by E64d (cf. Figs. 3 and 4).
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DISCUSSION |
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In this study, we have shown that the cytoplasmic domain of
integrin 3 subunit is cleaved during apoptosis in HUVECs
(Fig. 2). Cleavage was detected by loss of reactivity with an
anti-peptide antibody, anti-
3C, which recognizes the
3 C-terminal domain. We showed previously that
anti-
3C immunoreactivity was lost when the cytoplasmic
domain of
3 was cleaved by calpain (14). In apoptotic
endothelial cells, the loss of anti-
3C immunoreactivity was inhibited by two different calpain inhibitors, E64d and calpain inhibitor I (Fig. 3). As these calpain inhibitors do not inhibit caspases (4), caspases are unlikely to be responsible. Also, as these
calpain inhibitors do not inhibit DNA fragmentation of suspension-cultured endothelial cells (data not shown), it is unlikely
that their effect was due to inhibition of suspension culture-induced
apoptosis. Furthermore, cleavage of fodrin by calpain was also detected
in the suspension cultured endothelial cells, suggesting that calpain
is activated under these conditions (Fig. 4). Thus, the loss of
reactivity with anti-
3C is likely to result from
proteolytic cleavage of
3 by calpain. However, since we
showed only a partial inhibition of integrin and fodrin cleavage by
E64d, we cannot exclude the possibility that a different protease may
also be involved.
Cleavage of the integrin 3 cytoplasmic domain is linked
to the early phase of the convergent apoptosis signaling pathway. This
conclusion is supported by several lines of evidence. First, cleavage
of
3 was induced by two different apoptotic stimuli and
was accompanied by induction of the apoptotic phenotypes (DNA fragmentation and morphological changes), suggesting that cleavage may
be associated with the convergent apoptotic pathway. Second, cleavage
of integrin was blocked by the phosphatase inhibitor sodium
orthovanadate, an inhibitor that also blocks endothelial cell apoptosis
induced by either suspension culture (Fig. 5) (17) or serum starvation
(36), suggesting that the cleavage occurs downstream the activation of
protein-tyrosine phosphatase activity in the apoptosis signaling
pathway. Although it is not clear how protein phosphatases are
involved, it is known that survival of anchorage-dependent
cells requires signals initiated by adhesion receptors (e.g.
integrins) and growth factor receptors, both of which activate
protein-tyrosine kinases as early signaling mechanisms (for reviews,
see Refs. 16 and 37). Either abrogation of integrin binding to matrix
proteins or growth factor withdrawal may result in changes in the
balance between the protein-tyrosine kinase and phosphatase activities,
leading to dephosphorylation of intracellular proteins. Thus, the
tyrosine phosphatase inhibitor is likely to function in the early phase
of apoptosis pathway. Finally, the conclusion that calpain cleavage
occurs in the early phase of apoptosis is supported by our finding that
cleavage was initiated when cells were still able to readhere and
showed apparent normal morphology (Fig. 7).
Cleavage of the integrin 3 cytoplasmic domain may
negatively regulate integrin functions. Calpain cleavage sites in the
3 cytoplasmic domain have been identified in
vitro and in platelets; these sites flank the two NXXY
motifs (14). The NXXY motif is highly conserved among
different integrin
subunits, including
1 and
5 which are also found in endothelial cells. We have
evidence that the
1 cytoplasmic domain is also a calpain
substrate.2 The
NXXY motifs in
1 and
3 are
required for the localization of integrins to focal adhesion sites,
integrin-mediated tyrosine phosphorylation of signaling molecules such
as focal adhesion kinase and also required for regulating ligand
binding affinity (26, 28, 38). Truncation of the
3
cytoplasmic domain that removes C-terminal sequences containing the
NXXY motif abolishes formation of the integrin-focal
adhesion complex, abrogates integrin-mediated cell spreading, and
inhibits integrin-mediated tyrosine phosphorylation (27, 29, 39, 40).
Thus, it is possible that the cleavage of integrin
subunits, in
concert with the cleavage of other focal adhesion proteins, leads to
disruption of integrin-mediated adhesion, signaling, and cytoskeleton
organization. In support of this viewpoint, we have observed a
correlation between the extent of integrin cleavage and the ability of
apoptotic cells to reattach (Fig. 7). In addition, we found that
calpain inhibitor E64d significantly retarded the loss of cell
reattachment (Fig. 8). Integrin-mediated signaling activates
intracellular protein-tyrosine kinase and mitogen-activated protein
kinase pathways, which are required for proliferation and survival of
anchorage-dependent cells (41-45). Abrogation of integrin
signaling induces apoptosis in endothelial cells and other
anchorage-dependent cells (17-19, 21). Thus, it is also possible that
intracellular disruption of integrin survival signals by calpain
cleavage, if occurring to adherent anchorage-dependent
cells, serves as a feedback signaling mechanism that induces or
accelerates apoptosis.
Interestingly, we found that the suspension cultured cells will
re-adhere and spread when ~40% of 3 is cleaved, but
lose the capacity to re-adhere when ~70% of the integrin is cleaved (Fig. 7). This is very similar to results from studies of patients with
Glanzmann's thrombasthenia, a genetic deficiency in the integrin
IIb
3. Platelets obtained from
heterozygotes (50%
IIb
3 expression) showed no significant deficiency in adhesion while platelets expressing less than 30% of functional integrin were abnormal (46). Thus, it
appears that cell reattachment may require only a fraction of the total
integrins expressed. However, the finding that cells with ~40% of
integrin cleaved are still able to reattach does not suggest that the
cleavage of a small percentage of integrin will not affect integrin
function. Rather, it is possible that cleavage of a small population of
integrins, if localized to existing integrin-focal adhesion sites of
adherent cells, would abolish integrin signaling and cytoskeleton
association in those specific focal adhesion sites, and thus promote
cell rounding and detachment. Cleavage of integrin in existing focal
adhesion sites of adherent cells is likely since calpain is colocalized
in focal adhesion sites (47), and cleaves several focal adhesion
proteins (9, 10, 13).
One approach to further determine the roles of calpain cleavage of integrins during apoptosis is to inhibit calpain cleavage of integrin. Experiments using calpain inhibitors, however, are complicated by the fact that calpain cleaves several intracellular proteins and plays multiple roles in cell survival and growth. Calpain inhibitors inhibit the cleavage and clearance of p53, which is pro-apoptotic, and thus induce apoptosis and growth arrest (48, 49). We also have found that high levels of E64d and calpain inhibitor I are toxic. In addition, calpain inhibitors may have nonspecific effects such as inhibition of proteasome activity by calpain inhibitor I (50). Multiple roles of calpain in cells explain previous contradictory results that calpain inhibitors both inhibit and induce apoptosis in different experiments (5-7, 51-54). Thus, further elucidation of the roles of cleavage of integrin in the apoptosis pathway awaits the development of new tools to specifically inhibit integrin cleavage without affecting the other cellular roles of calpain.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mark H. Ginsberg and Dr. Martin Schwartz for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by Grant HL52547 from the National Institutes of Health and by a grant from the Campus Research Board of the University of Illinois.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.
§ Research Fellow of the American Heart Association California Affiliate.
** Established Investigator of the American Heart Association. To whom correspondence should be addresses: Dept. of Pharmacology (M/C868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-355-0237; Fax: 312-996-1225; E-mail: xdu{at}uic.edu.
1 The abbreviations used are: HUVEC, human umbilical vein endothelial cell; TUNEL, terminal deoxynucleotidyltransferase-mediated fluorescein-dUTP nick end labeling; EGM, endothelial cell growth medium; PBS, phosphate-buffered saline.
2 X. Du, unpublished data.
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REFERENCES |
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