From the Division of Toxicology, Leiden Amsterdam
Center for Drug Research, 2300 RA Leiden University, Leiden, The
Netherlands and the ¶ Department of Pathology, College of
Medicine, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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The relationship between focal adhesion protein
(FAK) activity and loss of cell-matrix contact during apoptosis is not
entirely clear nor has the role of FAK in chemically induced apoptosis been studied. We investigated the status of FAK phosphorylation and
cleavage in renal epithelial cells during apoptosis caused by the
nephrotoxicant dichlorovinylcysteine (DCVC). DCVC treatment caused a
loss of cell-matrix contact which was preceded by a dissociation of FAK
from the focal adhesions and tyrosine dephosphorylation of FAK.
Paxillin was also dephosphorylated at tyrosine. DCVC treatment activated caspase-3 which was associated with cleavage of FAK. However,
FAK cleavage occurred after cells had already lost focal adhesions
indicating that cleavage of FAK by caspases is not responsible for loss
of FAK from focal adhesions. Accordingly, although inhibition of
caspase activity with zVAD-fmk blocked activation of caspase-3, FAK
cleavage, and apoptosis, it neither affected dephosphorylation nor
translocation of FAK or paxillin. However, zVAD-fmk completely blocked
the cell detachment caused by DCVC treatment. Orthovanadate prevented
DCVC-induced tyrosine dephosphorylation of both FAK and paxillin;
however, it did not inhibit DCVC-induced apoptosis and actually
potentiated focal adhesion disorganization and cell detachment. Thus,
FAK dephosphorylation and loss of focal adhesions are not due to
caspase activation; however, caspases are required for FAK proteolysis
and cell detachment.
Apoptosis or programmed cell death is critical for normal
development and tissue homeostasis (1). However, uncontrolled apoptosis, as may occur after treatment with cytostatic chemicals, is a
pathophysiological process and is associated with the occurrence of
various human diseases (2, 3). Apoptosis is characterized by
fragmentation of the nucleus, activation of nucleases, and importantly,
activation of the caspase family of aspartate-directed proteases. The
latter cleave a diverse set of cellular proteins, such as PARP,
DNA-dependent protein kinase, protein kinase C- Maintenance of cell-matrix contact is an important cell survival factor
(16-21) and loss of cell matrix and cell-cell contact, or rounding up,
is a hallmark of apoptosis. Enforced loss of cell-matrix interactions
of endothelial and epithelial cells, is sufficient to initiate a form
of apoptosis that has been termed "anoikis" (18, 19). The fact that
apoptotic cells detach from their substratum raises the possibility
that the signaling events leading to anoikis are either the same as, or
may overlap with signals that lead to other forms of apoptosis, for
example, induced by chemicals or FasL/tumor necrosis factor.
Cell-matrix interactions occur at the closest contact between the cell
and the substratum: the focal adhesions. This is a complex network of
(cytoskeletal) proteins that links the filamentous actin (F-actin)
cytoskeletal network through cell adhesion molecules, so called
integrins, to the extracellular matrix (16, 17, 22, 23). Integrin
engagement by the extracellular matrix results in activation of focal
adhesion kinase (FAK). FAK is a 125-kDa protein tyrosine kinase that is
critical in transmission of signals from the focal adhesion to the
cytoplasm after cell attachment (24, 25). After activation, FAK
interacts with a variety of focal adhesion (signaling) macromolecules,
including the adaptor proteins paxillin and Grb-2, the cytoskeletal
proteins tensin and talin, and signal transduction molecules such as
Src, Fyn, and phosphoinositide 3-kinase (22, 23, 26-33). These
interactions link integrin-mediated adhesion to downstream signaling
cascades. For example, the SH2 domain of phosphoinositide 3-kinase
binds FAK at the tyrosine autophosphorylation site (Tyr397)
(34). Cell adhesion results in the activation of phosphoinositide 3-kinase and the downstream signaling molecule c-Akt/PKB (35, 36).
Adhesion also leads to activation of the mitogen-activated protein
kinase pathway, p70 ribosomal S6 kinase and protein kinase C (37-39).
Activation of Akt/PKB, mitogen-activated protein kinase, or protein
kinase C serve as anti-apoptotic stimuli (40-45). Although FAK is one
of the few proteins that is phosphorylated on tyrosine residues upon
cell adhesion, its role in the activation of all of the above signaling
pathways is still under investigation. Nonetheless, it seems clear that
FAK is important for focal adhesion signaling and cell attachment and migration.
Although the downstream effector molecules responsible for FAK
signaling are not completely known, there is increasing evidence that
FAK is involved in anoikis and, perhaps, other forms of apoptosis (12-15, 46-50). For example, microinjection of peptides that compete for FAK-integrin association, and also antibodies directed against FAK
itself can induce apoptosis in fibroblasts (48). Treatment of tumor
cells with FAK antisense oligonucleotides also leads to apoptosis (47).
Moreover, overexpression of constitutively active FAK prevents anoikis
(46, 51). Furthermore, during apoptosis FAK is cleaved both in adherent
and non-adherent cell lines (12-15), a process that depends on caspase
activation since zVAD-fmk blocks FAK cleavage. Recombinant FAK is also
cleaved by recombinant caspase-3 and -6 into fragments of approximately 44, 77, and 85 kDa corresponding to the fragments seen during FAK
cleavage in apoptotic cells (13, 15). Despite the fact that FAK
cleavage and/or inactivation provides an attractive explanation for the
loss of cell-matrix interactions that occur during apoptosis, there is
little or no evidence supporting this assumption, nor has the role of
FAK signaling been addressed in chemically induced models of apoptotic
cell death. Furthermore, the majority of studies have been performed in
immortalized or transformed cells; little is known about the
involvement of FAK signaling and cell detachment in vivo or
in primary cultures of normal epithelial cells. Therefore, we have
investigated the role of FAK in chemically induced apoptosis in primary
cultures of renal proximal tubular epithelial cells.
The renal proximal tubule epithelial cells (RPTE) are an important
target for a variety of nephrotoxic chemicals as well as ischemia/reperfusion injury (52, 53). Death of renal epithelial cells
is associated with detachment of viable RPTE from the extracellular matrix, both in vitro and in vivo, an effect that
is related to redistribution of integrins and loss of focal adhesion
organization (54-59). Conditions that prevent attachment of the renal
epithelial cell line Madin-Darby canine kidney also cause apoptosis,
suggesting that there is a relationship between cell detachment and
cell death (19, 46). Therefore, we investigated the role of FAK phosphorylation and its cleavage during loss of focal adhesion integrity and apoptosis of primary cultures of rat RPTE using the well
characterized nephrotoxicant
S-(1,2-dichlorovinyl)-L-cysteine (DCVC) (57, 58,
60-65). DCVC is metabolized by a To study the relationship between focal adhesion assembly, FAK
phosphorylation, and apoptosis and to investigate whether disturbances in FAK signaling may contribute to chemically induced apoptosis in
RPTE, we determined FAK localization and phosphorylation. These effects
were correlated with activation of caspases and the occurrence of
apoptosis after DCVC treatment. Apoptosis of RPTE was preceded by an
early tyrosine dephosphorylation of both FAK and the
FAK-associated protein paxillin. Dephosphorylation was also associated
with an early redistribution of FAK and paxillin from the
focal adhesion. The cleavage of FAK was a late event and
followed the activation of caspases. The general caspase inhibitor
zVAD-fmk blocked the DCVC-induced increase in caspase activity, FAK
cleavage, and apoptosis; however, FAK dephosphorylation and
translocation still occurred. The data suggest that FAK
dephosphorylation and dissolution of the focal adhesions precede
cleavage of FAK by caspases during chemically induced apoptosis of
renal epithelial cells.
Materials--
Fetal bovine serum was from Life Technologies
(Grand Island, NY). Dulbecco's modified Eagle's medium/Ham's F-12,
bovine serum albumin fraction V, cholera toxin, insulin, antibiotic
were from Sigma. Epidermal growth factor was from Upstate Biotechnology Inc. (UBI, Lake Placid, NY).
N,N'-Diphenyl-p-phenylenediamine (DPPD) was from
Kodak (Rochester, NY). Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) was purchased from Bachem (Bubendorf, Switzerland). Rhodamine-phalloidin was from Molecular Probes (Eugene, CA). DCVC was
synthesized as described (61).
Isolation, Culturing, and Treatment of Rat Renal Proximal Tubular
Epithelial Cells (RPTE)--
RPTE were isolated by collagenase
perfusion and separated by density centrifugation using Nycodenz as
described (63, 66). Cells were cultured on rat tail collagen
(Collaborative Research, Bedford, MA) coated dishes in Dulbecco's
modified Eagle's medium/Ham's F-12 containing 1% (v/v) fetal bovine
serum, 0.5 mg/ml bovine serum albumin, 10 µg/ml insulin, 10 ng/ml
epidermal growth factor, 10 ng/ml cholera toxin, and antibiotics as
described (57, 67). Cells were maintained at 37 °C in a humidified
atmosphere of 95% air and 5% carbon dioxide and fed every other day.
Cells were used after they had reached confluency 6 to 9 days after plating.
Confluent monolayers of RPTE in 24-well dishes containing coated glass
coverslips, 6-well or 10-cm dishes, were washed with Earle's balanced
salt solution (EBSS) twice. Thereafter cells were treated with DCVC in
EBSS in a final volume of 1, 2, or 10 ml, respectively, for 4 h.
To study events that are related to apoptosis, cells were treated with
DCVC in the presence of DPPD (20 µM; 10 mM
stock in dimethyl sulfoxide) which blocks necrotic cell death but
allows the onset of apoptosis (58, 65). The inhibitors zVAD-fmk (100 µM; 100 mM stock in dimethyl sulfoxide) or
Na3VO4 (5, 10, 25, or 50 µM; 100 mM stock in water) were added simultaneously with DCVC.
Following treatment with DCVC for 4 h, cells were allowed to
recover in complete medium containing DPPD (20 µM) with
or without the above inhibitors.
Cell Death and Caspase Activation Assays--
Cell death was
measured by the release of lactate dehydrogenase (LDH) in the culture
medium as described (62). Percentage of cell death was calculated from
the amount of LDH release caused by treatment with toxicants relative
to the amount released by 0.1% (w/v) Triton X-100, i.e.
100% release. To determine the percentage of cell detachment, floating
cells (supernatant) and adherent cells (obtained after trypsinization;
see below) were collected separately. Cells were counted using a
hemocytometer and the percentage of cell detachment was calculated as
floating cells/total cells times 100%. DNA laddering was determined by
agarose gel electrophoresis. Cells were harvested by scraping the
adherent cells which were then combined with floating cells present in
the culture medium. After centrifugation, the cell pellet was washed
once with ice-cold PBS by centrifugation, lysed with lysis buffer (10 mM Tris, 1 mM EDTA, and 0.2% (w/v) Triton
X-100, pH 7.4), and incubated on ice for 20 min. Cell debris was
removed by centrifugation; the supernatant was treated with RNase (60 µg/ml) for 1 h at 37 °C and then with proteinase K (120 µg/ml) for 1 h at 50 °C. The DNA was precipitated with 0.5 M NaCl and an equal volume of isopropyl alcohol and
separated by electrophoresis on 1% agarose gels. Nuclear fragmentation
was determined in cells cultured on collagen-coated glass coverslips.
After fixation with 3.7% (w/v) formaldehyde, cells were stained with 1 µg/ml Hoechst 33258 in PBS. After washing with PBS, coverslips were
mounted and viewed using a Nikon epifluorescence microscope. In some
experiments nuclear staining was combined with immunofluorescence
staining (see below).
Apoptosis was also determined by cell cycle analysis. Both floating and
adherent cells that were trypsinized were pooled and fixed in 90%
ethanol (
Caspase activity was assayed as follows. Briefly, attached and detached
cells were harvested and collected by centrifugation as above. The cell
pellet was taken up in lysis buffer (10 mM HEPES, 40 mM Immunoprecipitation and Western Blotting--
For
immunoprecipitation studies cells were plated in 10-cm Petri dishes
coated with collagen. After treatment with toxicant cells were
harvested in a Tris-sucrose-EGTA (TSE) buffer containing 10 mM Tris/HCl (pH 7.4), 150 mM sucrose, 1 mM EGTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotonin, 1 mM Na3VO4, and 50 mM sodium fluoride. After sonication, 500 µg of cell
protein was incubated overnight with 1 volume of RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1.0% (w/v) sodium deoxycholate, 1% (w/v) Triton
X-100, and 0.1% (w/v) sodium dodecyl sulfate), with protease and
phosphatase inhibitors as described for TSE buffer making a total
volume of 1 ml. Thereafter samples were cleared by centrifugation and
the supernatant was incubated with 4 µg of FAK antibody (Transduction
Laboratories, Lexington, KY) for 4 h followed by another 2-h
incubation with protein G-coated Sepharose 4B Fast Flow beads (Sigma).
Beads were pelleted by brief centrifugation and washed three times with
low-salt buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, and 0.1% (w/v) Nonidet P-40 (pH 7.4), including
protease and phosphatase inhibitors). Proteins were eluted from the
beads by boiling samples for 5 min in 40 µl of SDS-PAGE sample
preparation buffer containing Immunofluorescence and Imaging
Techniques--
Immunofluorescence studies were carried out using
cells cultured on collagen-coated glass coverslips in 24-well dishes.
After treatment with toxicants cells were fixated with 3.7%
formaldehyde for 10 min followed by 3 washes with PBS. After cell lysis
and blocking with PBS, 0.2% (w/v) Triton X-100, 0.5% (w/v) BSA (pH 7.4) (PTB) cells were stained for FAK, PAX, or PY using 10, 5, and 10 µg/ml monoclonal antibody diluted in PTB as described previously. During the staining period of the cells with secondary fluorescein isothiocyanate-labeled anti-mouse antibody the F-actin cytoskeletal network was labeled with rhodamine-phalloidin (Molecular Probes, Eugene, OR; 0.3 unit/ml). After a final wash of the cells with PBS the
DNA was stained with Hoechst 33258 as above. Cells were mounted on
glass slides using Aqua-Poly/Mount (Polysciences Inc., Warrington, PA).
Cells were viewed using a Bio-Rad 600 MRC confocal laser scanning
microscope. Representative images for the different treatment
conditions were printed using a Mitsubishi color video printer.
Statistical Analysis--
Student's t test was used
to determine if there was a significant difference between two means
(p < 0.5). When multiple means were compared,
significance was determined by a one-way analysis of variance (ANOVA;
p < 0.5). For ANOVA analysis, letter designations are
used to indicate significant differences. Means with a common letter
designation are not different; those with a different letter designation are significantly different from all other means with different letter designations. For example, a mean designated as A is
significantly different from a mean designated B, but neither is
different from a mean designated AB.
DCVC Causes Activation of Caspases and Cleavage of FAK in
RPTE--
Apoptosis is associated with the activation of caspases.
Treatment of RPTE with DCVC resulted in the cleavage of pro-caspase-3 (32 kDa) into the active caspase-3 fragment (17 kDa); this was associated with increased caspase-3 activity as determined using acetyl-DEVD-AMC as a substrate (Fig. 1).
Activation of caspase-3 preceded DNA fragmentation and cell death as
determined by formation of 180-200-base pair nucleosomal "ladders"
and release of LDH into the medium (Fig.
2). A general caspase inhibitor,
zVAD-fmk, completely blocked the DCVC-induced cleavage of caspase-3 and activation of DEVDase activity (Fig. 1), DNA fragmentation, and the
increase in apoptotic cells seen as a sub-G1/G0
population by flow cytometry (Fig. 2). Although zVAD-fmk clearly
blocked all the events that are characteristic for apoptosis, it did
not block the overall cell killing as determined by the release of LDH
from the cells (Fig. 2).
Since DCVC-induced apoptosis of RPTE is associated with impaired cell
adhesion (57, 58, 65), and because FAK is cleaved by caspases in
cell-free systems as well as in intact cells in vitro
(12-15), we determined whether DCVC-induced caspase activation is
associated with FAK cleavage in RPTE. A major increase in the cleavage
of FAK into two fragments with approximate sizes of 70 and 77 kDa was
observed 6 h after DCVC treatment, but not before (Fig.
3). Cleavage of FAK correlated with
activation of caspase-3 by DCVC (Fig. 1).
DCVC-induced Translocalization of FAK Precedes Caspase
Activation--
We next investigated the fate of FAK during
apoptosis at the cellular and biochemical level. First we
determined whether cleavage of FAK was associated with altered
localization of FAK by (immuno)staining of RPTE treated with DCVC for
FAK and F-actin. Although FAK cleavage and caspase activation is first
observed 6 h after DCVC treatment, FAK redistribution from the
focal adhesions to the cytoplasm was observed after 2 h and was
maximal 4 h after DCVC treatment (Fig. 4). Loss of FAK from focal adhesions was
accompanied by an increased FAK staining in the cytosolic compartment
and to some extent in the nucleus. Altered FAK localization was
temporally associated with disruption and collapse of the F-actin
cytoskeletal network, but FAK did not co-localize with F-actin (Fig.
4). Thus, altered FAK localization clearly preceded cleavage of FAK by
caspases, suggesting that an altered focal adhesion ultrastructure is
most likely not caused by caspase activation.
DCVC-induced Translocation of FAK Is Associated with Loss of
Tyrosine Phosphorylation--
The results indicated that DCVC-induced
loss of FAK from focal adhesions was independent of caspase activation;
therefore, other signals must be involved. Integrin engagement by
extracellular matrix results in autophosphorylation of FAK and
subsequent formation of focal adhesions (22-25). Since DCVC disrupts
focal adhesions in RPTE (57, 58), we determined whether DCVC-induced
loss of FAK from focal adhesions was associated with loss of tyrosine phosphorylation of FAK. Indeed, DCVC caused a
dose-dependent dephosphorylation of FAK at tyrosine as
demonstrated by immunoprecipitation of FAK followed by Western blotting
with anti-phosphotyrosine and anti-FAK antibody. FAK was almost
completely dephosphorylated after a 4-h treatment with 0.25 mM DCVC (Fig. 5), a time
point at which cleavage of FAK had not yet occurred (Fig. 3). The
increased intensity of FAK bands in the DCVC-treated cells (see Fig. 5)
may be explained by a translocation of FAK from the cytoskeleton
fraction to the cytosol, thus resulting in a higher recovery of FAK
from these samples for immunoprecipitation.
DCVC-induced Loss of FAK Phosphorylation Is Associated with Loss of
Paxillin Phosphorylation--
The data suggested that focal adhesion
disturbances and FAK dephosphorylation occur upstream of caspase
activation. Many focal adhesion-associated proteins are also
phosphorylated at tyrosine residues, and some are direct substrates for
FAK. To investigate whether disturbances in tyrosine phosphorylation
contributed to DCVC-induced impaired cell adhesion and cell death, we
first evaluated the alterations in total cell protein-phosphotyrosine
content. DCVC caused a dose dependent decrease in
protein-phosphotyrosine from proteins of ~60-70 kDa and proteins
with a molecular mass around 120-130 kDa (Fig.
6A). Paxillin is a 68-kDa
cytoskeletal adaptor protein that co-localizes with focal adhesions in
a variety of cell types and is heavily phosphorylated on tyrosine
residues. Immunoprecipitation of phosphotyrosine-containing proteins
with anti-phosphotyrosine antibody followed by electrophoresis and Western blotting for paxillin demonstrated that the broad protein band
of ~60-70 kDa contained the cytoskeletal adaptor protein paxillin
(Fig. 6B). Thus DCVC treatment causes a loss of protein phosphotyrosine from paxillin as well.
Next, we determined the effect of dephosphorylation on the cellular
localization of paxillin. DCVC caused a rapid loss of paxillin from
focal adhesions and an increase in paxillin staining in the cytosol
(Fig. 7). Intense staining of paxillin
was also observed in dots that co-localized with the collapsed F-actin cytoskeleton, suggesting that even though paxillin was lost from the
focal adhesion, it remained associated with F-actin (Fig. 7). To
further evaluate this translocation of paxillin to the F-actin network,
cells were permeabilized just prior to fixation to determine
cytoskeletal association. Within 2 h after DCVC treatment, co-localization of cytoskeleton-associated paxillin with the F-actin network was observed; after 4 h paxillin completely co-localized with large F-actin aggregates present in the cytosol (not shown). Paxillin did not co-localize with the cortical F-actin cytoskeletal network. A similar association with the F-actin network was observed with talin, another focal adhesion-associated cytoskeletal protein (data not shown). Although both paxillin and FAK co-localized in focal
adhesions in control cells, FAK and paxillin no longer co-localized
after DCVC treatment (compare Fig. 4 with Fig. 7).
Inhibition of Protein Tyrosine Phosphatases Increases FAK and
Paxillin Tyrosine Phosphorylation without Affecting Caspase Activation
and Apoptosis--
The results indicated that nephrotoxicants cause
dephosphorylation of both paxillin and FAK prior to loss of cell-matrix
contact and cell death. To investigate the role of phosphatases in
dephosphorylation of FAK and paxillin, cells were incubated with an
inhibitor of protein tyrosine phosphatases, orthovanadate. As expected,
treatment of RPTE with orthovanadate alone caused an overall increase
of protein tyrosine phosphorylation. In addition, immunoprecipitation experiments indicated that both FAK and paxillin had approximately 4-fold more phosphotyrosine after orthovanadate compared with control
cells (Fig. 8). Although DCVC alone
caused a complete loss of phosphotyrosine on paxillin and FAK, FAK
tyrosine phosphorylation after exposure to DCVC/orthovanadate remained
approximately the same as orthovanadate-treated control cells. Thus,
orthovanadate can block DCVC-induced dephosphorylation of both paxillin
and FAK suggesting that an imbalance in protein tyrosine kinase and phosphatase activity is responsible.
To determine if preventing DCVC-induced dephosphorylation of paxillin
and FAK was associated with loss of focal adhesion integrity or cell
adhesion, we determined the effect of orthovanadate on DCVC-induced
translocation of FAK and paxillin as well as cell attachment. Although
orthovanadate itself had only a minor effect on FAK localization, it
actually potentiated the DCVC-induced redistribution of FAK (Fig.
9). Similar results were obtained for the
redistribution of paxillin (data not shown). Because DCVC-induced translocation of FAK is associated with impaired cell adhesion, we
checked whether the potentiation of DCVC-induced FAK translocation by
orthovanadate was associated with enhancement of RPTE detachment. Indeed, orthovanadate potentiated the cell detachment caused by DCVC
(Table I). Thus, not only the
phosphorylation itself but rather the cellular localization of FAK, in
combination with tyrosine phosphorylation, seems important in the
regulation of cell adhesion.
Although maintaining the phosphorylation of both FAK and paxillin with
orthovanadate did not protect against cell detachment, the possibility
existed that orthovanadate protected against cell death. To investigate
this we determined the DCVC-induced caspase activation, a measure of
the activation of the apoptotic machinery, as well as cell death.
Orthovanadate did not inhibit the DCVC-induced activation of caspases
(Fig. 10). Moreover, DCVC-induced
release of LDH and formation of a population of cells with
sub-G0/G1 DNA content, i.e.
apoptotic cells, was unaffected (Fig. 10). Finally, although
orthovanadate increased tyrosine phosphorylation of FAK (see above),
this did not have an effect on the amount of FAK that was cleaved by
caspases (Fig. 10).
Caspases Mediate Cleavage of FAK and Cell Detachment Caused by
DCVC--
Having characterized the temporal relationship between FAK
and paxillin localization and dephosphorylation after DCVC treatment, we determined whether DCVC-induced activation of caspases is involved in the dephosphorylation of FAK and paxillin by determining the phosphotyrosine content of both proteins after DCVC treatment in the
presence or absence of zVAD-fmk. As might be predicted from the
disparity in the time dependence for FAK cleavage versus dephosphorylation, zVAD-fmk did not inhibit the DCVC-induced tyrosine dephosphorylation of cellular proteins (Fig.
11) including FAK and paxillin.
Next we determined whether the cleavage of FAK was dependent on caspase
activity. Therefore cells were treated with DCVC in the presence of
z-VAD-fmk. Inhibition of DCVC-induced caspase activation by z-VAD-fmk
(see Fig. 1) was associated with a complete inhibition of FAK cleavage
(Fig. 11). This indicates that the FAK cleavage is due to caspase activation.
A clear increase of floating RPTE was only observed after 8 h
treatment with DCVC. This was also a time point when caspase activity
and FAK cleavage was clearly increased. Therefore, the possibility
existed that cell detachment was dependent on caspase activity.
Inhibition of caspases with zVAD-fmk, which blocked the cleavage of
FAK, did not have an effect on the DCVC-induced rounding up of cells.
However, z-VAD-fmk clearly inhibited the cell detachment caused by DCVC
(Fig. 11). To determine whether this was related to a protection
against a disturbance of the focal adhesions, we analyzed the focal
adhesion organization after DCVC treatment in the presence of
z-VAD-fmk. Z-VAD-fmk did not block any of the DCVC-induced alterations
of the focal adhesions and F-actin network after 4 h treatment
(not shown). Thus, caspase-mediated proteolysis of cellular
(cytoskeletal) proteins, including FAK, does not mediate loss of the
focal adhesion and F-actin network organization during apoptosis.
These studies on the temporal relationship between FAK
dephosphorylation, translocation, and cleavage and the activation of the apoptotic machinery during apoptosis of primary cultured renal proximal tubular epithelial cells allow us to draw two important conclusions. First, FAK dephosphorylation caused by DCVC precedes activation of the apoptotic machinery, i.e. caspases, and is
independent of caspase cleavage. Dephosphorylation also precedes the
proteolysis of FAK. Although we have only described the effects of
DCVC, similar observations were made with another nephrotoxicant,
cis-platin, indicating that our observations are also important in
other forms of nephrotoxicant induced cytotoxicity
(59).2 FAK cleavage has been
reported before in other models of apoptosis (12-15), but the temporal
relationship between FAK dephosphorylation, FAK translocation, and FAK
cleavage has not been studied. Thus, to our knowledge, this is the
first report that indicates that FAK dephosphorylation precedes
activation of caspases and cleavage of FAK during apoptosis. Second, in
this model, dephosphorylation of FAK is closely associated with loss of
cell-matrix interactions, but even when FAK dephosphorylation is
complete, cells remain attached to the substratum. Although caspase
activity is not involved in the impairment of focal adhesion signaling,
it appears that cleavage of FAK and other cytoskeletal proteins is
required for irreversible detachment of cells from the substratum.
Possibly, cleavage of FAK and other cytoskeletal proteins not only
causes cell detachment but also prevents the reattachment and spreading of injured cells. This may have considerable consequences in renal injury, since blocking cell reattachment prevents the attendant tubular
obstruction that occurs after renal injury (54, 55). Thus, our studies
provide new insights into the role of focal adhesion signaling in renal injury.
Although disruption of focal adhesion signaling through FAK clearly
precedes cell detachment after chemically induced apoptosis, the
data also suggest that caspase activation is not responsible for the
former. Rather, the data indicate that toxicant-induced dephosphorylation of focal adhesion proteins including FAK may contribute to loss of focal adhesions and cell-matrix interactions. Forced loss of cell-matrix interactions of RPTE, by keeping trypsinized RPTE in suspension, also results in caspase activation and
apoptosis.3 Therefore, the
present observations fit a model in which loss of focal adhesion
architecture and signaling is a trigger for chemically induced
activation of caspases culminating in apoptosis; FAK may have a central
role. Indeed, FAK and related family members play an important role in
other models of apoptosis. Blocking the interaction of FAK with
Cell detachment during apoptosis is likely to occur in two steps: loss
of cell-to-cell contact and then loss of cell-substratum adhesion,
i.e. the reverse of the steps in organizing an
epithelial layer. Both steps seem important in controlling
apoptosis (19, 46, 68). Our results indicate that FAK
dephosphorylation and translocation as well as caspase-mediated FAK
cleavage were associated with renal cell detachment during apoptosis.
Yet, only caspase-mediated cleavage of FAK seems related to the final
renal cell detachment: zVAD-fmk blocked cell detachment completely
without affecting either the dephosphorylation and translocation of FAK
or the disruption of focal adhesions. Thus, our data suggest that FAK
dephosphorylation and loss of focal adhesions are closely associated
with loss of cell-matrix contact, but not irreversible detachment which
actually requires caspase activation since zVAD blocked detachment but not focal adhesion disruption. Thus, cells may still adhere to the
substratum even though they have completely disrupted focal adhesions.
This may be related to the fact that cell adhesion not only depends on
cell-matrix but also on cell-cell interactions. Besides FAK, other
cytoskeletal proteins that are involved in cell-cell interactions,
e.g. The implications of cell reattachment in metastasis of tumor cells are
well known. However, also during renal injury, it is clear that
reattachment of dying or injured cells, through integrin-mediated signaling, has important pathophysiological implications since it
contributes to the tubular obstruction that exacerbates renal injury
(54, 55). In this scenario, pharmacological intervention of caspase
activity could be used to block cell detachment after a renal insult
and, thereby, alleviate tubular obstruction. It would be expected that
such a treatment would reduce the severity of the renal injury. Thus,
our studies provide new insights into possible modulation of acute
renal injury.
Although it is clear that FAK dephosphorylation by itself is not
sufficient to cause cell detachment, the exact role of caspase-mediated cleavage of FAK in this process needs further investigation. We studied
the involvement of FAK in chemically induced apoptosis, however, also
deregulation of other focal adhesion-associated signaling molecules
that are implicated in focal adhesion regulation and/or control of cell
death, e.g. c-Src, Fyn, and Crk, may be involved. Regardless
of the role of other focal adhesion proteins, FAK cleavage during
apoptosis will be important to prevent focal adhesion reassembly of
cells that are otherwise supposed to die by apoptosis.
In summary, the present findings demonstrate that FAK dephosphorylation
precedes toxicant-induced activation of caspases and subsequent cleavage of FAK. The fact that focal adhesion-mediated signal transduction is fundamental for cell survival of normal epithelial cells and that many focal adhesion-associated molecules have
oncogenic potential, most likely by preventing anoikis (46), warrants
future investigation on the role of other focal adhesion-associated signaling molecules in the control of (chemically induced) apoptosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, gelsolin,
-catenin, GAS-2, fodrin, and
FAK1 (4-15). Presumably, the
change of function (gain or loss) caused by proteolysis of multiple
target proteins contributes to apoptosis. Thus, the proteolysis of
cytoskeleton-associated proteins is most likely involved in the
morphological alterations observed during apoptosis in vitro
and in vivo, i.e. membrane blebbing and
"rounding up" of cells.
-lyase to a reactive acylating
metabolite that covalently modifies cellular macromolecules (60-62);
DCVC induces apoptosis of RPTE in culture (58, 65). Cell death of
primary cultured RPTE caused by DCVC treatment is preceded by
disorganization of the F-actin cytoskeletal network and dissolution of
focal adhesions (57, 58). Thus, this compound is a useful agent to
study the role of focal adhesion disturbance and cell detachment in
apoptosis of primary cultures of kidney epithelial cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C). After washing cells twice with PBS/EDTA (1 mM), cells were resuspended in PBS-EDTA containing 10 µg/ml RNase A and 7.5 µM propidium iodide. After 30 min
incubation at room temperature the cell cycle was analyzed by flow
cytometry (FACScan, Becton Dickinson) and the percentage of cells
present in sub-G0/G1 was calculated using the
LYSIS software (Becton Dickinson).
-glycerophosphate, 50 mM NaCl, 2 mM MgCl2, and 5 mM EGTA) and
subjected to three cycles of freezing and thawing. Equal amounts of
cell proteins were used in a caspase assay using acetyl-DEVD-AMC (25 µM; Research Biochemicals Int., Natick, MA) as a
substrate. Fluorescence derived from release of the AMC moiety was
followed using a fluorescence plate reader (HTS 7000 Bioassay reader;
Perkin-Elmer, Norwalk, CT). Caspase activity was calculated as
picomole/min/mg cell protein using AMC as a standard.
-mercaptoethenol. Proteins in equal
volumes of sample were separated by SDS-PAGE followed by Western
blotting using standard techniques. In other experiments, cleared
samples were used for the immunoprecipitation of
tyrosine-phosphorylated proteins using agarose-conjugated
anti-phosphotyrosine (4G10) (Upstate Biotechnology, Lake Placid, NY).
Blots were probed with 1 µg/ml mouse monoclonal anti-FAK antibody,
0.25 µg/ml mouse monoclonal anti-paxillin (Transduction
Laboratories), or 1 µg/ml 4G10 anti-phosphotyrosine monoclonal
antibody (Upstate Biotechnology). For detection, horseradish
peroxidase-conjugated anti-mouse secondary antibody and the ECL method
were used (Amersham).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of zVAD on caspase activation by
DCVC. RPTE were treated with DCVC in EBSS containing DPPD (20 µM) and after 4 h cells were allowed to recover in
complete medium containing 20 µM DPPD for another 4 h (t = 8 h). At 2, 4, 6, or 8 h after the
addition of DCVC (0.25 mM) pro-caspase-3 cleavage into the
17 kDa caspase-3 fragment (A) and caspase activity
(B) were determined. The concentration-dependent
effect of DCVC on caspase cleavage (C) and caspase activity
(D) as well as the effect of zVAD-fmk (100 µM)
on the caspase activation by DCVC (0.25 mM) was determined
8 h after the start of the incubation. Caspase-3 cleavage and
overall caspase activity were determined as described under
"Experimental Procedures." Caspase-3 cleavage shown is
representative for four independent experiments. Caspase activity data
shown are mean ± S.E. of four independent experiments
(n = 4).
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Fig. 2.
Effect of zVAD on DCVC-induced
cytotoxicity. RPTE were treated with DCVC (0.25 mM) in
EBSS containing 20 µM DPPD in the presence or absence of
zVAD-fmk (100 µM) for 4 h followed by recovery. Cell
cycle analysis (A, left panel: without zVAD-fmk; right
panel, with zVAD-fmk) and DNA fragmentation (B) were
determined 12 h after the start of the incubation as described
under "Experimental Procedures." The data shown are representative
for four independent experiments. Cell death (C) was
determined by analyzing the relative release of LDH from cells in the
culture medium compared with total LDH present and was expressed as % cell death. Data shown are mean ± S.E. of four independent
experiments (n = 4).
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Fig. 3.
DCVC causes cleavage of FAK. RPTE were
treated with DCVC as described in the legend to Fig. 1 and FAK cleavage
was determined by Western blotting as described under "Experimental
Procedures." A, to determine the time dependent effect of
DCVC (0.25 mM) on FAK cleavage samples were taken at 2, 4, 6, and 8 h after start of the experiment. B, at 8 h after start of the experiment the concentration dependent effect of
DCVC on FAK cleavage was determined. Data shown are representative for
four independent experiments (n = 4).
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Fig. 4.
DCVC causes a redistribution of FAK.
RPTE were treated with DCVC (0.25 or 1 mM) as described in
the legend to Fig. 1. After 4 h cells were fixated and
double-stained for FAK and F-actin. Stained cells were viewed using a
Bio-Rad confocal laser scanning microscope. Data are representative for
three independent experiments (n = 3). Note that FAK
translocates to the cytosol and the nuclear compartment and does not
co-localize with the collapsed F-actin network.
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Fig. 5.
DCVC causes tyrosine dephosphorylation of
FAK. RPTE were treated with increasing concentration of DCVC (0.1, 0.25, 0.5, and 1.0 mM) in EBSS in the presence of DPPD (20 µM). After 4 h cells were harvested and equal
amounts of cell protein were immunoprecipitated with anti-FAK and
immunoprecipitated proteins were separated by SDS-PAGE followed by
Western blotting. Blots were probed for anti-phosphotyrosine (PY) and
anti-FAK (A). Intensity of the bands were scanned by
densitometry to determine the ratio of PY/pp125 FAK. The ratio was
expressed as fold over control (B). Data are representative
for three independent experiments (n = 3).
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Fig. 6.
DCVC causes a loss of paxillin
phosphorylation. RPTE were treated with increasing concentration
of DCVC (0.1, 0.25, 0.5, and 1.0 mM) in EBSS in the
presence of DPPD (20 µM). After 4 h cells were
harvested and total cell protein was separated by SDS-PAGE followed by
Western blotting for anti-phosphotyrosine (A). Equal amounts
of cell protein were immunoprecipitated with agarose-conjugated anti-PY
followed by SDS-PAGE and Western blotting. Blots were probed with
monoclonal anti-paxillin ( PAX) (B). Results
shown are representative of three independent experiments
(n = 3).
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Fig. 7.
DCVC causes a translocation of paxillin from
the focal adhesion to the F-actin stress fibers. RPTE were treated
with DCVC (0.25 or 1 mM) in EBSS in the presence of DPPD
(20 µM). After 4 h cells were fixed for 5 min with
3.7% formaldehyde. Cells were stained for paxillin and F-actin as
described under "Experimental Procedures." Stained cells were
viewed using a Bio-Rad confocal laser scanning microscope. Note the
exact co-localization of paxillin with the collapsed F-actin
network.
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Fig. 8.
Orthovanadate prevents DCVC-induced tyrosine
dephosphorylation of paxillin and FAK. RPTE were treated with DCVC
(0.25 mM) in EBSS containing 20 µM DPPD in
the presence or absence of Na3VO4 (25 µM). After 4 h cells were harvested. Total cell
protein was separated by SDS-PAGE followed by Western blotting; blots
were probed with anti-phosphotyrosine (PY). Note the
increase of total PY-protein after treatment with
Na3VO4 (A). Equal amounts of protein
were either immunoprecipitated with anti-PY or anti-pp125 FAK.
Immunoprecipitated PY-proteins were separated by SDS-PAGE followed by
Western blotting; blots were probed with anti-paxillin
( PAX) (B). Immunoprecipitated pp125 FAK was
also separated by SDS-PAGE followed by Western blotting; blots were
probed with anti-PY (
PY) and after stripping the blots
were re-probed with anti-pp125 FAK (
FAK) (C).
Intensity of the pp125 FAK after staining for either anti-PY or
anti-pp125 FAK, were scanned by densitometry, and the ratio PY/FAK was
calculated and expressed as fold over control (D).
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Fig. 9.
Orthovanadate does not prevent translocation
of FAK from the focal adhesions to the cytosol. RPTE treated with
DCVC (0.25 mM) in EBSS containing containing 20 µM DPPD in the presence or absence of
Na3VO4 (25 µM). After 4 h
cells were fixed and double-stained for pp125 FAK and F-actin. Stained
cells were viewed using a Bio-Rad confocal laser scanning microscope.
Data are representative for three independent experiments
(n = 3).
Effect of orthovanadate on DCVC-induced cell detachment of RPTE
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Fig. 10.
Orthovanadate does not inhibit DCVC-induced
cytotoxicity. RPTE were treated with DCVC (0.25 mM) as
described in the legend to Fig. 1 in the absence or presence of
Na3VO4 (25 µM). After 8 h
caspase-3 and FAK cleavage (A) and caspase activity
(B) were determined as described in the legends to Figs. 1
and 3. After 12 h the % cell death as determined by the relative
LDH release, and the % of cells present in the
sub-G0/G1 as measured by flow cytometry
analysis, was determined (C) as described under
"Experimental Procedures." The DCVC-induced DNA fragmentation was
also analyzed after 12 h (D). Data shown are
representative for four independent experiments (A an
D) or mean ± S.E. (n = 4;
C and D).
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Fig. 11.
zVAD-fmk blocks DCVC-induced FAK cleavage
and cell detachment without affecting tyrosine dephosphorylation.
RPTE were treated with DCVC (0.25 mM) as described in the
legend to Fig. 1 in the absence or presence of zVAD-fmk (100 µM). After 4 h total cell lysates were separated by
SDS-PAGE followed by Western blotting and blots were probed with
anti-phosphotyrosine (PY) (A). FAK cleavage was determined
after 8 h as described in the legend to Fig. 3 (B).
Shown are representative immunoblots of four independent experiments.
Cell detachment was quantified after 12 h as described under
"Experimental Procedures" and expressed as mean of % detached
cells ± S.E. (n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-integrin during cell adhesion induces apoptosis (48). Expression of
a constitutively active FAK, a CD2-FAK chimer that localizes to focal
adhesions, but not chimers containing FAK kinase-dead mutants, inhibit
anoikis (46, 51). In contrast to the anti-apoptotic role of FAK,
overexpression of proline-rich tyrosine kinase 2, also known as
cellular adhesion kinase
, a FAK related non-receptor protein
tyrosine kinase that also localizes at focal adhesion, induces
apoptosis (50). Thus, different FAK family members may have opposing
roles in regulating apoptosis. FAK activation seems a cellular
survival factor and FAK dephosphorylation may lead to decreased
activity of signaling cascades that otherwise would block the apoptotic
machinery. For example, cell adhesion results in activation of Akt/PKB,
which is involved in phosphorylation of the pro-apoptotic Bcl-2 family member Bad (35, 36, 40-42). This phosphorylation results in inactivation of the anti-apoptotic function of Bad and, as a
consequence, protection against apoptosis (40-42). The relationship
between chemically induced dephosphorylation and translocation of FAK and perturbation of downstream signal transduction cascades in the
control of apoptosis warrants further investigations.
-catenin and fodrin (non-erythroid spectrin), have
been identified as substrates for caspases (8, 11). Moreover, Gas2 and
gelsolin, whose functions are directly related to the organization of
the F-actin cytoskeletal network, are also caspase substrates (7, 9).
Thus, the caspase-mediated cleavage of several cytoskeletal proteins,
including FAK, may be required for the complete loss of cell-matrix as
well as cell-cell interactions during apoptosis. Cleavage of these
proteins may actually ensure that the cell does not reattach and
proliferate at a distant site. Moreover, FAK cleavage will prevent
(re)activation of anti-apoptotic signaling cascades, for example,
through Akt/PKB, derived from focal adhesions thereby preventing
survival of cells that are otherwise destined to die. Indeed,
overexpression of the C-terminal fragment of FAK prevents
autophosphorylation of endogenous FAK upon cell adhesion (15), and FAK
cleavage is associated with loss of interaction between FAK and
paxillin (14). Thus, these data indicate that FAK cleavage is linked to
inhibition of focal adhesion-related signaling events as well as loss
of protein-protein interactions.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Susan Jaken, and Russel Bowes as well as other members of the laboratory for discussion and helpful suggestions and Gerard Mulder for critically reading the manuscript. We thank Dr. Donald Nicholson for providing anti-CPP32/caspase-3 antibodies.
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FOOTNOTES |
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* This work was supported by a Colgate Palmolive Fellowship and a Talent Stipend and Grant 902-21-208 from the Dutch Organization for Scientific Research (to B. v. d. W) and National Institutes of Health Grants DK46267 and ES07847 (to J. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Div. of Toxicology, LACDR, Leiden University, P. O. Box 9503, 2300 RA Leiden, The Netherlands. Tel.: 31-71-5276223; Fax: 31-71-5276292; E-mail: water_b{at}LACDR.LeidenUniv.nl.
2 B. Van de Water and J. L. Stevens, unpublished observations.
3 B. Van de Water, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: FAK, focal adhesion kinase; RPTE, renal proximal tubule epithelial; DCVC, S-(1,2-dichlorovinyl)-L-cysteine; DPPD, N,N'-diphenyl-p-phenylenediamine; EBSS, Earl's balanced salt solution; LDH, lactate dehydrogenase; PBS, phosphatebuffered saline; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amino-4-methylcoumarine.
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