From the Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510
Received for publication, December 17, 2002
, and in revised form, March 26, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The direct pro-apoptotic action of TNF on various cell types generally
results from the ligand-dependent assembly of a death-inducing signaling
complex (DISC), so called for the ability of this complex to initiate caspase
activation. For TNF, the formation of a DISC is dependent on ligand-binding to
TNF receptor type I (TNFR-1, also designated CD120a), which leads to the
recruitment of the cytosolic adapter protein TNFR-1 associated death domain
protein (TRADD) (1). The
association between TNFR1 and TRADD involves the "death domains"
(DD) of these proteins. DDs are homologous regions of 80 amino acids that
mediate protein-protein interaction and are also found in other receptors such
as Fas (CD95) (2). TRADD may
subsequently recruit Fas-associated death domain protein (FADD) through DD
interactions (3). FADD contains
both a DD and a death effector domain, the latter of which can interact with
either pro-caspase-8 (also known as FLICE) or with cellular FLICE inhibitory
protein (c-FLIP) (4,
5). FADD-associated
pro-caspase-8 undergoes autocatalytic activation by proteolysis, liberating
the active enzyme from the pro-form
(6). c-FLIP may inhibit this
process.
Activated caspase-8 dissociates from the DISC and acts on various cytosolic substrates. For example, caspase-8 may proteolytically activate the effector caspase-3. Activated caspase-3, in turn, cleaves a variety of substrates, resulting in apoptotic cell death (7). Alternatively, caspase-8 may proteolytically activate a cytosolic protein called Bid (8). Bid is a pro-apoptotic Bcl-2 family member containing a single Bcl-2 homology (BH) domain designated BH3. Proteolytically activated forms of "BH3-only" family members, such as Bid and Bad, bind to mitochondrial-associated "BH13" or "multidomain" proteins, such as Bax and Bak, causing supramolecular openings of the outer mitochondrial membrane (9, 10, 11, 12). These openings allow the release of cytochrome c from the mitochondria (13). Within the cytosol, cytochrome c associates with Apaf-1, and this complex further recruits pro-caspase-9 forming an assembly sometimes called an apoptosome. The apoptosome mediates ATP-dependent autocatalytic processing of caspase-9 and activated caspase-9, like caspase-8, can catalyze the proteolytic activation of caspase-3 resulting in apoptotic cell death (14). Thus caspase-8-mediated cleavage of Bid activates an amplification pathway for mitochondrial-dependent activation of caspase-3. Mitochondrial openings may also release other proteins, such as apoptosis-inducing factor that can initiate caspase-independent cell death (15). The requirement for the Bid/cytochrome c/Apaf1 amplification pathway differs among various cell types and correlates with the extent to which active caspase-8 is generated by the DISC (8, 16). DISC activity may be positively regulated by the expression levels of FADD and pro-caspase-8 (17) or negatively regulated by the levels of c-FLIP (5).
IL-1, like TNF, initiates the activation of signal transduction cascades by the recruitment of adapter proteins to its receptor. In EC, IL-1 may also initiate apoptotic cell death. To date, the precise components of the IL-1 receptor-associated DISC have not been defined. A number of adapter proteins involved in IL-1 signal transduction, namely MyD88 and IRAK (18), contain DDs and are possible mediators of caspase recruitment and activation.
Most untransformed cell types are not sensitive to the pro-apoptotic
actions of TNF or IL-1, unless mRNA translation or protein synthesis is
blocked. This observation has been explained by the capacity of TNF to
stimulate the activation of NFB, resulting in the up-regulation of
anti-apoptotic gene products such as c-FLIP, XIAP, c-IAP 1, and c-IAP 2
(19,
20,
21). The expression levels of
several anti-apoptotic genes, such as c-FLIP and IAP 1 are also regulated by
the proteasome (22,
23). In the presence of
cycloheximide (CHX), levels of c-FLIP are rapidly diminished, favoring
DISC-dependent activation of caspase-8
(19,
22). Reduction of c-FLIP by
antisense oligonucleotides mimics the effect of CHX and similarly sensitizes
cells to death (22).
Although apoptosis is generally associated with caspase activation, either through a DISC or through an apoptosome, caspase-independent cell death has been observed with the generation of apoptotic-like features in a variety of cell types (24, 25, 26). In many instances, these variant forms of apoptosis are mediated by non-caspase proteases such as the cysteine protease cathepsin family, calpains, serine proteases, or the proteasome complex. Of particular interest, the activation of cathepsin B has been shown to play a central role in the generation on TNF-mediated cell death in fibrosarcoma cells (25). Furthermore, TNF-mediated apoptosis has been shown to be strongly reduced in hepatocytes from cathepsin B-deficient mice (27). This pathway has not been described in a normal (untransformed) human cell type.
While most TNF activities on EC result in inflammation and/or apoptosis, we
have shown in EC that TNF and IL-1 also activate the anti-apoptotic
phosphatidylinositol 3-kinase/Akt pathway
(28). PI3K converts plasma
membrane phosphatidylinositol 4,5-bisphosphate into phosphatidylinositol
3,4,5-trisphosphate, catalyzing the recruitment of several enzymes, such as
PI3K-dependent protein kinase and Akt to the plasma membrane. Akt is a
serine/threonine kinase that exerts an anti-apoptotic action by the
phosphorylation of a number of substrates containing the phosphorylation
consensus RXRXX(S/T). Akt-mediated phosphorylation
inactivates the pro-apoptotic Bcl-2 homologue Bad
(29), the apoptosis-initiating
enzyme caspase-9 (30), and the
forkhead family transcription factor FKHRL1
(31), which mediates
transcription of pro-apoptotic gene products. In some cells Akt may regulate
the activity of NFB either through direct phosphorylation and
activation of IKK
(32)
or through regulation of the transactivation capacity of Rel A
(33). We have shown that
activation of PI3K in EC does not contribute to the activation of NF
B
or have any significant effect on NF
B-dependent inflammatory responses
(28). Similarly, Akt is
reported not to have any effect of NF
B activation in HeLa cells
(34).
In the present study we have examined the role of PI3K and Akt activation in the regulation of apoptosis induced by TNF and IL-1. To do so, we either inhibited PI3K with LY294002 or inhibited Akt by retroviral transduction with an inactive (K179M), dominant-negative form of Akt. We report that inhibition of PI3K but not of Akt sensitizes EC to the apoptotic actions of TNF and IL-1 and that the cell death caused by this pathway could not be blocked by caspase inhibition with zVADfmk but instead appears to be mediated through cathepsin B.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MaterialsRecombinant human TNF was purchased from R&D, and IL-1 was purchased from Peprotech Inc (Rocky Hill, NJ). LY294002, CHX, and Z-Arg-Arg-amidomethylcoumarin (Z-Arg-Arg-AMC) were purchased from Calbiochem (San Diego, CA). Propidium iodide, Hoescht reagent, 4',6-diamidino-2-phenylindole HCl (DAPI), JC-1, calcein-AM, and LysoTracker red were purchased from Molecular Probes (Eugene, OR). RNase A and digitonin were purchased from Sigma. The cathepsin B inhibitor CA-074-Me was purchased from Peptides International (Louisville, KY). Complete protease inhibitor mixture tablets and Pefabloc were purchased from Roche Applied Science (Indianapolis, IN). Mouse anti-FLIP antibody was a gift from Dr. Peter Krammer (DFKZ, Heidelberg, Germany). Rabbit anti-Bid antibody was purchased from BD Pharmingen (San Jose, CA). Mouse anti-cathepsin B was purchased from Oncogene Research Products (San Diego, CA). CaspaTag fluorescein broad range (VAD), caspase-3 (DEVD), caspase-8 (LETD), and caspase-9 (LEHD) activity assay kits were purchased from Serologicals (Norcross, GA). Mouse anti-Bax antibody was purchased from Transduction Laboratories (San Jose, CA). Rabbit anti-FKHR and phospho-FKHR antibodies were purchased from Cell Signaling (Beverly, MA). Mouse anti-hemagglutinin (HA) was purchased from Roche Applied Science (Indianapolis, IN). Horseradish peroxidase-conjugated secondary antibodies for Western blotting were purchased from Jackson ImmunoResearch (Westgrove, PA).
ImmunoblottingFor immunoblots, each well of a six-well plate containing a confluent HUVEC monolayer was washed twice in ice-cold PBS and lysed by the addition of 100 µl of lysis buffer (50 mM Tris-Cl, pH 6.8, 150 mM NaCl, and 1% Triton X-100) supplemented with Pefabloc (1 mM) and complete protease inhibitor mixture. For the measurement of phospho-proteins, NaF (10 mM) and Na3VO4 (1 mM) were also included in the lysis buffer to reduce phosphatase activity. After 20 min on ice, lysates were harvested by scraping. Where indicated, detached EC were harvested by centrifugation, washed in PBS, and pooled with the lysate of the attached EC from the same sample. For each sample, an equal amount of protein was separated by SDS-PAGE (35) then transferred electrophoretically to a polyvinylidene difluoride membrane (Immobilon P, Millipore, Milford, MA) and immunoblotted with primary and horseradish peroxidase-conjugated secondary antibodies. Detection of the bound antibody by enhanced chemiluminescence was performed according to the manufacturer's instructions (Pierce Chemical Co., Rockford, IL).
Cell Cycle and Hypodiploid DNA AnalysisEC grown to confluence on 12-well plates were treated as described in the text. At the indicated time after treatment, floating EC were collected and pooled with residual attached EC suspended by trypsin treatment. The pooled EC were washed once in PBS and fixed by resuspension in 70% ethanol for 15 min. After fixation, EC were washed once more in PBS before incubation in PBS containing propidium iodide (50 µg/ml) and RNase A (1 mg/ml) for 0.252 h. The DNA content of EC was then determined by FACS analysis using Cell Quest software (FACSort, BD Biosciences, San Jose, CA).
DAPI Staining of ECEC were grown to confluence on
gelatin-coated 12-well plates and treated as described in the text. After
treatment, attached EC were harvested with trypsin and combined with floating
EC harvested from the same sample. Cells were washed in PBS, and 1
x 105 cells were adhered to a glass coverslip by spinning in
a cyto-centrifuge (Shandon, Pittsburgh, PA). Slides were air-dried and dipped
in a chamber containing MeOH and DAPI (1 µg/ml). After rinsing in PBS, a
drop of Gel Mount (Biomeda Corp., Foster City, CA) and a coverslip was placed
over the cells. Specimens were examined by immunofluorescence microscopy using
a Nikon diaphot microscope with a 360-nm filter.
Quantitation of EC Adherence and ReplatingTo quantify the
number of cells that remained adherent, EC plated on gelatin-coated 96-well
plates were treated as indicated in the text. After experimental manipulation
the medium was removed and cells were washed twice in PBS. The residual
attached cells were fixed and stained by the addition of 70% ethanol
containing 100 µg/ml Hoescht 33258 reagent (Molecular Probes, Eugene, OR)
for 30 min at room temperature. Cells were again washed twice with PBS, and
the residual fluorescence was recorded (ex = 360 nm,
em = 460 nm) using a fluorescence plate reader (Perspective
Biosystems Inc, Framingham, MA). To assess the viability of detached
versus adherent cells, EC were grown to confluency in 12-well plates
and treated as indicated in the text. Detached EC were harvested, washed in
Hanks' balanced salt solution, and re-seeded onto gelatin-coated plates. EC
that remained substrate-attached were similarly re-plated following harvest
with trypsin. Viability was assessed as replating efficiency 18 h later
quantified by Hoescht staining as above.
Caspase Activity AssaysFor experimental manipulation EC were plated on 12-well plates and treated at confluency as indicated in the text. After described treatment the CaspaTag peptide (FAM-VAD-fmk for broad range caspase activity, FAM-LETD-fmk for caspase-8, FAM-LEHD-fmk for caspase-9, or FAM-DEVD-fmk for caspase-3) was added to each well and incubated a further 1 h according to the manufacturer's instructions. Subsequent to incubation with the peptide, floating EC were harvested and combined with attached EC from the same well harvested with trypsin. Caspase activity was demonstrated by the generation of a second peak or shoulder on FL-1 that results from peptide binding to active caspase by FACS.
Mitochondrial Membrane Potential ()
AnalysisAfter experimental manipulation of EC seeded on 12-well
plates, floating EC were harvested by centrifugation and combined with
remaining substrate-attached EC harvested with trypsin. The pooled EC were
washed 1x in PBS containing 1% bovine serum albumin before resuspension
in 200 µl of PBS/bovine serum albumin containing JC-1 (10 µg/ml). After
15 min of incubation at 37 °C, EC were washed, re-suspended in PBS, and
analyzed by FACS.
Retroviral TransductionHA-tagged murine K179M Akt (a gift from Dr. W. Sessa, Yale University) was Topo®-cloned using EcoRI and NotI into the retroviral LZRS expression vector (a gift from Dr. A. L. M. Bothwell, Yale University), and the construct was verified by sequencing. The caspase-resistant Bcl-2 retroviral construct (a gift from A. L. M. Bothwell) has been described previously (36). The amphotropic Phoenix packaging cell line was transfected with either the empty vector LZRS, LZRS-K179M Akt, or LZRS-Bcl2 using LipofectAMINE (Invitrogen) and selected for gene expression 24 h after transfection using puromycin (1 µg/ml). Puromycin-resistant cells were used to derive conditioned medium to provide a retroviral stock for HUVEC transduction. For transduction of primary HUVECs, M199 containing ECGF was removed, and cells were washed and incubated 58 h with retroviral conditioned media containing Polybrene (8 µg/ml, Sigma). After incubation, retrovirus was removed and replaced with normal growth medium overnight. The transduction process was repeated a further three times with intermittent cell passage as required. Using this protocol the percentage of HUVECs expressing the transgene is routinely >95%.
Preparation of Cytosolic Extracts for the Analysis of Cathepsin BMeasurement of cytosolic cathepsin B was determined using methodology similar to Foghsgaard et al. (25). Endothelial cells were pretreated with LY294002 (50 µM) for 3 h in complete M199 prior to the addition of cytokine for a further 3 h. After treatment media were removed and cells were washed twice in PBS prior to the addition of extraction buffer (50 µg/ml digitonin, 250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM Pefabloc, pH 7.5) and incubation on ice for 20 min. (The conditions that allow for the selective permeabilization of the plasma membrane by digitonin without perturbation of lysosomes were determined in preliminary experiments using EC pre-loaded both with calcein-AM and LysoTracker red.) After incubation, the cytosolic extract was collected. Samples were analyzed for cathepsin B either by Western blotting or with a cathepsin B activity assay.
Measurement of Cathepsin B ActivityA 50-µl volume of
cytosolic extract was added to an equal volume of cathepsin reaction buffer
(50 mM sodium acetate, 4 mM EDTA, 8 mM
dithiothreitol, 1 mM Pefabloc, pH 6.0). Cathepsin B activity was
measured by the addition of 20 µM Z-Arg-Arg-AMC
(Calbiochem). Liberated AMC was measured (ex = 360 nm,
em = 460 nm) using a fluorescence plate reader immediately
following the addition of the peptide substrate (T0) and
following a 60 min incubation at 37 °C (T60). Activity
was determined by subtracting the background activity at
T0 from activity at T60 and correcting
for the amount of protein in each sample.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Inhibition of Akt Does Not Account for Sensitization to Cytokine-mediated ApoptosisTo determine whether the sensitization to TNF- or IL-1-mediated cell death upon inhibition of PI3K with LY294002 was dependent upon inhibition of Akt, EC were subjected to retroviral transduction with a dominant-negative inactive kinase form of Akt (K179M Akt). The functional expression of K179M Akt was confirmed by the inhibition of the TNF-dependent phosphorylation of the forkhead transcription factor (FKHR) (Fig. 2A). Surprisingly, expression of K179M Akt had no effect on cell survival following treatment with TNF or IL-1. Similarly, transduction with K179M Akt did not result in potentiation of cell death observed in response to LY294002 either alone or in combination with cytokine treatment. These experiments suggest that inhibition of Akt is not sufficient to explain the death response observed in response to cytokine following inhibition of PI3K. K179M Akt expression did cause a significant increase in the death response observed following treatment with cytokine plus CHX (Fig. 2B) suggesting that active Akt can limit caspase-induced cell death. The remainder of this study focused on the effects of LY294002 treatment.
|
Activation of Caspase-8, -9, and -3 following Inhibition of PI3K or Treatment with CycloheximideThe most proximal caspase activated by formation of a DISC is caspase-8. Activated caspase-8 may directly activate caspase-3 or, via its actions on the mitochondria, generate the apoptosome and activate caspase-9 (7, 8). We analyzed the effects of cycloheximide and PI3K inhibition on TNF- or IL-1-mediated activation of caspase-8 and subsequent downstream caspases-9 and -3. Using a living cell, fluorometric caspase assay (38) we observed activation of caspases-8, -9, and -3 within 1h of treatment with cytokine plus CHX, which increased to a maximal activity at around 6 h (Fig. 3A). We could not clearly discern by this method sequential activation of these caspases, although caspase-8 activation was in some experiments detected slightly earlier than activation of caspase-9 or -3 (not shown). Activation of caspases-8, -9, and -3 could also be observed in response to cytokine following inhibition of PI3K. In this case, however, caspase activation was slower and less extensive. No activation was observed for the first 6 h, and peak activity occurred only after an overnight incubation of 18 h or more (Fig. 3B).
|
Treatment of EC with Cycloheximide but Not LY294002 Decreases the
Expression of c-FLIPSensitization of EC to cytokine-mediated
apoptosis by TNF or IL-1 has been reported to result from the inhibition of
NFB-dependent transcription of anti-apoptotic genes coupled with
degradation of the apoptosis inhibitor protein c-FLIP
(19,
22). We confirm that treatment
of EC with CHX results in a rapid decrease in the expression of c-FLIP
(Fig. 4A). However, in
contrast to the effect observed with CHX, inhibition of PI3K by treatment with
LY294002 had no effect on the expression of c-FLIP, nor did it prevent TNF- or
IL-1-induced up-regulation of c-FLIP (Fig.
4B).
|
Caspase Activation Is Not Required for Cytokine-initiated Cell Death
following Inhibition of PI3KMany of the features of programmed
cell death such as detachment, DNA degradation, loss of mitochondrial membrane
potential (), and nuclear condensation or fragmentation are
dependent on the activation of effector caspases. To assess the role of
caspase activation in the TNF- or IL-1-mediated death pathways activated
following inhibition of PI3K or CHX treatment, we employed the broad range
caspase inhibitor, zVADfmk. Titration of zVADfmk from 0 to 50 µM
resulted in a dose-dependent inhibition of caspase activity in response to TNF
in the presence of either CHX or LY294002 (not shown) with full inhibition
being achieved with zVADfmk at 25 µM
(Fig. 5A). This
concentration was used in all further studies. Although zVADfmk effectively
blocked the detachment of EC observed in response to TNF or IL-1 in the
presence of CHX, the inhibitor had no effect or actually increased the
detachment of EC in response to cytokine treatment following inhibition of
PI3K (Fig. 5B).
(Detached ECs were confirmed as being dead by the inability to re-attach to
substrate following removal from the apoptotic stimulus (data not shown).)
|
The disparity between the ability of zVADfmk to block cell death in
response to cytokine plus CHX but not following inhibition of PI3K suggested
the existence of a caspase-independent death pathway in the latter case. To
further characterize the death pathway activated in response to TNF or IL-1
following inhibition of PI3K, we examined the effect of zVADfmk on some of the
parameters associated with apoptosis, i.e. DNA degradation, nuclear
condensation/fragmentation, and loss of . Caspase inhibition
blocked DNA degradation observed both in response to TNF or IL-1 following
inhibition of PI3K or treatment with CHX assessed by PI staining and FACS
analysis (Fig. 5C).
Nuclear morphology by DAPI staining confirmed that treatment with TNF or IL-1
plus LY294002 resulted in the generation of fragmented nuclei
(Fig. 1, bottom panel,
E and F). Caspase inhibition completely prevented nuclear
condensation associated with TNF or IL-1 plus CHX
(Fig. 1, top panels, H
and I). However, in the presence of zVADfmk, cytokine plus LY294002
still resulted in nuclear condensation but did not progress to fragmentation
(Fig. 1, bottom panel,
H and I). Treatment with TNF or IL-1 in the presence of CHX or
following inhibition of PI3K resulted in loss of
as observed by
an increase in fluorescence (FL-1) with the dye JC-1
(Fig. 6A). Caspase
inhibition with zVADfmk blocked loss of
in response to cytokine
plus CHX but had no effect on the loss of
in response to cytokine
following inhibition of PI3K (Fig.
6B). These data suggested that, in the presence of
LY294002, TNF and IL-1 activated a mitochondrial death pathway that was not
dependent upon caspase activation. However, in the absence of zVADfmk, caspase
activation did occur and the activation of caspases altered the morphological
and biochemical features of cell death.
|
Bcl2 Expression Blocks Cytokine-initiated Cell Death following
Inhibition of PI3K but Not Protein SynthesisTo further examine the
role of the mitochondria in the death pathways evoked by TNF or IL-1 following
inhibition of PI3K or treatment with CHX, EC were subjected to retroviral
transduction with a caspase-resistant form of Bcl-2. Four rounds of infection
with either empty vector (LZRS) or LZRS-Bcl-2 resulted in over 95% expression
of the transgene in EC as confirmed by intracellular FACS staining (described
previously in Ref. 36). We
observed that transduction with Bcl-2 protected EC from cytokine-initiated
cell death and detachment following inhibition of PI3K but had no effect or
actually increased the killing observed in the presence of CHX
(Fig. 7). Similarly, Bcl-2
expression effectively blocked both the activation of caspase-8 and loss of
observed following inhibition of PI3K but had no effect on the
activation of caspase-8 or loss of
in response to cytokine in the
presence of CHX (Fig. 8, A and
B). Together these results support the observation that
caspase activation in response to cytokine treatment following inhibition of
PI3K results from rather than causes alterations in the mitochondria. In
contrast, cytokine plus CHX-induced cell death occurs through a typical Type I
death pathway that is completely caspase-dependent. Changes in the
mitochondria of EC in this pathway occur downstream of caspase-8 activation
and are not required for cell death.
|
|
Cathepsin B Activation Is Required for Cytokine-initiated Death
following Inhibition of PI3KWe explored the possibility that the
non-caspase protease cathepsin B could be the signal upstream of the
mitochondria to initiate caspase-independent cell death following inhibition
of PI3K using the specific cathepsin B inhibitor CA-074-Me. CA-074Me (30
µM) effectively blocked all of the features of cell death in
response to cytokine following inhibition of PI3K such as increased EC
detachment, DNA fragmentation, caspase activity, and loss of
(Figs. 9, AC, and 10B). In
contrast, inhibition of cathepsin B had little or no effect on the same
parameters observed in response to cytokine in the presence of CHX (Figs.
9, AC, and
10A). Direct evidence
that the activation of cathepsin B occurred in response to cytokine treatment
following inhibition of PI3K was provided by translocation of active cathepsin
B to the cytosol. Following only 3 h of treatment with LY294002 with or
without TNF (i.e. at an earlier time than we could detect caspase
activation) we observed an increase in immunoreactive cathepsin B in the
cytosol using Western blotting (Fig.
11A). The appearance of cytosolic cathepsin B was
paralleled by an increase in the cytosolic activity of the enzyme that was
completely blocked in the presence of CA-074-Me (30 mM)
(Fig. 11B).
|
|
|
To identify the mechanism through which an increase in cathepsin B activity
might effect mitochondria to result in a loss of and activation
of downstream effector caspases, we examined the effect of zVADfmk on the
cleavage of Bid observed in response to cytokine plus CHX or following PI3K
inhibition. In the presence of CHX or following inhibition of PI3K, TNF, and
IL-1 resulted in a clear decrease in the levels of the 22-kDa inactive form of
Bid. Following caspase inhibition with zVADfmk, the decrease in p22 Bid was
blocked in both cases (data not shown). Similarly, it was also possible to
observe dimerization of Bax in response to cytokine following inhibition of
PI3K, and this was also blocked by caspase inhibition despite continued cell
death (data not shown). These data indicate that treatment with TNF or IL-1
following inhibition of PI3K results in the activation of cathepsin B and that
this activation accounts for the loss of mitochondrial function in a manner
that is not dependent on the cleavage of Bid and dimerization of Bax.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EC could also be sensitized to the pro-apoptotic actions of TNF and IL-1
following inhibition of PI3K with LY294002. This death pathway occurred with
features typical of apoptosis such as DNA degradation, and nuclear
condensation and fragmentation. However, caspase activation was delayed and
less complete, and death could be blocked by overexpression of Bcl-2. These
data initially suggested a Type II amplification pathway of caspase-3
activation. However, nuclear condensation, loss of , and cell
death were still observed during complete caspase inhibition by zVADfmk as
detected in an activity assay. Moreover, although it did not prevent death,
zVADfmk still caused inhibition of nuclear fragmentation and of DNA
degradation. These data suggest that caspases were activated and contributed
to some of the morphological and biochemical features of death but that
caspases were not necessary for death to occur. It is, however, formally
possible that some degree of caspase activation occurs in the presence of
zVADfmk that is too slight to measure yet still contributes to cell death.
Given the effectiveness of inhibition of cytokine plus CHX treated by zVADfmk,
this appears highly unlikely.
Caspase-independent cell death has previously been observed in a variety of
other cell types. Caspase inhibition has been ineffective in suppressing
anti-CD2 or staurosporine-induced death of activated T lymphocytes
(24), TNF- plus CHX-mediated
death of NIH3T3 cells (26), or
TNF-mediated cell death of WEHI-S fibrosarcoma cells
(25). The activation of
non-caspase proteases such as cathepsins, calpains, or granzymes is now
emerging as an alternate means to induce cell death
(39,
40). Cathepsins are lysosomal
proteases primarily thought to be involved in the degradation of proteins
within the lysosomal compartment. However, several studies have shown
translocation of cathepsins from the lysosome to the cytosol during cell death
indicating that cathepsins may be able to gain access to cytosolic proteins
(41,
42). The implication of
cathepsin B activation during TNF-mediated cell death in mouse hepatocytes and
human fibrosarcoma cells in vitro
(25,
27,
41) prompted us to consider
the role of cathepsin B in TNF- and IL-1-mediated cell death observed
following inhibition of PI3K in EC. We observed that inhibition of cathepsin B
with CA-074-Me prevented loss of , caspase activation, DNA
fragmentation, and cell detachment observed in response to cytokine treatment
following inhibition of PI3K. This inhibition occurred without significant
effect on the same parameters activated in response to cytokine plus CHX. We
also observed translocation of cathepsin B to the cytosol following inhibition
of PI3K prior to the activation of caspases. Together, these results indicate
that cathepsin B plays a critical role in a caspase-independent,
mitochondrial-dependent death pathway in cytokine-treated EC.
We do not know the mechanism for cathepsin B activation in cells treated
with TNF or IL-1 plus LY294002. Cathepsin B was liberated from the lysosomes
by the actions of LY294002 alone. This agent produced some degree of cell
death, but the presence of TNF or IL-1 markedly enhances this effect. We do
not know how either LY294002 or cytokine work in this system. Because EC could
not be sensitized to cytokine-mediated cell death following inhibition of Akt
with K179M Akt, it is unlikely that Akt is involved in this pathway.
Guicciardi et al.
(41) reported that activation
of cathepsin B by TNF plus actinomycin D was likely to be caspase-dependent,
because recombinant caspase-8 could cause the release of cathepsin B from
purified lysosomes. Our data do not support this conclusion. We observed that
loss of was not blocked by inhibition of caspases, that
transduction of EC with Bcl-2 prevented cytokine-dependent caspase-8
activation, and that the treatment of EC with LY294002 either alone or in
combination with cytokine treatment failed to decrease levels of c-FLIP. We
therefore conclude that the TNF- or IL-1-mediated death pathway activated in
EC following inhibition of PI3K results from cathepsin B activation in a
caspase-independent manner. A recent report has shown that selective lysosomal
permeabilization by TNF in hepatocytes is paralleled by sphingosine-mediated
lysosomal permeabilization
(42). Inhibition of PI3K may
sensitize EC to generate sphingosine either by the activation of
sphingomyelinase (43) or by
de novo biosynthesis
(44).
How cathepsin B may result in the activation of a mitochondrial death pathway generating a phenotype with partial nuclear condensation is also undefined. Guicciardi et al. (41) allude to the possibility that cathepsin B activation might result in activation of a mitochondrial death pathway by the cleavage and activation of a cytosolic protein such as Bid. Stoka et al. (45) have also demonstrated cleavage of recombinant mouse Bid following treatment with isolated lysozymes. In contrast to these reports, we observed that caspase inhibition was able to prevent Bid depletion that occurred in response to cytokine treatment either following inhibition of PI3K or protein synthesis. Caspase inhibition was also completely effective in preventing the dimerization of Bax. These observations indicate that Bid cleavage and Bax dimerization are most likely consequences of caspase activation downstream of the mitochondria and not required to initiate cell death. We also failed to detect any increase in p53 as a result of treatment with cytokine plus LY294002.2 Identification of cathepsin substrates that are pertinent to the caspase-independent death pathway will be an important direction for future studies.
![]() |
FOOTNOTES |
---|
Supported by an American Heart Association Scientist Development grant.
Supported by National Institutes of Health Training Grant GM07205.
¶ To whom correspondence should be addressed: Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Tel.: 203-737-2292; Fax: 203-737-2293; E-mail: Jordan.Pober{at}yale.edu.
1 The abbreviations used are: EC, endothelial cell; ,
mitochondrial membrane potential; CHX, cycloheximide; c-FLIP, FLICE inhibitory
protein; DD, death domain; DISC, death-inducing signaling complex; FADD,
Fas-associated death domain protein; IL-1, interleukin-1; NF
B, nuclear
factor
B; PI3K, phosphatidylinositol 3-kinase; TNF, tumor necrosis
factor; TNFR1, TNF receptor 1; TNFR2, TNF receptor 2; TRADD, TNF
receptor-associated death domain protein; BH, Bcl-2 homology domain; FKHR,
forkhead transcription factor; AMC, amidomethylcoumarin; DAPI,
4',6-diamidino-2-phenylindole HCl; HA, hemagglutinin; PBS,
phosphate-buffered saline; FACS, fluorescence-activated cell sorting; HUVEC,
human umbilical vein endothelial cell; zVADfmk,
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; CA-074-Me,
[L-3-trans(propylcarbamoyl)oxirane-2-carboxyl]-L-isoleucyl-L-proline
methylester.
2 L. A. Madge and J. S. Pober, unpublished observations.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|