Published online before print June 21, 2001, doi: 10.1161/hh1301.092506
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Phosphatidylinositol 3-Kinase/Akt Signaling Controls Endothelial Cell Sensitivity to Fas-Mediated Apoptosis via Regulation of FLICE-Inhibitory Protein (FLIP)
From the Division of Cardiovascular Research (T.S., T.M., K.W.), St. Elizabeth’s Medical Center, and the Program in Cell, Molecular, and Developmental Biology (B.E.O., K.W.), Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Mass, and The First Department of Medicine (T.M.), Osaka University School of Medicine, Suita, Osaka, Japan.
Correspondence to Kenneth Walsh, PhD, Division of Cardiovascular Research, St. Elizabeth’s Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail kwalsh@world.std.com and kwalsh{at}opal.tufts.edu
Abstract
Abstract—Fas is constitutively expressed on endothelial cells, but in contrast to smooth muscle and other cell types, endothelial cells are highly resistant to Fas-mediated apoptosis. In this study, we examined the role of the serine/threonine kinase Akt/PKB in controlling the sensitivity of endothelial cells to Fas-mediated apoptosis. Serum deprivation inhibited expression of the caspase-8 inhibitor FLICE-inhibitory protein (FLIP), which functions downstream from Fas. FLIP expression levels were restored when serum-depleted cells were treated with vascular endothelial growth factor. Treatment with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002 or infection of the adenoviral construct expressing dominant-negative Akt (Adeno-dnAkt) also inhibited the expression of FLIP in endothelial cells, whereas the MEK inhibitor PD98059 had no effect. Conversely, adenovirus-mediated transfection of a constitutively-active Akt gene abolished the wortmannin- and LY294002-mediated downregulation of FLIP. Suppression of PI 3-kinase signaling sensitized endothelial cells to Fas-mediated apoptosis. Under conditions of suppressed PI 3-kinase signaling, restoration of FLIP expression reversed the induced sensitivity of endothelial cells to Fas-mediated apoptosis. These data suggest that inhibition of Fas-mediated apoptosis, via promotion of FLIP expression, is a mechanism through which Akt signaling can promote endothelial cell survival.
Key Words: apoptosis • cell signaling • signal transduction • growth factors • cytokines
Fas (also called APO-1 or CD95) is a type I membrane protein belonging to the TNF receptor family that functions to transmit a death signal to the cell.1 Activation of this pathway requires receptor cross-linking with Fas ligand (FasL) or anti-Fas antibodies.2 Fas-mediated apoptosis is an essential mechanism for the maintenance of homeostasis in multicellular organisms, and disruption of the Fas/FasL system can lead to lymphoproliferative disorders3 4 and accelerate autoimmune diseases.5 Conversely, its overactivation can cause pathological tissue destruction.6 With regard to the vasculature, expression of Fas has been detected in both the normal and diseased vessel wall. Furthermore, it has been proposed that Fas-mediated apoptosis of vascular cells is a feature of atherogenesis,7 atherosclerotic plaque vulnerability,8 and allograft arteriopathy.9
Fas ligation induces the recruitment of the proapoptotic proteins Fas-associated death domain (FADD) and procaspase-8, to form a complex in which the proteolytic activation of caspase-8 leads to the generation of an apoptotic cascade.10 The antiapoptotic FLICE-inhibitory protein (FLIP) is a cytoplasmic protein that is homologous to caspase-8 (also referred to as FLICE).11 FLIP acts in a dominant-negative manner to inhibit caspase-8 because it lacks a cysteine residue at the active site that is essential for proteolysis. FLIP expression is elevated during early stages of T-cell activation and during macrophage differentiation, and this is believed to make these cells resistant to Fas-mediated apoptosis.12 13 Multiple isoforms of FLIP (two of which are designated FLIP-L [long isoform] and FLIP-S [short isoform]) result from alternative splicing.12 Endothelial cells express abundant FLIP-L, but the level of FLIP-S protein is very low or undetectable.14 Endothelial cells are naturally resistant to Fas-mediated apoptosis,7 15 16 17 and specific stimuli are thought to sensitize endothelial cells to Fas ligation via downregulation of FLIP or upregulation of Fas expression.14 18
The serine/threonine protein kinase Akt/PKB promotes viability in various cell types, including endothelial cells.19 Activation of Akt involves the binding of phosphatidylinositol 3-kinase (PI 3-kinase)–generated inositol lipids to Akt via its pleckstrin homology domain.20 PI 3-kinase–dependent activation of Akt also involves PDK1-mediated phosphorylation of threonine 308, leading to the autophosphorylation of serine 473.21 Akt-mediated viability is dependent, at least in part, on the ability of Akt to phosphorylate and inactivate proapoptotic proteins.20
In the present study, we examined the role of Akt/PKB signaling in controlling sensitivity to Fas-mediated apoptosis in endothelial cells. We found that like serum deprivation, suppression of either PI 3-kinase or Akt signaling induced downregulation of FLIP expression and sensitized endothelial cells to Fas-mediated apoptosis. Under these conditions, adenovirus-mediated FLIP gene transfer protected endothelial cells from apoptosis. These data suggest that Akt signaling controls the sensitivity of cells to Fas-mediated apoptosis via the regulation of FLIP expression.
Materials and Methods
Cell Culture and Reagents
Human umbilical vein endothelial
cells (HUVECs) were isolated as
described.22 Human vascular
smooth muscle cells (VSMCs) were isolated from an internal mammary
artery obtained during coronary bypass surgery. The human
T-cell leukemia cell line, Jurkat clone E6-1, was obtained from the
American Type Culture Collection. HUVECs were
grown to confluence on gelatin-coated 10-cm dishes, 6-well plates, or
slide chambers with medium containing 2% FBS. Medium was replaced with
fresh medium, with or without serum, typically at the time reagents
were added. Mouse monoclonal antibody against human FLIP (NF6) was a
gift from Dr Marcu E. Peter (German Cancer Research Center,
Heidelberg, Germany). Caspase-8 activity was determined with a
colorimetric assay from R&D
Systems.
Adenoviral Constructs
AdTet-FLIP was constructed by inserting a DNA
cassette containing seven consecutive tetracycline-responsive elements
(TRE), a CMV minimal promoter, and SV40 polyA into the multicloning
site of adenovirus shuttle vector p
E1sp1A (Microbix Biosystems) to
create p1ATRE. A DNA fragment containing the FLAG-tagged
protein-coding sequence of human FLIP-L was then inserted downstream
from the TRE repeats and CMV minimal promoter of p1ATRE. Recombinant
E1-E3–deleted adenovirus constructs were generated by homologous
recombination with adenovirus genome plasmid PJM17 in human embryonic
kidney 293 cells. The adenoviral vector AdTet-LacZ encoding
ß-galactosidase (ß-gal) was generated by using p1ATRE, as
described previously.23 The
adenoviral vector AdCMV-rtTA encodes a chimeric transcription factor
composed of a mutant tetracycline repressor fused to the VP16
trans-activator
under control of the CMV promoter/enhancer. Replication-defective
adenovirus vectors expressing dominant-negative and constitutively
active forms of murine Akt from the CMV promoter have been described
previously.24 The
dominant-negative Akt mutant (Adeno-dnAkt) has alanine residues
substituted for threonine at position 308 and serine at position 473.
This protein functions as a dominant negative for
endogenous
Akt.25 The constitutively
active Akt (Adeno-myrAkt) has an in-frame fusion of the
c-src myristoylation sequence
to the N-terminus of the wild-type Akt coding sequence, thereby
targeting the fusion protein to the membrane. All recombinant Akt
vectors are fused in frame to the hemagglutinin (HA) epitope.
Ad-ß-gal expresses the LacZ
gene from the CMV
promoter.26 All viral
constructs were purified by CsCl gradient
ultracentrifugation. HUVECs were typically infected
with adenoviral constructs overnight, followed by replacement with
fresh medium with or without serum or test agent (wortmannin, LY294002,
or PD98059). After 6 hours in fresh medium, cells were harvested to
assay caspase-8 cleavage and activity. After 24 hours, cells were
harvested for RT-PCR and Western blot analyses (except for
caspase-8), and apoptosis was assessed at 48
hours.
X-Gal Staining for ß-Gal Expression
HUVECs were cotransfected with AdCMV-rtTA and
AdTet-LacZ with or without doxycycline (Dox, 300 ng/mL) at a
multiplicity of infection (MOI) of 2 or 10 for 24 hours. After
transfection, cells were washed with PBS twice and fixed with 2%
formaldehyde and 0.2% glutaraldehyde for 30 minutes
and then stained with
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
(X-Gal) solution for 2 hours at 37°C.
Western Immunoblot
Analysis
Cells were washed with PBS twice and harvested by
scraping. Cell lysates were prepared in cell lysis buffer (50
mmol/L Tris-HCl, pH 8.0, 20 mmol/L EDTA, 1% SDS, and 100
mmol/L NaCl). Protein concentration was determined by using a protein
assay kit (Bio-Rad). Ten to 20 µg of protein
extract was fractionated by SDS-PAGE and transferred to a
polyvinylidine difluoride membrane
(Immobilon-P, Millipore).
The membrane was blocked with T-PBS (1x PBS and 0.3%
Tween 20) containing 3% dry milk and incubated
with primary antibody overnight at 4°C. After three washes with
T-PBS, the membrane was incubated with secondary antibody (anti-mouse
IgG HRP conjugate) for 1 hour and then washed with 0.05%
Tween 20 in PBS. The immune complexes were
detected by enhanced chemiluminescence methods (ECL,
Amersham).
Cell Viability Assays
HUVECs were grown on gelatin-coated 2-well chamber
slides (Nalge Nunc International) at a density
of 2x105 cells per well. At the conclusion
of the treatment period, cells were washed with cold PBS twice and then
fixed with 3.7% formaldehyde in PBS for 20 minutes. After fixation,
the cells were washed with PBS twice and stained with Hoechst 33342.
Cells were analyzed for the appearance of pyknotic nuclei by
using a Nikon Diaphot microscope. Alternately,
apoptosis was monitored by measuring hypodiploid DNA content.
After various treatments, attached and floating
endothelial cells were harvested and fixed in cold 90%
ethanol for 20 minutes and then resuspended in staining buffer
consisting of 1 mg/mL RNaseA, 20 µg/mL propidium iodide, and 0.01%
NP40. DNA content was analyzed by flow cytometry on an FL-2
channel, and gating was set to exclude debris and cellular aggregates.
Ten thousand events were counted for each
analysis.
Caspase-8 Activity
Caspase-8 activity was measured by using the
caspase-8 colorimetric assay kit according to the
directions of the manufacturer (R&D Systems).
Cell lysates (from 1x106 cells) were
incubated with 5 mmol/L DTT and the caspase-8
colorimetric substrate that was conjugated to
IETD-p-nitroanaline in
reaction buffer for 2 hours at 37°C. Cleavage of the substrate was
determined by an increase in absorbance at 405
nm.
Results
VEGF Promotes FLIP Expression via PI
3-Kinase Signaling
Endothelial cells, such as HUVECs, are
normally resistant to Fas-mediated
apoptosis.7 14 15 16
HUVECs express high levels of FLIP protein compared with human VSMCs or
Jurkat cells
(Figure 1A
), which are more susceptible to Fas-mediated
apoptosis.15 As
shown in
Figure 1B, serum deprivation of HUVECs for 12
hours results in decreased FLIP protein expression. Exposure to
vascular endothelial growth factor (VEGF, 100 ng/mL)
restores FLIP expression levels to those seen in cultures incubated in
complete media, whereas coincubation with the PI 3-kinase
inhibitor wortmannin (200 nmol/L) abolishes the ability of
VEGF to promote FLIP expression
(Figure 1B). Wortmannin treatment in the absence of VEGF or
serum did not lead to a further reduction in FLIP expression (data not
shown). Because it has been proposed that VEGF promotes cell growth and
survival via both PI
3-kinase19 and MAP kinase
(MAPK)27 pathways, we
examined the effect of the MEK inhibitor PD98059 on FLIP
expression. As shown in
Figure 1C, PD98059 (3 µmol/L) had no effect on the ability
of VEGF to promote FLIP expression. PD98059 also had no effect on basal
FLIP expression levels.
|
Akt Signaling Regulates FLIP Expression and
Susceptibility to Fas-Mediated Apoptosis
To elucidate the role of Akt signaling on FLIP
expression, HUVECs were transfected with adenovirus vectors expressing
either the constitutively active Akt (Adeno-myrAkt) or the
dominant-negative Akt mutant (Adeno-dnAkt). Western
immunoblots were performed to assess levels of FLIP,
adenovirus-encoded Akt construct (anti-hemagglutinin [HA]), and
tubulin. Under conditions in which FLIP expression is reduced by
incubation with wortmannin, FLIP levels were restored when cells were
infected with Adeno-myrAkt
(Figure 2A). Similarly, myrAkt restored FLIP levels in HUVECs
exposed to serum-free medium or the PI 3-kinase inhibitor
LY294002
(Figure 2B). In contrast, suppression of Akt signaling by
infection with Adeno-dnAkt led to a decrease in basal FLIP expression,
whereas the control vector expressing ß-gal had no effect
(Figure 2C). In the absence of wortmannin, infection with
Adeno-myrAkt increased FLIP expression above basal levels
(Figure 2C).
|
To determine the role of PI 3-kinase/Akt signaling in
endothelial cell sensitivity to Fas-mediated
apoptosis, HUVEC survival after treatment with agonistic
anti-Fas antibody was assessed
(Figure 3). In these experiments, cultures were treated with
IFN-
to upregulate endogenous levels of cell surface Fas
expression.15 16
This cytokine alone does not affect endothelial
FasL expression (data not shown), nor does it promote Fas-mediated
apoptosis under normal culture conditions
(Figure 3). In contrast, treatment with wortmannin induced
apoptosis, and the frequency of apoptosis was further
increased when cells were incubated with wortmannin and anti-Fas
antibody. Similarly, transduction of dominant-negative Akt sensitized
the HUVECs to apoptosis in the presence of anti-Fas antibody
(data not shown). Transduction with myrAkt protected cells from
apoptosis induced by wortmannin and anti-Fas antibody (Figure 3).
Suppression of PI 3-kinase/Akt also sensitized the HUVEC cultures to
Fas-mediated apoptosis in the absence of IFN-, but the
overall level of apoptosis was less (data not shown). In
contrast, the MAPK inhibitor PD98059 did not promote cell
death, nor did it synergize with anti-Fas antibody to induce
apoptosis
(Figure 3). Collectively, these data suggest that inhibition
of PI 3-kinase/Akt signaling sensitizes endothelial
cells to Fas-mediated apoptosis.
|
Tetracycline-Regulated FLIP Expression
System
To examine the role of FLIP in the Fas-mediated
apoptosis of endothelial cells, a
tetracycline-inducible FLIP expression system was developed by using a
binary-adenovirus strategy
(Figure 4A). The first replication-defective adenovirus
encodes the transgene, either
FLIP-L or
LacZ, under the transcriptional
control of seven tetracycline operator sites (AdTet-FLIP). The second
vector expresses a chimeric transcription factor composed of a mutant
tetracycline repressor fused to the VP16
trans-activator
domain from the CMV promoter/enhancer (AdCMV-rtTA). This factor does
not efficiently
trans-activate
tetracycline operator sites under basal conditions, but the addition of
Dox, an analogue of tetracycline, results in maximal transgene
expression. The AdTet-LacZ vector, expressing ß-gal from the
tetracycline-regulated promoter, was used to evaluate inducible
expression with this system by using X-Gal
staining.23
|
FLIP expression from the AdTet-FLIP vector was
analyzed by Western blot analysis. As shown in
Figure 4B
, coinfection of HUVEC cultures with AdTet-FLIP and
AdCMV-rtTA, each at an MOI of 2, for 12 hours in the presence of 300
ng/mL Dox increased the level of FLIP that was slightly above the level
of endogenous expression. Increasing the levels of the
AdTet-FLIP vector by 5-fold (MOI 10) resulted in a dramatic
overexpression of FLIP under these assay conditions. Expression of
transgene-encoded FLIP was dependent on the level of Dox in the media,
and little or no expression was detected in the absence of this
drug.
To assess transduction efficiency on a cell-by-cell
basis, ß-gal expression from the AdTet-LacZ vector was monitored by
X-Gal staining of endothelial cells coinfected with
AdTet-LacZ (MOI 2 or 10) and AdCMV-rtTA (MOI 10). As shown in
Figure 4C, coinfection of AdTet-LacZ (MOI 2) and AdCMV-rtTA
(MOI 10) produced little or no detectable ß-gal expression in the
absence of Dox, but the addition of Dox led to the detection of
transgene expression in the majority of cells. In contrast, coinfection
of both vectors at an MOI of 10 led to detectable ß-gal expression in
some cells even in the absence of Dox, whereas robust expression was
observed in the majority of cells in the presence of
Dox.
FLIP Restoration Protects
Endothelial Cells Against Fas-Mediated
Apoptosis Under Conditions of Suppressed PI 3-Kinase
Signaling
To examine the role of PI 3-kinase signaling in the
control of Fas-mediated apoptosis in
endothelial cells, HUVEC cultures were assessed for the
number of pyknotic nuclei present after incubation with wortmannin
(200 nmol/L) or the agonist anti-Fas antibody (CH11)
(Figure 5). In these experiments, all cultures were
pretreated with INF- because this factor increases cell surface Fas
expression.15 16
As can be seen in the control lanes of
Figure 5, agonistic anti-Fas antibody did not promote
apoptosis in HUVECs, consistent with previous
reports,15 16
whereas treatment with wortmannin produced levels of cell death that
were significantly higher than those observed in control cultures. Of
particular importance, coincubation of cultures with wortmannin plus
anti-Fas antibody induced higher levels of apoptosis than were
observed in the presence of wortmannin alone. Similarly, higher levels
of apoptosis were detected in the presence of anti-Fas antibody
when HUVECs were infected with Adeno-dnAkt (data not shown).
Collectively, these data show that inhibition of PI 3-kinase/Akt
signaling will potentiate Fas-mediated death signals in
endothelial cells.
|
To examine the functional significance of FLIP
downregulation under conditions of suppressed PI 3-kinase and Akt
signaling, HUVECs were infected with AdTet-FLIP or AdTet-LacZ (MOI 2)
in the presence of AdCMV-rtTA (MOI 10) and Dox
(Figure 5). Restoration of FLIP expression had no effect on
the frequency of cell death induced by wortmannin alone. However,
exogenous FLIP completely blocked the increased apoptosis
observed when cells were incubated with anti-Fas antibody under
conditions of suppressed PI 3-kinase signaling. Parallel experiments
with AdTet-LacZ did not affect apoptosis frequencies in the
presence of wortmannin with or without anti-Fas antibody relative to
control, demonstrating that FLIP expression, and not the viral
expression system itself, was responsible for inhibiting Fas-mediated
apoptosis in HUVECs under conditions of suppressed PI 3-kinase
signaling.
FLIP Restoration Inhibits Depletion of
Procaspase-8 When Cells Are Treated by Wortmannin and Fas
Agonist
Cleavage of procaspase-8 is essential for
Fas-mediated
apoptosis.28
Therefore, depletion of procaspase-8 was assessed to corroborate that
FLIP restoration inhibits the Fas-mediated apoptosis pathway
under conditions of suppressed PI 3-kinase signaling. As shown in
Figure 6A, treatment with wortmannin or anti-Fas antibody
alone did not substantially change procaspase-8 levels, as assessed by
Western blot analysis. In contrast, the
simultaneous treatment of HUVECs with wortmannin and
anti-Fas antibody led to a marked decrease in procaspase-8 levels,
consistent with the propagation of a Fas-mediated death
signal. However, prior infection of HUVECs with AdenoTet-FLIP (MOI 2)
and AdenoCMV-rtTA (MOI 10 for each) blocked the depletion of
procaspase-8 by the combination of wortmannin and anti-Fas antibody.
The caspase-8 cleavage data were confirmed by a
colorimetric assay of caspase-8 activity
(Figure 6B). In this assay, wortmannin sensitized the cells
to caspase-8 activation by anti-Fas antibody, and this activation was
blocked when cells were transduced with
FLIP.
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Discussion
The PI 3-kinase/Akt signaling pathway is of central importance in endothelial cell biology. Recent reports have shown that this pathway is essential for endothelial cell differentiation,29 migration30 and NO production,31 32 33 key features of the angiogenic response. In addition, PI 3-kinase/Akt signaling also confers survival to endothelial cells in response to angiogenic cytokine stimulation,19 34 fluid shear stress,35 and matrix attachment signals.19 It is generally assumed that PI 3-kinase/Akt signaling promotes endothelial cell survival by suppressing the mitochondrial pathway of apoptosis via Akt-mediated phosphorylation of Bad and procaspase-9.20 In the present study, it is shown that this signaling pathway also blocks Fas-mediated apoptosis in endothelial cells.
It is well established that endothelial cells are normally resistant to Fas-mediated apoptosis.7 15 16 The present study demonstrates that Akt signaling is an important determinant of endothelial sensitivity to Fas-mediated death signals through its ability to modulate the expression of the caspase-8 inhibitor FLIP. It was shown that FLIP is downregulated under conditions that lead to diminished PI 3-kinase/Akt signaling in endothelial cells, including serum deprivation, wortmannin and LY294002 treatment, or transduction with dominant-negative Akt. Importantly, the downregulation of FLIP induced by treatment with PI 3-kinase inhibitors could be reversed by transduction of constitutively active Akt. In contrast, modulation of Akt signaling had no detectable effect on cell surface Fas expression on HUVECs, whereas FasL expression was modestly upregulated and downregulated by dominant-negative and constitutively active Akt, respectively (data not shown). The functional significance of PI 3-kinase/Akt signaling in endothelial cell sensitivity to Fas-mediated apoptosis was indicated by the observation that cells become sensitive to killing by a Fas agonist antibody in the presence of wortmannin or dominant-negative Akt. Similarly, the functional significance of FLIP downregulation under these conditions was demonstrated by experiments showing that FLIP transduction prevented caspase-8 activation and endothelial cell apoptosis under conditions of PI 3-kinase inhibition.
There has been considerable confusion and controversy
regarding the function of cellular FLIP. Confusion results, at least in
part, from the isolation of multiple FLIP isoforms that arise from
alternative splice patterns of a single gene. Moreover, the functions
of these proteins has been controversial, with some groups reporting
that they act as death activators and others finding that
they inhibit
apoptosis.12 36 37
Most of these studies have evaluated FLIP function by overexpressing
FLIP by transient transfection experiments, in which levels of FLIP
overexpression in the individually transduced cells were not assessed.
To address this issue, we established a tetracycline-regulated
adenoviral expression system the allowed us to assess the effects of
systematically varying FLIP expression levels on cell viability. In
this system, one adenoviral vector expresses FLIP downstream from seven
tetracycline operator sites, whereas the second adenoviral vector
expresses the tetracycline-dependent
trans-activator.
Under optimal induction conditions, this system can produce far more
robust transgene expression than is produced by conventional adenoviral
vectors that express transgenes from the CMV promoter, yet it is
relatively silent under noninduction
conditions.23 Relatively low
levels of FLIP overexpression, achieved with a viral titer of MOI 2,
protected endothelial cells from Fas-mediated
apoptosis under conditions of suppressed PI 3-kinase signaling.
Under these conditions, the majority of the cells were transduced with
adenovirus, and levels of exogenous FLIP were similar to
endogenous levels. Furthermore, exogenous FLIP did not
induce toxicity at these viral titers. However, higher levels of FLIP
expression (AdTet-FLIP MOI 
10) produced apoptosis in both
HUVECs and human VSMCs (data not shown). Under conditions of
superphysiological FLIP expression, toxicity was
associated with cleavage of procaspase-8 (data not shown). Taken
together, these data suggest that excessive FLIP overexpression may
lead to activation of caspase-8 because of forced aggregation and
cleavage of procaspase-8 molecules, which display a low level of
intrinsic caspase
activity.38
These data provide a mechanistic rationale that may explain previous observations of antagonism between Akt/PI 3-kinase and Fas signaling pathways. For example, Gibson et al39 have shown that epidermal growth factor stimulation protects epithelial cell lines from Fas-mediated caspase activation and apoptosis. In addition, PTEN heterozygous mutant mice display impaired Fas-mediated apoptosis and develop a polyclonal autoimmune disorder.40 Because PTEN encodes a phosphatase that opposes the PI 3-kinase reaction, these data implicate PI 3-kinase/Akt signaling as a negative regulator of Fas-mediated apoptosis. Further investigation is required to determine whether the regulation of FLIP can account for the PI 3-kinase/Akt–dependent suppression of Fas-induced apoptosis in these systems, although it has recently been shown that Akt signaling regulates FLIP expression in tumor cells.41
Our observations are also relevant for endothelial cell biology because multiple angiogenic factors activate PI 3-kinase/Akt signaling,29 30 34 and endothelial cells are normally resistant to Fas-mediated apoptosis, although they express functional FasL on their cell surface.15 16 17 Thus, decreases in FLIP expression, which are due to diminished PI 3-kinase/Akt signaling, may lead to blood vessel regression. For example, it has been shown that the matrix-derived angiogenesis inhibitor canstatin specifically induces apoptosis in endothelial cells, and this toxicity has been correlated with a downregulation of FLIP.42 Furthermore, FLIP expression is diminished when endothelial cells are exposed to oxidized lipids,14 a condition that is associated with impaired blood vessel growth.43 44 45 46 Therefore, the regulatory mechanism described in the present study may be significant for maintenance of the endothelium and blood vessel growth.
Acknowledgments
This manuscript was supported by grants AG-15052, AR-40197, and HD-23681 to Dr Walsh. We thank Roy C. Smith for helpful comments.
Footnotes
Original received December 15, 2000; revision received April 30, 2001; accepted May 1, 2001.
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