© 1999 American Heart Association, Inc.
Original Contribution |
Regulation of Vascular Smooth Muscle Cell Apoptosis
Modulation of Bad by a Phosphatidylinositol 3-Kinase–Dependent Pathway
From the Molecular Vascular Cell Biology Research Laboratory, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St, Boston, Mass.
Correspondence to Gary H. Gibbons, MD, Cardiovascular Research Institute, Morehouse School of Medicine, 720 Westview Dr SW, Atlanta, GA 30310-1495.
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Abstract |
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Abstract—Our objective was to define the signaling mechanisms by which mitogens such as insulin-like growth factor-I (IGF-I) regulate vascular smooth muscle cell (VSMC) apoptosis. We confirmed that IGF-I inhibits serum withdrawal–induced apoptosis of cultured VSMCs in a dose-dependent and time-dependent fashion. To test the hypothesis that the phosphatidylinositol (PI) 3-kinase signaling pathway regulates VSMC survival, we examined the relationship between PI 3-kinase activity and cell fate. PI 3-kinase was elevated in viable VSMCs maintained in serum-containing medium, declined significantly in response to serum withdrawal, and increased in response to IGF-I–induced survival. Moreover, blockade of PI 3-kinase with 2 structurally dissimilar inhibitors (wortmannin or LY294002) abolished the capacity of IGF-I to maintain VSMC viability. Similarly, transient transfection of a dominant-negative
p85 PI 3-kinase mutant construct abrogated
the capacity of IGF-I to prevent VSMC death. Thus, PI 3-kinase is a
critical antiapoptotic signal in VSMCs. To define the distal
element of the antiapoptotic cascade, we tested the hypothesis
that IGF-I inhibits the influence of the proapoptotic gene Bad.
Indeed, IGF-I stimulates increased expression of the inactive,
phosphorylated form of Bad by a PI 3-kinase–dependent
pathway. Moreover, the proapoptotic effect of Bad was
attenuated by the stimulation of IGF-I. Thus, growth factors appear to
prevent VSMC death by activating signal transduction pathways linked to
apoptotic regulatory genes.
Key Words: vascular smooth muscle cell • insulin-like growth factor-I • phosphatidylinositol 3-kinase • programmed cell death • Bad
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Introduction |
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The pathogenesis of various forms of vascular disease involves an abnormal accumulation of cells within the intimal space. Although many studies have focused on alterations in the regulation of cell growth as a fundamental feature in the pathogenesis of vascular disease,1 it is becoming clear that perturbations in the regulation of cell death may be equally important.2 3 4 5 6 7
An emerging theme suggests that many of the regulatory mechanisms originally characterized as governing cell growth also modulate cell death.2 3 It is postulated that the increased expression of growth factors observed within vascular lesions may promote an increase in the vascular smooth muscle cell (VSMC) population by preventing apoptosis as well as enhancing cell cycle progression. Indeed, recent studies by Bennett et al8 have documented that growth factors such as insulin-like growth factor-I (IGF-I) and platelet-derived growth factor-BB are potent inhibitors of VSMC death. However, the molecular basis by which growth factors prevent VSMC death remains to be further defined.
A recent report has demonstrated that phosphatidylinositol (PI) 3-kinase is involved in mediating the antiapoptotic effects of nerve growth factor in rat pheochromocytoma cell lines.9 However, given the cell-type specificity of apoptosis regulation, it remains unclear whether PI 3-kinase plays a role in regulating VSMC fate. Moreover, the downstream elements of this antiapoptotic signaling pathway remain to be further defined. It is postulated that the signal transduction apparatus of membrane receptors must be coupled to the regulation of apoptosis regulatory genes in the Bcl-2 family. Cell fate is determined by the relative balance of proapoptotic members of the Bcl-2 family, such as Bad, versus antiapoptotic mediators, such as Bcl-XL.10 One current working model suggests that cell suicide is induced when the proapoptotic mediator Bad forms heterodimers with Bcl-XL and thereby inactivates the antiapoptotic functions of Bcl-XL. The signal transduction pathway that regulates the dimerization of apoptosis regulatory genes remains to be further elucidated. The present study tests the hypothesis that the regulation of VSMC survival is mediated by a PI 3-kinase–dependent signal transduction pathway that modulates the proapoptotic effects of Bad.
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Materials and Methods |
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Cell Culture
Human umbilical artery smooth muscle cells were purchased from Clonetics Corp and were grown according to the manufacturer's instructions. Passages 5 to 7 were used for experimentation. The clonal embryonic rat thoracic VSMC line A7r5 was obtained from American Type Culture Collection. In addition, we have established several polyclonal lines of neomycin-selected A7r5 transfected with a retroviral construct to constitutively overexpress Bcl-2.11 These Bcl-2 stable transfectants were used in the experiments involving transient transfection of the Bad expression construct. VSMCs were maintained in 75-cm2 polystyrene flasks in DMEM/F-12 HAM (GIBCO-BRL) supplemented with 10% heat-inactivated FBS (GIBCO-BRL), penicillin (100 U/mL), streptomycin (100 mg/mL), and 25 mmol/L HEPES buffer.
Experimental Protocols
Human recombinant IGF-I (R&D Systems) was supplemented to the
serum-free medium at various concentrations (0, 0.1, 1, 10, and 100
ng/mL). In the experiments to determine the time window of the
stimulus, IGF-I or a vehicle control was added at different time points
(0, 4, and 8 hours) after serum withdrawal. Two structurally dissimilar
inhibitors of PI 3-kinase were used to selectively inhibit
the PI 3-kinase activity, wortmannin (Sigma Chemical Co) and LY294002
(Biomol). Previous studies have established the specificity of PI
3-kinase blockade with these agents in the dose range used in this
study.9 12 13 In pilot studies, we have also confirmed
blockade of VSMC PI 3-kinase in our model system in this dose range. On
the basis of these studies, we observed that the optimal
inhibitory concentrations were 100 nmol/L wortmannin and
50 µmol/L LY294002 administered 30 minutes before the addition
of IGF-I in VSMCs. Each drug was dissolved in DMSO, and the final
concentrations of DMSO did not exceed 0.1%. This concentration of DMSO
had no direct effect on cell viability. In some experiments with
wortmannin, it was added every 4 hours to the medium to achieve
sustained blockade given the short half-life of its biological
efficacy.12 In each experiment, cells were treated with a
DMSO vehicle control. As an additional control in the experiments with
LY294002, comparisons were also made with cells treated with the
structurally related but inactive analogue LY298619 (kindly provided by
Lilly Research Laboratory).13
Determination of Apoptosis
Nuclear chromatin morphology analysis and caspase-3
activity assay were used as quantitative indices of apoptosis.
Nuclear chromatin morphology analysis has been extensively
validated and cross-correlated with other complementary assays of
apoptotic cell death in our laboratory, including time-lapse
video microscopy, DNA electrophoresis, terminal
deoxynucleotidyltransferase-mediated
dUTP nick-end labeling (TUNEL) staining, caspase-3 activity assays, and
fluorescence-activated cell sorting
analysis.14 Cells to be analyzed for
apoptosis by nuclear chromatin morphology were stained with
H33342 and PI (Molecular Probes, Inc) and viewed under
fluorescence microscopy at 12 hours after serum withdrawal.
Using both membrane-permeable (H33342) and -impermeable (PI) dyes in
the assay allows for the determination of cell viability and plasma
membrane integrity. The proportion of cells rescued by IGF-I treatment
(% rescue) was calculated as the percentage of apoptotic cells
under serum-free conditions minus the percentage of the
apoptotic cells after IGF-I treatment divided by the percentage
of apoptotic cells under serum-free conditions.
Caspase-3 activity assay was performed according to the manufacturer's protocol (Clontech Laboratory, Inc). The caspase-3 activity was determined by assay of each sample normalized to the basal activity in the viable cells maintained in serum-containing medium.
PI 3-Kinase Assay
The measurement of PI 3-kinase activity was performed as
previously described.15 VSMCs were incubated with cell
lysis buffer (in mmol/L, HEPES [pH 7.5] 25, NaCl 50, NaF 50,
EDTA 5, okadaic acid 10, sodium orthovanadate 1, and
phenylmethylsulfonyl fluoride 1; 1% NP-40; and 1 µg/mL each
of antipain, aprotinin, and leupeptin) for 10 to 15 minutes on ice.
Insoluble material was removed by centrifugation at
12 000g for 20 minutes. The amount of cell lysate was
normalized by protein content in each experiment (Bio-Rad
Laboratories). The cell lysate (0.5 mg of protein) was incubated with
an antibody against the p85
subunit of PI 3-kinase (2 µg/mg of
protein) (Upstate Biotechnology) with constant agitation for 3 hours
and then further incubated with 25 µL of protein A/G plus agarose
(Santa Cruz Biotechnology) with agitation for 1 hour. The beads
containing the immunoprecipitates were washed 4 times with lysis buffer
and washed once with buffer (0.1 mol/L NaCl, 1 mmol/L EDTA, and
20 mmol/L Tris-HCl, pH 7.5). The washed pellets were resuspended
in 50 µL of kinase reaction buffer (in mmol/L, Tris-HCl [pH
7.5] 20, NaCl 100, and EGTA 0.5) and incubated at 25°C for 10
minutes with 0.5 mL of 20 mg/mL PI dissolved in chloroform to make
micelles of PI. Assays were initiated with the addition of 5 µL of
ATP solution (0.4 µmol/L ATP, 0.1 mol/L
MgCl2, and 1 µCi/mL
[
-32P]ATP) and incubated at room temperature
for 30 minutes. The reaction was stopped after the addition of 100 µL
of chloroform, methanol, and 11.6N HCl (100:200:2). After
centrifugation, the lower organic phase was taken for
thin-layer chromatography on silica gel plates
(Aldrich Chemical Co) and developed in chloroform, methanol, 25%
ammonium hydroxide, and water (43:38:5:7). The plate was exposed to
Kodak X-Omat film at –70°C with an intensifying screen and
quantified by computer-enhanced video densitometry using the NIH image
analysis program.
Plasmid Constructs and Transfection
To determine whether PI 3-kinase is an essential signaling
molecule mediating the inhibition of apoptosis, a
dominant-negative mutant bovine 85-kDa subunit of PI 3-kinase (p85)
that lacks the binding site for the 110-kDa subunit of PI 3-kinase was
overexpressed in VSMCs by transient transfection. An expression vector
containing a promoter, which is composed of the simian virus 40
(SV40) early promoter and the R segment and part of the U5 sequence of
the long terminal repeat of human T-cell leukemia virus type I (SR),
and the p85 PI 3-kinase mutant construct was kindly provided by Dr
W. Ogawa (Kobe University, Japan). Several previous studies have
documented that overexpression of the dominant-negative p85 PI
3-kinase mutant selectively interferes with endogenous PI
3-kinase function.16 17 To investigate the
proapoptotic effect of Bad, 6-amino acid epitope of the bovine
papilloma virus capsid protein (AU1)-tagged wild-type Bad
construct subcloned into a pCDNA3 vector (a gift from Dr G. Nunez,
University of Michigan, Ann Arbor)18 was transiently
transfected into VSMCs stably expressing Bcl-2. The A7r5 cells stably
transfected with a retroviral construct containing Bcl-2 exhibit normal
viability for >24 hours despite serum withdrawal and therefore provide
a means to assess the effects of Bad on cell fate under serum-free
conditions in the presence or absence of IGF-I stimulation.
Transient transfection was performed with 2 µg of total DNA per 35-mm
dish and LipofectAMINE (GIBCO-BRL) according to the manufacturer's
instructions (DNA:LipofectAMINE ratio, 1:3). Pilot studies have
documented a transfection efficiency of 
15% to 25% in A7r5 VSMCs
using this approach. Serum withdrawal was performed to induce
apoptosis in the presence or absence of IGF-I 48 hours after
transfection. Cells transfected with an expression vector containing
the same promoter but without the subcloned transgene cassette (empty
expression vector) served as a control. The effect of the transgene on
cell viability was determined by assaying the activity of a
cotransfected reporter gene19 20 21 or nuclear morphology
with H33342 in the cells coexpressing the reporter gene, as previously
described.22 23 Briefly, VSMCs were cotransfected with an
expression vector containing the reporter gene luciferase driven by an
SV40 promoter (Promega), along with transfection of either the p85
PI 3-kinase mutant transgene or the wild-type Bad expression construct,
versus the control vector at a 3:1 transgene:reporter ratio. Cells that
were successfully transfected and remained viable were assayed by
measurement of luciferase activity using standard methods with a
luminometer. In pilot studies conducted in VSMCs stably transfected
with Bcl-2, we confirmed that serum withdrawal failed to have a direct
inhibitory effect on reporter gene activity in viable cells
using this constitutively active viral promoter construct. This assay
of luciferase activity provides an objective, sensitive, and
quantitative index of cell viability among the subpopulation of
transfected cells under these conditions. The proportion of viable
transfected cells after serum withdrawal was determined by assay of
luciferase activity in this group in relation to the basal level of
luciferase activity expressed in transfected cells maintained in
serum-containing medium. We also used the expression vector containing
the reporter gene green fluorescent protein (GFP) driven by the
cytomegalovirus promoter (GIBCO-BRL) to confirm that the loss of
reporter gene expression in response to serum withdrawal in transfected
VSMCs is due to apoptotic cell death, as assessed by nuclear
chromatin morphology with H33342.22 23 The
characterization of nuclear morphology in GFP-positive cells provides
an assessment of the effect of the transgene under study on cell fate
using a single-cell analysis approach.
Immunoblot Analysis
Cells were harvested 6 hours after serum withdrawal in the
presence or absence of IGF-I (100 ng/mL) and lysed in a modified
radioimmunoprecipitation assay buffer (in mmol/L, NaCl 150, Tris
[pH 7.4] 10, EDTA 1, and PMSF 1, as well as 1% Triton X-100 and 1%
deoxycholic acid, with the addition of a complete protease
inhibitor mixture [Boehringer Mannheim]). To
assess the role of PI 3-kinase, cells were pretreated with wortmannin
(100 nmol/L), LY294002 (50 µmol/L), or a DMSO vehicle at the
time of serum withdrawal. Protein concentrations were determined and
normalized as above. Cell lysates (40 µg protein/sample) were run in
a 15% SDS-PAGE under DTT-reducing conditions. Proteins were then
transferred to a nitrocellulose filter by semidry blotting in (in
mmol/L) Tris 25, glycine 200, and SDS 1.3, and 20% methanol (pH 8.3)
at 160 mA for 3 hours. The filters were stained with 10% Ponceau S
solution (Sigma) for 10 minutes to verify equal loading and transfer
efficiency. The filters were then blocked in 10% dry milk for 1 hour
at room temperature and probed with polyclonal antibodies against Bad
(catalog No. sc-941, Santa Cruz Biotechnology) (1 µg/mL)
overnight at 4°C. After washing with TBS-T (20 mmol/L Tris,
137 mmol/L NaCl, and 0.1% Tween 20), signals for Bad expression
were detected by horseradish peroxidase–conjugated secondary antibody
(Transduction Laboratories) for 1 hour at room temperature. The
specificity of the signal was confirmed by the absence of signal when
membranes were incubated with nonimmune IgG. After extensive washing in
TBS-T, electrochemical luminescence was developed (Amersham), and
membranes were exposed to Hyperfilm-ECL (Amersham) and quantified by
computer-enhanced video densitometry using the NIH image
analysis program.
Bad Phosphorylation
Previous studies have established that Bad is
phosphorylated on serine residues and that this
phosphorylated form of Bad migrates as a retarded band
on gel electrophoresis relative to the
nonphosphorylated form.20 21 22 We observed
both the phosphorylated and
nonphosphorylated forms of Bad endogenously
expressed in VSMCs maintained in serum as determined by a gel mobility
shift assay. To confirm that the retarded band on gel electrophoresis
reflected serine phosphorylation, we assessed the
effect of in vitro treatment of cellular extracts with a protein
phosphatase (Lambda Protein Phosphatase, New England Biolabs Inc) at
37°C for 30 minutes, in accord with previous reports in nonvascular
cells.20 21 22 Furthermore, as an additional verification,
we also analyzed VSMCs overexpressing Bad after transient
transfection of AU1-tagged Bad constructs. Modulation of Bad
phosphorylation in VSMCs was determined by sequential
immunoprecipitation with an anti-AU1 antibody (BAbCO Co) and
immunoblotting with a phosphospecific (serine 136)
anti-Bad antibody (New England Biolabs, Inc). The anti-Bad monoclonal
antibody (Transduction Laboratories) was used to determine the total
Bad protein expression.
Statistical Analysis
Each experimental condition was tested in triplicate, and
each experiment was repeated a minimum of 3 times. Statistical
analyses were performed by ANOVA or unpaired 2-tailed Student
test. Data are presented as mean±SD.
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Antiapoptotic Effect of IGF-I on VSMCs
First, we confirmed a previous report that IGF-I is a potent antiapoptotic factor for human VSMCs.8 In addition to the morphological features of apoptotic cell death, we observed that human VSMCs maintained in serum-free medium showed a marked increase of caspase-3 activity within 24 hours (5-fold increase of caspase-3 activity in serum-free versus serum control; P<0.001). However, the addition of IGF-I (100 ng/mL) in the serum-free medium significantly inhibited caspase-3 activation (1.8±0.6, with IGF-I, versus 5.1±0.3, serum free; P<0.001). To further elucidate the molecular mediators regulating VSMC fate, we initiated our studies by establishing an in vitro model system using a phenotypically stable, clonal cell line that was amenable to the use of recombinant DNA approaches. We observed that the rat A7r5 clonal VSMCs grown to near confluence in 10% FBS undergo apoptosis when switched to serum-free medium. Using a quantitative nuclear chromatin morphology assay, we determined that 20% of cells underwent cell suicide within 12 hours under these conditions (Figure 1
). However, the
addition of IGF-I at the time of serum withdrawal inhibited
apoptosis in a dose-dependent manner, with significant effects
noted at nanomolar concentrations (Figure 1).
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P<0.01, IGF-I 10 ng/mL vs 1 ng/mL;
P<0.01, IGF-I 100 ng/mL vs 10 ng/mL. The results
shown are representative (mean±SD) of 3 independent
experiments.
We postulated that a critical time window exists in which the
activation of a countervailing antiapoptotic signaling cascade
is sufficient to prevent cellular suicide. We therefore examined
whether the survival-promoting effects of IGF-I are a time-dependent
phenomenon. Accordingly, cell viability was assessed in response to
adding IGF-I or a vehicle control at different time points after serum
withdrawal as shown in Figure 2. The
addition of IGF-I (100 ng/mL) at 4 hours after serum withdrawal had
survival-promoting effects similar to those of IGF-I administration at
the time of serum withdrawal (time 0). However, the addition of IGF-I
at 8 hours after serum withdrawal failed to rescue a significant
portion of VSMCs committed to cell suicide compared with IGF-I
administration at 4 hours after serum withdrawal. These findings
suggested that VSMC survival may depend on the activity level of an
antiapoptotic signaling pathway during a critical time window
between 4 and 8 hours after serum withdrawal.
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IGF-I Induces PI 3-Kinase Activation in VSMCs
To further define the molecular mediators of VSMC survival, we
tested the hypothesis that the activity of the PI 3-kinase pathway
determines VSMC viability. According to our analysis of the
time course kinetics of VSMC death, we postulated that an
antiapoptotic signal active in the presence of serum may decay
in activity over the course of 4 to 8 hours after serum withdrawal.
Indeed, as shown in Figure 3, analysis of PI 3-kinase activity by thin-layer
chromatography documented high levels in
serum-containing medium and a substantial decline that is evident
within 4 hours after serum withdrawal. Moreover, the addition of IGF-I
stimulated a 5.6-fold increase in PI 3-kinase activity within 5 minutes
of administration compared with control (n= 5, P<0.01). The
IGF-I–induced activation of PI 3-kinase activity was maintained for at
least 40 minutes. Similar results were obtained in both human and rat
VSMCs. These results indicate a striking association between VSMC
survival and the activity of the PI 3-kinase signaling pathway.
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Antiapoptotic Signaling Role of PI 3-Kinase:
Pharmacological Blockade
On the basis of this apparent association between VSMC survival
and PI 3-kinase activity, we directly tested the hypothesis that the
survival-promoting effects by IGF-I are mediated via the activation of
a PI 3-kinase–dependent signal transduction pathway. We used 2
structurally dissimilar pharmacological probes (wortmannin and
LY294002)12 13 to selectively inhibit PI 3-kinase activity
and define the role of this cellular mediator as a determinant of
IGF-I–stimulated VSMC survival.
As compared with the induction of apoptosis in control rat VSMCs exposed to serum withdrawal, IGF-I treatment rescued 78±14% stimulated to undergo cell suicide. However, IGF-I treatment failed to rescue a substantial portion of VSMCs from apoptotic death in the context of PI 3-kinase pathway blockade with wortmannin (78±14% rescue in DMSO vehicle-treated cells versus 28±7% rescue with wortmannin treatment; P<0.001). To further confirm that these findings reflect a specific response to blockade of PI 3-kinase, we also used the structurally dissimilar PI 3-kinase inhibitor LY294002. In accord with the results obtained with wortmannin, blockade of PI 3-kinase activation with LY294002 significantly inhibited the capacity of IGF-I to promote VSMC survival (67±16% rescue in vehicle-treated cells versus 37±16% rescue with LY294002 treatment, P<0.01). As an additional experimental control to confirm the specificity of the response, we observed that an inactive analogue of LY294002, LY298619, did not have any effect on IGF-I–induced VSMC survival.
In addition, we confirmed that the antiapoptotic effect of
IGF-I and the reversal by pharmacological blockade of PI 3-kinase were
also observed in human VSMCs. As shown in Figure 4, IGF-I significantly inhibited the
activation of caspase-3 activity in response to serum withdrawal
(5.1±0.3-fold increase of caspase-3 activity in vehicle-treated
serum-free conditions versus 1.8±0.6-fold with IGF-I;
P<0.01). However, the blockade of PI 3-kinase completely
abolished the antiapoptotic effect of IGF-I on caspase-3
activity (1.8±0.6-fold with IGF-I versus 5.7±0.8 with
IGF-I+wortmannin; P<0.01). Although there was a slight but
significant potentiating effect of wortmannin on the induction of
caspase activity in response to serum withdrawal (5.1±0.3-fold in
vehicle-treated serum-free conditions versus 6.4±0.7 with wortmannin;
P<0.05), the level of caspase-3 activation was not
significantly different in wortmannin-treated cells exposed to IGF-I
compared with wortmannin-treated vehicle controls (5.7±0.8-fold with
IGF-I+wortmannin versus 6.4±0.7-fold in vehicle-treated serum-free
conditions with wortmannin; P>0.05). These data indicate
that IGF-I–induced survival-promoting effects are mediated by PI
3-kinase in both human and rat VSMCs.
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Dominant-Negative PI 3-Kinase Mutant Expression Inhibits
IGF-I–Induced Antiapoptotic Effects
To further confirm the mediator role of PI 3-kinase as a
determinant of VSMC survival induced by IGF-I, we also used a gene
transfer strategy to selectively block the activity of the PI 3-kinase
pathway. Previous studies have established that overexpression of the
dominant-negative p85 mutant construct of PI 3-kinase results in the
suppression of endogenous PI 3-kinase
function.15 16 To assess the effect of this transgene on
VSMC fate, we used a cotransfection protocol with an expression vector
containing the reporter gene luciferase to focus on the assessment of
cell viability among the subpopulation of successfully transfected
cells. The comparison of reporter gene expression between VSMCs
cotransfected with the PI 3-kinase p85 mutant construct versus a
control expression vector provides a means of assessing the role of
this signaling pathway on cell viability in response to serum
withdrawal.
Rat VSMCs cotransfected with the reporter gene luciferase and a control
vector exhibited preserved cell viability, as indicated by high
luciferase activity in serum-containing medium. However, the withdrawal
of serum induced cell death, as reflected by a significant decrease in
luciferase activity of 58% (Figure 5).
The induction of cell death in these control vector-transfected cells
was attenuated by the administration of IGF-I as indicated by the
significantly higher level of luciferase activity in IGF-I–treated
cells compared with those in the serum-free condition (58±11% in
serum-free conditions versus 79±8% with IGF-I; n=9,
P<0.01). In contrast to the antiapoptotic effect of
IGF-I stimulation observed in cells transfected with the control
vector, cotransfection with the dominant-negative p85 mutant PI
3-kinase abolished the capacity of IGF-I to preserve VSMC viability
under serum-free conditions.
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Taken together, these findings using a gene transfer experimental strategy complement and confirm the pharmacological studies and provide corroborating evidence that IGF-I promotes VSMC survival via a PI 3-kinase–dependent signaling pathway. However, the link between this signal transduction pathway and the downstream elements of the cell death program governing VSMC fate remained to be defined.
IGF-I Phosphorylates Bad via PI
3-Kinase–Dependent Pathway
It is postulated that extracellular factors may promote cell
survival by stimulating the phosphorylation of Bad,
reduce Bad–Bcl-2 heterodimer formation by inducing this
post-translational modulation, and thereby potentiate the effects of
antiapoptotic mediators such as Bcl-2.
Given that the expression pattern of members of the Bcl-2 family is
cell specific, we initially explored whether VSMCs express Bad. As
shown in Figure 6A, immunoblots of Bad protein expression confirmed that VSMCs
maintained in serum-containing growth medium exhibited a doublet of
24 to 25 kDa, which is consistent with the retarded mobility
of the phosphorylated form as well as a
faster-migrating nonphosphorylated form of Bad, as
previously described in nonvascular cells.21 We confirmed
that the upper band reflected retarded mobility because of protein
phosphorylation in the samples stably expressing Bad
phosphorylation by in vitro treatment of cellular
extracts with a protein phosphatase. As shown in Figure 6B, in
vitro treatment with the phosphatase eliminated the retarded band
signal.
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In contrast to the cells in serum, VSMCs maintained in serum-free
medium for 6 hours exhibited substantially less expression of the
inactive, phosphorylated form of Bad (Figure 6A
). Moreover, IGF-I treatment of VSMCs in serum-free medium
increased the inactive, phosphorylated form of Bad to
the levels comparable with the serum-stimulated baseline. On the basis
of the evidence that PI 3-kinase mediates the IGF-I–induced
antiapoptotic signals in VSMCs, we tested the hypothesis that
this signaling pathway is coupled to the
phosphorylation of Bad. Indeed, blockade of PI 3-kinase
activity with either wortmannin or LY294002 attenuated the increase in
expression of the phosphorylated form of Bad in
response to IGF-I stimulation (Figure 6A). The densitometric
analysis revealed that IGF-I stimulation increased the
expression of the phosphorylated form of Bad by 160%
(n=4, P<0.05) but had no significant effect on the overall
protein expression of Bad.
We further explored the capacity of extracellular stimuli to regulate
VSMC fate by modulating the proapoptotic activity of Bad using
a gene transfer experimental approach. As a complementary strategy to
assess the effect of IGF-I on Bad phosphorylation, we
transiently transfected rat A7r5 VSMCs stably overexpressing Bcl-2 with
an epitope-tagged Bad construct that enabled us to perform sequential
immunoprecipitation using the anti-AU1 antibody followed by
immunoblot with the phosphospecific anti-Bad antibody that
selectively recognizes the phosphorylated epitope at
serine 136. In addition, total Bad expression was assessed by
immunoblot using the same anti-Bad antibody used in Figure 6A and 6B. As shown in Figure 6C, IGF-I treatment of
transfected cells under serum-free conditions induced significant
phosphorylation of Bad as demonstrated by the increased
retarded mobility of the doublet. Furthermore, blockade of the PI
3-kinase signaling pathway attenuated the IGF-I–induced
phosphorylation of Bad. This observation was confirmed
using the phosphospecific anti-Bad antibody. As shown in Figure 6D, IGF-I treatment induced serine
phosphorylation of Bad, and blockade of the PI 3-kinase
signaling pathway attenuated the IGF-I–induced serine
phosphorylation of Bad. We confirmed that neither
stimulation with IGF-I nor blockade of PI 3-kinase activity modulated
the total expression levels of Bad protein. Overall, these data provide
further evidence that serine phosphorylation of Bad is
regulated in a PI 3-kinase–dependent manner in VSMCs.
IGF-I Modulates the Proapoptotic Effect of Bad
The working hypothesis poses that VSMC survival depends on the
interplay between the proapoptotic mediator Bad and
antiapoptotic factors such as Bcl-2. As noted above, treatment
of VSMCs with IGF-I is associated with the serine
phosphorylation of Bad by a PI 3-kinase–dependent
pathway. However, it remained to be determined whether the
IGF-I–induced phosphorylation of Bad influences the
proapoptotic activity of Bad on the fate of VSMCs. To examine
whether the proapoptotic activity of Bad is modulated by
extracellular stimuli such as IGF-I, we assessed the effect of
transient transfection of Bad in rat A7r5 cells stably overexpressing
Bcl-2. This experimental strategy takes advantage of the fact that
these Bcl-2 stable transfectants do not undergo apoptosis under
serum-free conditions for 24 hours,11 and therefore the
modulatory influence of IGF-I on Bad-induced cell death can be
determined. These experiments used a cotransfection protocol with the
GFP reporter gene to quantify apoptosis among the subpopulation
of successfully transfected cells. Figure 7A illustrates GFP staining in a
microscopic field and concomitant analysis of nuclear chromatin
morphology. In VSMCs stably overexpressing Bcl-2, we observed preserved
cell viability after 24 hours of serum withdrawal to levels comparable
with those of cells maintained in serum. However, transfection with the
Bad expression vector construct abrogated the survival-promoting
influence of Bcl-2 and induced apoptosis in 31% of cells under
serum-free conditions (31±4% Bad transfected in serum-free conditions
versus 5±1% Bad transfected in serum; P<0.01).
Furthermore, the proapoptotic effect of Bad transfection was
significantly attenuated by IGF-I treatment (31±4% Bad transfected in
serum-free conditions versus 15±2% Bad transfected with IGF-I; n=6,
P<0.01). Thus, the IGF-I-induced serine
phosphorylation of Bad is associated with an
attenuation of its proapoptotic effects on VSMC fate.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we have characterized a signal transduction pathway involved in regulating VSMC apoptosis. Our findings indicate that activation of PI 3-kinase plays a critical mediator role in preventing VSMC death. Moreover, we document for the first time that VSMCs express the proapoptotic mediator Bad and that the inactivation of Bad by serine phosphorylation is mediated by a PI 3-kinase–dependent pathway in these cells. Overall, these data provide evidence for an antiapoptotic signal transduction pathway in VSMCs in which protein kinases involved in generating second messengers are coupled to apoptosis regulatory genes that govern cell fate.
A rapidly emerging body of evidence suggests that vascular remodeling and lesion formation are determined in large part by the balance between cell proliferation and apoptotic cell death.2 4 5 6 We have previously shown that vasoactive substances such as angiotensin II may be important determinants of VSMC fate by modulating programmed cell death.14 Similarly, Bennett et al8 have documented that classic peptide mitogens such as IGF-I and platelet-derived growth factor-BB are also potent inhibitors of VSMC death. The present study has confirmed this observation in both human and rat VSMCs at physiological concentrations in a dose-dependent and time-dependent manner. However, the intracellular signaling pathways by which growth factors or vasoactive substances promote VSMC survival remain to be elucidated.
Several recent studies in neuronal and hematopoietic cell lines have suggested that a signal transduction pathway that includes the lipid kinase PI 3-kinase plays an important role in regulating cell death.9 24 However, it has become clear that the regulation of apoptosis is cell specific and contextual, and the role of these pathways as determinants of VSMC fate remained unclear. PI 3-kinase is a heterodimeric protein composed of 85- and 110-kDa subunits that catalyze the phosphorylation of the D3 portion of PI.25 The regulatory p85 subunit of PI 3-kinase has no intrinsic catalytic activity but forms complexes with activated growth factor receptors as well as adapter proteins such as IRS-1 and Shc via SH2 domains. The resultant association leads to a translocation of the catalytic p110 subunit from the soluble fraction to a vesicular fraction enriched in Golgi membranes. This maneuver enhances the catalytic activity of p110 and leads to the production of PI3-phosphate, PI(3,4)-biphosphate, and PI(3,4,5)-triphosphate. These lipids subsequently serve as second messengers for specific downstream signal transduction events such as receptor endocytosis, intracellular vesicular trafficking, and the regulation of cell survival.25
The present study has examined the IGF-I–induced
survival-signaling cascade in VSMCs and documented that it is mediated
by a PI 3-kinase–dependent pathway according to the following lines of
evidence: (1) IGF-I induces activation of PI 3-kinase, (2) 2
structurally dissimilar PI 3-kinase inhibitors abolished
IGF-I–induced survival effects, and (3) overexpression of a
dominant-negative p85 mutant construct of PI 3-kinase abolished the
capacity of IGF-I to preserve VSMC viability under serum-free
conditions. Taken together, these findings provide substantial evidence
that PI 3-kinase is an important antiapoptotic signaling
pathway in VSMCs. However, the downstream mediators of this survival
signal remained unclear.
Recent studies provide a rapidly growing body of evidence that indicates that Akt/PKB (protein kinase B) is a critical downstream effector of PI 3-kinase that promotes cell survival in nonvascular cells.19 26 Akt/PKB, the cellular homologue of the viral oncogene v-akt, encodes a serine/threonine protein kinase that is ubiquitously expressed and of which the catalytic domain is closely related to the catalytic domain of all the members of protein kinase C family. Akt/PKB differs in that it contains an N-terminal domain related in part to the pleckstrin homology domain.27 28 We speculate that Akt may be the critical distal element of the PI 3-kinase–dependent VSMC antiapoptotic signaling pathway. Studies are underway to determine the potential role of Akt/PKB as a determinant of VSMC fate.
It is now well established that members of the Bcl-2 family are critical regulators of apoptosis in a variety of cell types. The expression pattern and role of different members of the family appears to be cell specific.29 Our laboratory has recently demonstrated that vascular lesion formation is associated with an upregulation of the antiapoptotic gene Bcl-XL within intimal VSMCs in animal models and human specimens of vascular disease. Moreover, we have demonstrated that downregulation of Bcl-XL expression within intimal cells using antisense oligonucleotides induces VSMC apoptosis and regression of vascular lesions.30 These findings indicate that Bcl-XL is a critical determinant of intimal VSMC viability and lesion formation.
Bcl-XL and Bcl-2 appear to inhibit cell suicide by several mechanisms that include the following: binding to the caspase CED-4 (Apaf-1) to prevent the activation of the proteolytic caspase-mediated cell execution cascade, altering mitochondrial membrane potential, and inhibiting cytochrome c.31 32 33 The antiapoptotic activity of mediators such as Bcl-XL is modulated in part by the formation of heterodimers with proapoptotic members of the Bcl-2 family such as Bad, Bak, and Bax.10 It is postulated that these proapoptotic mediators may promote cell death in part by sequestering the antiapoptotic mediators such as Bcl-XL or Bcl-2. Recent studies have demonstrated that formation of Bad-Bcl-XL or Bad-Bcl-2 heterodimers is regulated by post-translational modifications of Bad. The phosphorylation of Bad at 2 sites, serine 112 and serine 136, substantially reduces its capacity to bind to Bcl-XL or Bcl-2. In addition, this serine phosphorylated form of Bad becomes associated with the docking protein 14-3-3.20 21 Thus, the phosphorylation of Bad promotes its sequestration from the apoptosis regulatory machinery and thereby plays a permissive role in promoting the antiapoptotic effect of Bcl-XL or Bcl-2. In hematopoietic cells, the cytokine interleukin-3 stimulates cell survival in association with phosphorylation of Bad at serine 112 and serine 136.21 Mutagenesis studies indicate that phosphorylation of these serine residues is a necessary condition for the antiapoptotic effect of interleukin-3 and that Akt/PKB is one of the serine kinases that may induce Bad phosphorylation.18 34 The present study has documented that VSMC survival is associated with phosphorylation of Bad and that the inactivation of Bad by this post-translational modification is mediated by a PI 3-kinase–dependent pathway in VSMCs. During the preparation of this manuscript, this observation was confirmed by reports using nonvascular cells.18 34 Furthermore, we observed that Bad inhibits the antiapoptotic influence of Bcl-2 in VSMCs and that this proapoptotic influence of Bad can be modulated by extracellular stimuli such as IGF-I.
In summary, the present study has defined an antiapoptotic signal transduction pathway that couples extracellular stimuli to the activity of apoptosis regulatory genes in VSMCs. Our data suggest a working model in which VSMC fate is critically dependent on a cell-survival signaling cascade mediated by PI 3-kinase, the generation of phosphoinositols, and activation of the serine kinase Akt. This receptor-activated, antiapoptotic signaling pathway is linked to the cell death program by its capacity to phosphorylate Bad and thereby reduce its capacity to inhibit antiapoptotic mediators such as Bcl-XL or Bcl-2. The findings of these in vitro experiments combined with our in vivo studies30 suggest that factors that modulate the expression level and/or activity of antiapoptotic genes such as Bcl-XL may play a fundamental pathogenic role in vascular disease by regulating VSMC apoptosis.
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Acknowledgments |
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The authors gratefully acknowledge grant support from the NIH, the American Heart Association, and the Pew Charitable Trusts.
Received January 15, 1999; accepted May 19, 1999.
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References |
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