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INTRODUCTION |
Angiotensin II (AngII)1
exerts its biological effects via the activation of two different
receptors known as AT1 and AT2 receptors both
belonging to the G protein-coupled receptor family (1, 2). The high
levels and transient expression of AT2 receptors in many
neonatal tissues and some pathological states such as vascular injury
and myocardial infarction have led to the postulate that this receptor
has a role in the cardiovascular development and remodeling.
AT1 receptor stimulates proliferation, whereas AT2 receptor exerts growth inhibitory effects both in
cultured cells as well as in vivo (3-6).
One of the growth inhibitory effects of AT2 receptor is
apoptosis (4). Despite growing interest in AT2
receptor-mediated apoptosis, relatively little is known about the
molecular basis of this process. It has been shown that ERK
inactivation by AT2 receptor results in Bcl-2
dephosphorylation, which may contribute to apoptosis (6). The potential
significance of AT2 receptor-induced apoptosis led us to
explore further the signaling mechanism of this process in this study.
Several lines of evidence suggest that sphingolipids are important
mediators of apoptosis (7). We therefore considered the possibility
that SLs would play a role in the proapoptotic signaling of
AT2 receptor. We found that an increase in ceramide levels
was followed by protease activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result
from the degradation of complex SLs such as sphingomyelin or
glycosphingolipids. Based on the measurement of serine
palmitoyltransferase activity and use of inhibitors, the activation of
de novo sphingolipid biosynthesis plays a key role in
AT2 receptor-mediated apoptosis. Taken together, our
current data suggest that AT2 receptor induces the
activation of de novo pathway, which then leads to an
accumulation of a sphingolipid metabolite, possibly ceramide, that
results in apoptosis.
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EXPERIMENTAL PROCEDURES |
Materials--
Murine nerve growth factor (NGF) and Dulbecco's
modified Eagle's medium (DMEM) were from Life Technologies, Inc. ECL
detection system and Hybond N membranes were from Amersham Pharmacia
Biotech. Fumonisin B1 (FB1),
-chloro-L-alanine (
CA), pertussis toxin (PTX), sodium
orthovanadate, bovine brain ceramide, and angiotensin II were purchased
from Sigma. Terminal transferase was from Roche Molecular Biochemicals.
125I-[Sar1,Ile8]AngII,
[methyl-3H]choline chloride (75 mCi/mmol),
L-[3-14C]serine, [1-14C]stearic
acid (55 mCi/mmol), [
-32P]ATP (3,000 Ci/mmol), and
[
-32P]dideoxyATP (3,000 Ci/mmol) were from NEN Life
Science Products. C2-ceramide was purchased from Biomol.
Escherichia coli diacylglycerol kinase and
octyl-
-D-glucopyranoside were from Calbiochem. PD98059 and anti-phospho-ERK antibody (recognizing rat, mouse, and human phospho-ERK 1 and 2) were from New England Biolabs. The anti-ERK antibody (recognizing rat and mouse ERK 1 and 2) was from Upstate. All
other reagents were from Sigma or were of the highest available research quality.
Cell Culture--
PC12W, a subline of PC12 rat pheochromocytoma
cell line that expresses high levels of AT2 receptor but no
AT1 receptor, were cultured as described previously (4, 8).
Briefly, PC12W cells were maintained in DMEM supplemented with 10%
fetal calf serum, 5% horse serum, 100 units/ml penicillin, and 1 mg/ml
streptomycin in humidified atmosphere of 95% O2 and 5%
CO2, and cells were used in passages 10-20.
AT2 receptor binding was analyzed every five passages and
no changes in receptor expression was observed. In each experiment,
cells were grown in DMEM until they reached 70-80% confluence. In our
experiments, cells were grown in 10% fetal calf serum and 5% horse
serum before switching to serum-free medium containing NGF (10 ng/ml)
with or without AngII. Cardiomyocytes were prepared from the hearts of
neonatal Sprague-Dawley rats (postnatal day 1) according to published
methods (9). Ventricles were cut and then digested with collagenase II.
The cells obtained from a 10-min digestion period were collected and
preplated in DMEM supplemented with 20% calf serum at 37 °C for 90 min. Then the nonadhering cells, collected as a cardiomyocyte-rich
population, were seeded (2 × 105/cm2) and
maintained for the subsequent 2 days in DMEM with 7% calf serum.
Experiments were conducted in 0.5% calf serum. In cardiomyocytes, AngII receptor expression was (AT1 5.9 ± 0.5, AT2 4.0 ± 0.9 fmol/106 cells,
n = 4). Cell viability was assessed by trypan blue
exclusion assay.
Angiotensin II Receptor Whole Cell Ligand Binding
Assay--
Whole cell ligand binding assay was performed on cultured
cells seeded in 12-well plates. AT1 and AT2
receptor binding was assessed by incubating the cells for 1 h at
37 °C with 0.2 nM of
125I-[Sar1, Ile8]AngII in the
presence or absence of 1 µM of PD123319 or losartan as a
competitor, respectively. Cells were then washed twice with ice-cold
phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin
and lysed in 0.5 N NaOH and gamma counted.
Extraction of Lipids and Ceramide Assay--
Ceramide was
quantified as described previously (10). In brief, cells were washed
twice with ice-cold PBS, frozen with liquid nitrogen, and scraped.
Lipids were extracted according to the method described by Bligh and
Dyer (11). Lipids were alkaline hydrolyzed for 1 h with 0.1 N methanolic KOH and reextracted. Ceramide levels were
determined in mixed micellar conditions and quantitatively converted to
ceramide-1-phosphate by E. coli
diacylglycerol kinase. The labeled ceramide-1-phosphate was
resolved by thin layer chromatography (TLC) (silica gel 60 plates) with
a solvent system consisting of chloroform/methanol/acetic acid
(65:15:5, v/v/v). Quantification of ceramide was performed using a
standard curve of known amounts of ceramide. Cell number was determined by hemacytometer.
Metabolic Labeling by [1-14C]Stearic
Acid--
[1-14C]stearic acid (10 µCi) was dried under
N2, resuspended in 100:l of the medium, sonicated, and
added to the cells. Cells were prelabeled for 24 h before AngII
stimulation, and [1-14C]stearic acid (10 µCi) was also
present during AngII stimulation. At the indicated times, cells were
washed with ice-cold PBS, frozen with liquid nitrogen, and then
scraped. The samples were extracted and alkaline hydrolyzed as
described above. 1-14C-labeled ceramide was resolved by TLC
on silica gel 60 plates using a solvent system of
chloroform/methanol/acetic acid/water (85:4.5:5:0.5) and were detected
by comigration of ceramide standards (12, 13).
Sphingomyelin Determination--
Changes in sphingomyelin levels
were measured by [3H]choline (75 Ci/mmol) labeling as
described previously (14). PC12W cells were incubated with
[3H]choline (1 µCi/ml in cell culture medium) for three
passages. The procedures of extraction and alkalinic hydrolysis were
identical to those used for ceramide determination. Sphingomyelin was
resolved by TLC on silica gel 60 plates using a solvent system
consisting of chloroform/methanol/acetic acid/water (50:30:8:3) and was
quantified by liquid scintillation counting.
Sphingomyelinase Assay--
Cells were washed in ice-cold PBS,
pelleted, and homogenized in 20 mM Tris/HCl, pH 7.5, and 1 mM EDTA containing 10 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM
aprotinin. The homogenate was centrifuged at 4,000 × g. Neutral sphingomyelinase activity was assayed with 20 nM [methyl-14C]sphingomyelin in
0.2% Triton X-100, 80 mM MgCl2, and 100 mM Tris/HCl, pH 7.4, as described previously (15). TNF-
(30 ng/ml) and boiled samples were used as positive and negative
controls, respectively. After 1 h of incubation, the reaction was
stopped by 1.5 ml of chloroform/methanol (2:1), vortexed, and
centrifuged to separate phases. The upper phase was counted by liquid
scintillation counting. Acid sphingomyelinase activity was measured as
above except that 100 mM sodium acetate, pH 5.5, replaced
Tris/HCl.
Sphingosine Assay--
Sphingosine was quantified as described
previously (16). The enzyme was prepared from Swiss 3T3 fibroblasts. In
brief, cells were washed twice with ice-cold PBS, frozen with liquid
nitrogen and scraped. Lipids were extracted according to the method
described by Bligh and Dyer (11). Assay was performed in lipids
solubilized in bovine serum albumin. Sphingosine was quantitatively
converted enzymatically to sphingosine 1-phosphate and then the labeled sphingosine 1-phosphate was resolved by TLC with a solvent system consisting of 1-butanol/methanol/acetic acid/water (80:20:10:20, v/v/v)
or chloroform/methanol/acetic acid/water (90:90:15:6, v/v/v). Quantification was performed by adding known amounts of sphingosine.
Labeling and Analysis of Glycolipids--
PC12W were prelabeled
with [14C]serine (1 µCi/ml) in serine-free medium for
24 h followed by AngII stimulation in the presence of
[14C]serine (1 µCi/ml) for 24 h. Cells were washed
with ice-cold PBS, scraped, and centrifuged at 6,000 rpm for 10 min.
Total lipids were extracted from the pellet with 3 ml of
chloroform/methanol/water/pyridine (60:30:6:1) for 48 h at
50 °C (17). The extract was treated with methanolic KOH 100 mM for 1 h at 37 °C. Then lipids were desalted by
reverse-phase chromatography (18), applied to TLC plates, and
chromatographed with chloroform/methanol/0.22% aqueous CaCl2 (60:35:8, v/v/v). TLC plates were exposed for 5-7
days in
70 °C.
Serine Palmitoyltransferase Assay--
Enzymatic activity was
determined by measuring the incorporation of
L-[3-14C]serine into 3-ketosphinganine.
The samples were processed and the assay was performed as described
previously (19). The assay was performed in 0.1 M Hepes (pH
7.4), 5 mM dithiothreitol, 5 mM EDTA, 50 µM pyridoxal 5'-phosphate, 0.15 mM
palmitoyl-CoA, and 1 mM 14C-labeled
L-serine in a total volume of 0.1 ml at 37 °C for 10 min
and was terminated by 1.5 ml of chloroform/methanol 1:2. Boiled samples, samples without palmitoyl-CoA, and samples containing 10 mM
-chloro-L-alanine served as negative
controls. To extract 3-ketosphinganine, 25 µg of sphinganine was
added as a carrier. The phases were separated, and the lower phase was
washed twice with water. The chloroform soluble material was either
scintillation counted or run on TLC using silica gel plates with
chloroform/methanol/2 N NH4OH (40:10:1) as a solvent.
Phospho-ERK Immunoblot--
PC12W were serum starved for 24 h. Then these cells were stimulated by 10 ng/ml NGF for 10 min with or
without PD98059, washed two times with ice-cold PBS, frozen in liquid
nitrogen, and scraped. Phosphorylated ERK was detected using
anti-phospho-ERK antibodies (New England Biolabs), visualized by
enzyme-linked chemiluminescence (Amersham Pharmacia Biotech), and
quantified using scanning densitometry.
Apoptosis Assay--
The PC12W cells were seeded into 6-well
plates at 5 × 105 cells/well. Morphological changes
in the nuclear chromatin of apoptotic cells were determined by
staining with bis-benzimide (Hoechst 33342) and propidium
iodide. A minimum of 500 cells were scored for the apoptotic chromatin
changes (condensation of chromatin, its compaction into periphery of
the nucleus, and segmentation of the nucleus into more than three
fragments). Data are expressed as percentage of apoptotic cells in
total counted cells.
Oligosomal fragmentation of PC12W genomic DNA was measured using cells
seeded on 6-well plates as described (20). The amount of
[
-32P]dideoxyATP incorporated into low molecular
weight (<20 kilobase pairs) DNA fraction was quantified by cutting the
respective fraction of DNA from the dried gel and scintillation counted.
Changes in caspase 3 activity were measured using synthetic
tetrapeptide Asp-Glu-Val-Asp (DEVD) coupled to
p-nitroaniline as a substrate (Apoalert kit,
CLONTECH). PC12W cells were seeded on 6-well plates
and for each caspase 3 activity determination, 2 × 106 cells were used. At appropriate time points, cells were
washed with ice-cold PBS and frozen in liquid nitrogen. The samples
were processed according to the manufacturer's instructions.
Statistics--
Results are expressed as mean ± S.E. and
were obtained by combining data from three to six independent
experiments. Statistical significance was assessed by Student's
t test.
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RESULTS |
Apoptosis Induced by AT2 Receptor Stimulation--
We
used PC12W cells that express high levels of AT2 receptor
but no AT1 receptor. PC12W cells were maintained in
serum-free medium in the presence of 10 ng/ml NGF, which protects these
cells from apoptosis. Incubation of PC12W cells with AngII for 48 h resulted in the appearance of typical morphological changes of apoptosis such as nuclear condensation and chromatin fragmentation, and
the number of apoptotic cells increased from 3.1 ± 1.1% to 11.9 ± 2.5% (n = 4, p < 0.01).
The apoptotic effect of AngII (10
7 M) in
these cells was blocked by 10
5 M of the
AT2 receptor-specific antagonist PD123319 but not by the
AT1 receptor antagonist losartan (10
5
M). The effect of endogenously produced angiotensin II on
apoptosis was controlled by checking whether PD123319
(10
5 M) would affect the basal level of
apoptosis. No effect could be observed suggesting that the endogenously
produced AngII has no major functional significance. The time-course of
DNA fragmentation revealed that an increase in apoptosis became
apparent at 24 h after the addition of AngII (10
7
M). At maximum, nearly 4-fold increase in DNA fragmentation
was observed 48 h after AngII stimulation. In control cells
maintained under NGF without AngII, almost no DNA fragmentation was
detected (Fig. 1A). Moreover,
the stimulation of AT2 receptor resulted in the activation
of caspase 3 that preceded the onset of DNA fragmentation (Fig.
1B).

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Fig. 1.
Time-course of AngII-induced apoptosis in
PC12W cells. A, representative autoradiograph of the
time-course of AngII-induced DNA fragmentation. Cells were maintained
in serum-free medium containing 10 ng/ml NGF and were stimulated by
AngII (10 7 M) for the indicated times.
Genomic DNA was extracted from these cells and analyzed for DNA
fragmentation as described under "Experimental Procedures."
B, time-course of caspase 3 activity in response to
AT2 receptor stimulation. * p < 0.05 and
** p < 0.01 versus no AngII, respectively.
Experimental conditions are as described under "Experimental
Procedures" (mean ± S.E., n = 3).
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AT2 Receptor-induced Increase in Ceramide
Levels--
We observed that the addition of membrane permeable
C2-ceramide (100 µM) led to approximately
30% apoptosis after 24 h (data not shown), suggesting that
ceramide in fact exerts direct proapoptotic effect in PC12W cells. To
test the hypothesis that ceramide is involved in the mechanism of
AT2 receptor-induced apoptosis, we measured the time-course
of ceramide production after AngII stimulation. Early time points saw
no substantial changes in ceramide formation by AngII stimulation.
Accumulation of ceramide was detectable 18 h after AngII
stimulation, reached a peak at 24 h, and was sustained for at
least 48 h (Fig. 2). Compared with
control cells, AngII (10
7 M) stimulation
increased cellular ceramide content 4-fold at 24 h. In addition,
metabolic labeling experiments were performed in which cells were
prelabeled with [3H]stearic acid for 24 h before
AngII stimulation. In accordance with diacylglycerol kinase assay,
AT2 receptor-induced ceramide increase after 24 h of
AngII stimulation was 3.7-fold. Sphingosine levels were only modestly
affected by 24 h of AngII stimulation, and sphingosine increased
from 25.8 ± 7.0 to 41.6 ± 9.2 pmol/106 cells
(n = 3, p = not significant). To study
whether AngII-induced ceramide increase occurs in other cell systems,
we studied rat neonatal cardiomyocytes. AngII (10
7
M) in the presence of losartan (10
5
M) causes (297 ± 51% of control, n = 4) increase in DNA laddering, and no increase occurred in the presence
of AngII and PD123319 (10
5 M). In
cardiomyocytes, 24 h of AngII stimulation increased ceramide levels 4-fold. Elevation in intracellular ceramide in PC12W preceded caspase 3 activation and the appearance of DNA fragmentation. The
evidence presented above indicates that there is a temporal association
between AT2 receptor-induced ceramide accumulation and
apoptosis.

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Fig. 2.
Changes in ceramide levels in response to
AngII. Solid bar represents control cells that were
maintained in serum-free medium containing NGF (10 ng/ml);
hatched bar represents cells that were stimulated by
10 7 M AngII. * p < 0.05 and
** p < 0.01 versus no AngII. At indicated
time points, lipids were extracted and assayed with diacylglycerol
kinase. Lipids were separated by TLC, and radioactive spots were
visualized by autoradiography and ceramide 1-phosphate was quantified
as described under "Experimental Procedures" (mean ± S.E.,
n = 3).
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The Mechanism of AT2 Receptor-induced Ceramide
Production--
Ceramide can be derived from complex SLs or from
de novo biosynthesis. We first tested the hypothesis that
AT2 receptor induces an elevation in intracellular ceramide
by activating sphingomyelin hydrolysis. We measured both acidic and
neural sphingomyelinase activity in cell lysates at various time points
and could not detect any effect by AT2 receptor activation
(Fig. 3A).
Sphingomyelin/ceramide molar ratio in our system is approximately 7:1,
and therefore ceramide increase from 50 to 200 pmol/106
cells would require approximately 40% turnover of sphingomyelin pool.
In accordance with sphingomyelinase data, no sphingomyelin breakdown
was detected by AT2 receptor stimulation in
[3H]choline-labeled PC12W cells up to 24 h (Fig.
3B). In addition, we analyzed the effect of 24 h of
AngII stimulation on glycolipids. Cellular lipids were prelabeled with
[14C]serine for 24 h before AngII stimulation and
during treatment. Short chained glycosphingolipids such as
glucosylceramide and lactosylceramide were well separated, and no
changes in the levels of these lipids were observed. In addition, no
changes in the levels of sphingomyelin or various gangliosides were
observed (data not shown). Taken together, our results suggest that
ceramide is not derived from complex SLs. These findings prompted us to explore the de novo biosynthetic pathway by measuring serine
palmitoyltransferase activity that is the rate-limiting reaction in
de novo sphingolipid synthesis (21, 22). We observed 2-fold
increase in the activity of this enzyme after 24 h of stimulation
(Fig. 3C). To block the de novo pathway, we
measured the ceramide levels at 24 h after AngII stimulation in
the presence or absence of 5 mM
CA and 10 µM FB1. At these concentrations, both
CA
and FB1 completely block the de novo pathway
(23-25). Ceramide measurement demonstrated that both
CA and
FB1 blocked the effect of AT2 receptor on
ceramide production (Fig. 4A).
Interestingly, similar results using 10:M FB1 was observed
in rat neonatal cardiomyocytes where AT2 receptor-induced ceramide production was blocked by FB1 suggesting that the
ceramide pathway is not cell-type specific. Treatment of PC12W cells
with doses of FB1 and
CA and up to 10 µM
and 5 mM, respectively, in the absence of AngII for as long
as 48 h, did not affect cell viability (data not shown), agreeing
with the results of other studies (14, 23).

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Fig. 3.
Changes in sphingomyelinase activity,
sphingomyelin levels, and serine palmitoyltransferase activity in
response to AngII stimulation. A, the activity of
acidic (tall bars) and neutral (short bars)
sphingomyelinase measured in cell lysates at pH 5.0 and 7.4, respectively, using [methyl-14C]sphingomyelin
as a substrate. B, sphingomyelin levels in cells. At
indicated time points, lipids were extracted and separated by TLC.
Spots corresponding to sphingomyelin were scraped and counted for their
radioactivity (mean ± S.E., n = 3). C,
AT2 receptor stimulates serine palmitoyltransferase
activity in PC12W cells. Solid bars represent controls that
were maintained in serum-free medium containing NGF (10 ng/ml),
hatched bars represent PC12W that were stimulated by AngII
(10 7 M). Synthesis of 3-ketosphinganine was
measured as described under "Experimental Procedures" using
L-[3-14C]serine as a substrate. **
p < 0.01 versus control cells.
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Fig. 4.
The effect of FB1 and
CA on AngII-induced ceramide accumulation and
apoptosis. A, ceramide was measured at 24 h after
AngII (10 7 M) stimulation with or without 10 µM FB1 or 5.0 mM CA. **
p < 0.01 versus control. Lipids were
extracted, and ceramide content was measured as described under
"Experimental Procedures" (mean ± S.E., n = 4). B, effects of FB1 and CA on DNA
fragmentation. PC12W were kept in serum-free medium supplemented with
10 ng/ml NGF and stimulated with AngII (10 7
M) for 48 h with 0.1 to 10 µM
FB1 or 0.5 to 5.0 mM CA. The amount of
[32P]dideoxyATP incorporated into low molecular weight
(<20 kilobases) DNA fraction was quantified by cutting the respective
fraction of agarose gel (mean ± S.E., n = 3 for
each condition). * p < 0.05 and ** p < 0.01 versus control.
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The evidence presented above indicates that there is a correlation
between ceramide accumulation and apoptosis. To examine the role of
ceramide in AT2 receptor-mediated apoptosis, we
investigated whether AT2 receptor-generated apoptotic
signals could be interrupted by
CA or FB1.
AT2 receptor-mediated increase in DNA fragmentation was
attenuated in a concentration-dependent manner and abolished by
10 µM FB1 and by 5 mM
CA (Fig.
4C). We performed nuclear staining and observed that
FB1 decreased the number of apoptotic nuclei in an
analogous manner to DNA fragmentation, and the number of apoptotic
cells decreased from (3.1 ± 1.1%, no AngII) 11.9 ± 2.5%, 5.5 ± 0.23%, 4.2 ± 0.20%, 4.1 ± 0.60% (mean ± S.E., n = 6 for each condition) by 0, 0.1, 1, and 10 µM FB1, respectively. Interestingly, similar
results using 10 µM FB1 was observed in rat
neonatal cardiomyocytes where AT2 receptor-induced
apoptosis was blocked by FB1 suggesting that the
activation of de novo biosynthesis of SLs may mediate apoptosis also in neonatal cardiomyocytes.
Effects of Pertussis Toxin, Orthovanadate, and Okadaic
Acid--
Our previous studies (4) have established that in PC12W
cells the AngII-induced apoptosis is PTX- and vanadate-sensitive. We
therefore examined whether these compounds block the apoptosis by
modulating the ceramide response. In agreement with the previous studies, AngII stimulation did not induce apoptotic nuclear changes in
PC12W cells pretreated with 200 ng/ml PTX for 24 h (Fig.
5). Ceramide levels were also measured in
PTX-treated cells at 24 h after AT2 receptor
stimulation, and no elevation in ceramide levels was detected
(Fig. 5).

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Fig. 5.
The effect of pertussis toxin and
orthovanadate on AT2 receptor-induced apoptosis and
ceramide production. Effect of PTX and orthovanadate on ceramide
accumulation (solid). After 24 h of AngII stimulation,
lipids were extracted and ceramide levels were measured. Hatched
bars show the quantification of apoptotic cells by chromatin
staining following incubation with AngII (10 7
M) and PTX (200 ng/ml) or orthovanadate (100 µM) for 48 h (mean ± S.E., n = 3). ** p < 0.01 versus control cells.
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Given that the AT2 receptor-induced apoptotic cell death is
mediated by a tyrosine phosphatase-dependent mechanism, we
treated PC12W cells for 24 h with 100:M orthovanadate before AngII
(10
7 M) stimulation and confirmed that AngII
did not induce any apoptotic nuclear changes (Fig. 5). As shown in Fig.
5, orthovanadate almost completely blocked AngII-induced elevation in
cellular ceramide levels. In contrast, okadaic acid did not demonstrate
any effect on apoptosis induced by AT2 receptor nor did it
influence AT2 receptor-induced ceramide generation (data
not shown).
Effect of ERK Inhibition on Ceramide Production--
ERK
inhibition by AT2 receptor results is a central part of
AT2 receptor signaling (4, 26). Therefore, we examined the possibility that ERK inhibition induces ceramide accumulation in PC12W
cells maintained under NGF (10 ng/ml). In ERK phosphorylation assay 1, 10, and 100 µM PD98059 reduced NGF (10 ng/ml) stimulated phosphorylation by 20, 45, and 65%, respectively (Fig.
6). Inhibition of ERK by 45% had no
effect, and 65% inhibition caused a 2-fold increase in DNA
fragmentation. Interestingly, PD98059 did not cause any changes in
ceramide levels (Fig. 6).

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Fig. 6.
The effect of PD98059 on ERK, apoptosis, and
ceramide levels. Top panel, effect of PD98059 on
NGF-stimulated ERK (10 ng/ml) activity after 10 min of stimulation.
Middle panel, effect of 10 and 100 µM PD98059
on DNA fragmentation after 24 h of incubation. Lower
panel, effect of PD98059 on ceramide levels. After 24 h,
lipids were extracted, and ceramide levels were measured.
Autoradiographs of three independent experiments are represented.
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DISCUSSION |
Time-course analysis of AT2 receptor-stimulated
ceramide accumulation revealed no early increase, rather a late
ceramide increase starting 18 h after AT2 receptor
stimulation that was followed by caspase 3 activation and DNA
fragmentation. This is in accordance with a large body of evidence
suggesting that ceramide can be placed upstream of the executioner
caspase 3-like proteases (27). Interestingly, previously published
studies (13, 28) have reported that Fas ligand induces delayed ceramide
accumulation that precedes the onset of apoptosis. The mechanism of
ceramide accumulation following AT2 receptor stimulation
does not appear to be secondary to the sphingomyelinase pathway nor to
glycosphingolipid breakdown. Therefore, we examined if the de
novo pathway of sphingolipid synthesis would be involved (23, 29).
We found serine palmitoyltransferase, the rate-limiting step of
de novo pathway, to be activated by AT2 receptor
suggesting that the accelerated de novo sphingolipid synthesis could mediate AT2 receptor-induced apoptosis.
Daunorubicin and in some instances TNF-
have been shown to cause
apoptosis by accelerating ceramide synthesis via the activation of
de novo pathway (14, 30). We examined the effects of two
inhibitors of de novo pathway,
CA (inhibitor of serine
palmitoyltransferase) and FB1 (inhibitor of ceramide
synthase). FB1 is a specific inhibitor de novo
sphingolipid synthesis, but it results in the accumulation of
sphinganine. Sphinganine has been reported to be biologically active
and could potentially modulate apoptosis (31, 32). However, it has been
shown that in contrast to sphingosine 1-phosphate, sphinganine
1-phosphate appears not to be antiapoptotic in PC12 cells (33).
CA
does not cause accumulation of SL metabolites (25). Both of these
inhibitors strongly attenuated the generation of ceramide induced by
AT2 receptor stimulation, suggesting that the activation of
de novo SL synthesis is required for ceramide accumulation.
We tested the functional significance of de novo SL
synthesis in AT2 receptor signal transduction by using
FB1 and
CA and found that both inhibited the
AT2 receptor-induced apoptosis in a
dose-dependent manner, and the required concentrations were
in accordance with the IC50 of FB1 or
CA
(23, 25). Thus, AT2 receptor stimulation induced an
acceleration of de novo sphingolipid synthesis leading to an
accumulation of ceramide or some other sphingolipid metabolite, which
then promotes apoptotic cell death. The effect of AT2
receptor stimulation on SL is not cell type specific as similar effects
were observed in rat neonatal cardiomyocytes.
Both pertussis toxin- and vanadate-inhibited AT2 receptor
stimulated ceramide accumulation thus suggesting that both G protein and tyrosine phosphatase activation are essential in controlling the
accelerated ceramide synthesis (4, 6, 34). The alternative mechanism
for the effect of vanadate is ceramidase activation that may
independently of AT2 receptor activation reduce ceramide levels (35). Our group and others have previously identified ERK as one
of the downstream effectors for AT2 receptor (4). ERK
inhibition by AT2 receptor is a rapid response that occurs within minutes after receptor activation and returns back to baseline within 30 min, thus occurring before an increase in ceramide levels. In
PC12 cells, the inhibition of ERK is an essential step in growth factor
deprivation-induced apoptosis (36). We used MEK inhibitor PD98059 to
attenuate NGF-stimulated ERK activity and found that it did not cause
any increase in ceramide levels, suggesting that ERK inhibition alone
is not sufficient to enhance ceramide production. Although
AT2 receptor-induced apoptosis and ceramide accumulation correlate well, AT2 receptor-induced apoptosis could be
mediated by another sphingolipid metabolite. This could also explain
why PD98059 does not induce ceramide accumulation but induces apoptosis.
Our principal findings are that AT2 receptor activation
induces the activation de novo biosynthesis of SLs and an
accumulation of ceramide. Our data support the hypothesis that
sphingolipid metabolite(s) is (are) involved in AT2
receptor-induced apoptosis. The changes in ceramide correlate well with
apoptosis, however, this does not directly prove that ceramide is the
true mediator. The data presented here is the first evidence to show
that an activation of a G-protein coupled receptor can activate the
de novo sphingolipid synthesis leading to programmed cell death.