Activation of the de novo Biosynthesis of Sphingolipids Mediates Angiotensin II Type 2 Receptor-induced Apoptosis*

Jukka Y. A. Lehtonen, Masatsugu Horiuchi, Laurent DavietDagger , Masahiro Akishita, and Victor J. Dzau§

From the Department of Medicine, Cardiovascular Research, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2 receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, beta -chloro-L-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and beta -chloro-L-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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), beta -chloro-L-alanine (beta 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), [gamma -32P]ATP (3,000 Ci/mmol), and [alpha -32P]dideoxyATP (3,000 Ci/mmol) were from NEN Life Science Products. C2-ceramide was purchased from Biomol. Escherichia coli diacylglycerol kinase and octyl-beta -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-alpha (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 beta -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 [alpha -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

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).

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 beta CA and 10 µM FB1. At these concentrations, both beta CA and FB1 completely block the de novo pathway (23-25). Ceramide measurement demonstrated that both beta 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 beta 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 beta 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 beta 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 beta 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 beta 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.

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 beta 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 beta 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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha 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, beta 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). beta 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 beta 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 beta 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.

    FOOTNOTES

* This study was supported by National Institutes of Health Grants HL46631, HL35252, HL35610, HL48638, HL07708, and HL58616 and a grant from the Longwood Foundation for Translational Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a postdoctoral fellowship from the American Heart Association Massachusetts Affiliate and of a research fellowship from INSERM.

§ Recipient of National Institutes of Health MERIT Award HL35610. To whom correspondence should be addressed: Dept. of Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-732-8911; Fax: 617-975-0995; E-mail: vdzau{at}bics.bwh.harvard.edu.

    ABBREVIATIONS

The abbreviations used are: AngII, angiotensin II; AT1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; beta CA, beta -chloro-L-alanine; FB1, fumonisin B1; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; NGF, nerve growth factor; PBS, phosphate-buffered saline; PTX, pertussis toxin; SL, sphingolipid; TNF-alpha , tumor necrosis factor-alpha ; C2-ceramide, N-acetylsphingosine; C6-ceramide, N-hexanoylsphingosine; TLC, thin layer chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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