Diacylglycerol and ceramide formation induced by dopamine D2S
receptors via G
-subunits in Balb/c-3T3 cells
Gele
Liu,
Mohammad H.
Ghahremani,
Behzad
Banihashemi, and
Paul R.
Albert
Ottawa Health Research Institute (Neuroscience), University
of Ottawa, Ottawa, Canada K1H 8M5
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ABSTRACT |
Diacylglycerol (DAG)
and ceramide are important second messengers affecting cell growth,
differentiation, and apoptosis. Balb/c-3T3 fibroblast cells
expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a
model of G protein-mediated cell growth and transformation. In Balb-D2S
cells, apomorphine (EC50 = 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at
1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating
Gi/Go proteins. To address which G proteins are
involved, Balb-D2S clones expressing individual PTX-insensitive
G
i proteins were treated with PTX and tested for
apomorphine-induced responses. Neither PTX-insensitive G
i2 nor G
i3 rescued D2S-induced DAG or
ceramide formation. Both D2S-induced DAG and ceramide signals required
G
-subunits and were blocked by inhibitors of phospholipase
C
[1-(6-[([17
]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione
(U-73122) and partially by D609]. The similar G protein specificity of
D2S-induced calcium mobilization, DAG, and ceramide formation indicates
a common G
-dependent phospholipase C-mediated pathway. Both D2
agonists and ceramide specifically induced mitogen-activated protein
kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of
D2S-induced ERK1/2 activation, leading to cell growth.
phospholipase C; G protein; mitogen-activated protein
kinase
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INTRODUCTION |
THE
DOPAMINE-D2 RECEPTOR GENE encodes two splice
variants of the receptor, long and short forms (D2L and D2S), that are
pharmacologically and functionally equivalent with both forms coupling
equivalently to Gi/Go "inhibitory" proteins
(10, 28). In pituitary cells, the dopamine D2S receptor
inhibits ERK1/2 activation and cell proliferation (1, 13, 32,
37). Oppositely, in Balb/c-3T3, Chinese hamster ovarian (CHO),
and C6 mesenchymal cells, the D2S receptor stimulates cell
proliferation and induces tumor formation, involving calcium
mobilization and ERK1/2 activation (8, 16, 20, 26, 31).
These actions are blocked by pertussis toxin (PTX), implicating
Gi/Go proteins. However, the signaling pathways that mediate Gi/Go-induced regulation of cell
proliferation remain poorly defined.
Ceramide is a novel and important second messenger involved in a
wide variety of signal transduction pathways that mediate cell-specific
biological responses such as cell growth, differentiation, inflammation, and apoptosis (11, 12, 19, 22).
Ceramide is produced from many sources, such as the action of
sphingomyelinase (SMase) on sphingomyelin (SM) (33), de
novo synthesis (27), or metabolism of other lipids
(7, 17). There is also a close dynamic relationship
between the biosynthetic pathways for diacylglycerol (DAG) and ceramide
via SM synthase, which interconverts ceramide/phosphatidylcholine (PC)
into SM/DAG (22). Thus this work focused on the
relationship between DAG and ceramide levels and their regulation by
D2S receptor activation in signaling to cell proliferation.
To address whether different G proteins mediate divergent
D2S-induced responses, we have expressed antisense or PTX-insensitive mutant G
i/G
o constructs in GH4 pituitary
or Balb/c-3T3 fibroblast cells (15, 16, 23, 24).
PTX-insensitive G
mutants were generated by a conservative
Cys-to-Ser substitution and retain coupling to a variety of effectors
(9, 16, 38), including G
i-mediated
inhibition of adenylyl cyclase, G
-induced inhibition of
calcium channels, and G
-induced ERK1/2 activation. By pretreating cells with PTX to block endogenous Gi/Go
proteins, the specific contribution of individual G proteins can be
assessed in cells transfected with individual PTX-resistant G
proteins. In Balb-D2S cells (Balb/c-3T3 cells transfected with D2S
receptor cDNA), G
i2/G
mediated D2S-induced
ERK1/2 activation and DNA synthesis, and G
i3 was
required for D2S-induced transformation (16). These results indicate that whereas ERK1/2 activation is linked to
D2S-mediated DNA synthesis, G
i3-dependent signaling to
transformation did not require ERK1/2 and utilized unknown messengers.
We therefore addressed the question of whether D2S receptor activation
regulates other second messengers, such as DAG or ceramide, and which G proteins are required.
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MATERIALS AND METHODS |
Materials.
Apomorphine, dopamine, Staphylococcus aureus SMase, PTX,
1,2-dioleoyl-rac-glycerol (C18:1[cis]-9), DAG,
U-73122 and all other drugs, standards, and salts were purchased from
Sigma. Escherichia coli DAG kinase (13 U/mg protein) and
D609 were purchased from Calbiochem. [
-32P]ATP
(specific activity: >3,000 Ci/mmol) was supplied by Amersham. [
-32P]dCTP was from Amersham Sera. Media and geneticin
(G418) were obtained from Life Technologies. Thin-layer chromatography
(TLC) plates (0.25 mm) were from Whatman. Solvents were supplied by BDH. Phospho-SAPK/JNK (Tyr183/tyr185) G9
monoclonal antibody, phospho-p38
(Tyr180/tyr182) monoclonal antibody, and the
phosphoplus p44/42 MAP kinase antibody kit were purchased from New
England Biolabs (Mississauga, Ontario, Canada).
Cell culture and transfection.
Balb-D2S cells and derivative clones were cultured in Dulbecco's
modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS).
Balb-D2S (clone 11) cells were generated as described, and specific D2S
receptor density was 143.6 ± 35.9 fmol/mg protein by saturation
binding analysis with [3H]spiperone (16).
PTX-insensitive mutant G
i/G
o subunit
constructs (Gi2-PTX and Gi3-PTX) and GRK-ct
were constructed previously (15) and transfected
individually (30 µg) into Balb-D2S (clone 11), and the cells were
selected in medium containing G418 (700 µg/ml) for 2-3 wk
(16). Antibiotic-resistant clones of each transfection were picked (24 clones/transfection) and tested for expression of the
corresponding G
i/G
o proteins using
Northern blot and Western blot analyses.
Treatment and lipid extraction.
Equivalent numbers of cells were cultured in 10 × 10 cm plates
with DMEM plus 10% FBS in a humidified atmosphere of 5%
CO2 at 37°C, growing to 80-90% confluence. Before
experiments, the cells were cultured in serum-free medium for 16 h. For PTX treatment, the cells were treated with 50 ng/ml PTX for
16 h before experimentation. Cells were treated with apomorphine
at the designed concentration and time and with S. aureus
SMase (0.1 U/ml) for 30 min. After treatment, twice washing with
ice-cold phosphate-buffered saline (PBS) terminated the reaction. The
lipid extraction method was based on that of Bligh and Dyer
(5). After centrifugation at 500 g for 1 min at
4°C, the supernatants were aspirated and the cells were lysed with
0.5 ml of chloroform-methanol-HCl (20:40:1 vol/vol/vol). Extracts were
sonicated each 5 s × 6 times on ice, rinsed with 1 ml of
chloroform and 0.3 ml 1 M NaCl, and spun at 14,000 g for 15 min at 4°C. The upper aqueous layer was discarded, and the lower
lipid-containing layer was transferred to a 1-ml glass Chrompack vial,
dried under a stream of O2-free N2 gas, and
redissolved in 200 µl of chloroform. The samples were stored at
80°C until analysis. The particulate protein interface was air-dried, dissolved in 0.5 ml 2 M NaOH, and assayed for protein according to Lowry's method.
Quantitation of DAG and ceramide.
DAG and ceramide were quantified in parallel using the DAG kinase
method, as described by Preiss et al. (34) and modified by
Wright et al. (44). Briefly, after treatment and solvent extraction of cell lipids, the chloroform of samples was then evaporated under N2. A blank tube and standard ceramide/DAG
tubes were included as controls. For each sample, 10 µl of DAG kinase (20 mU), 50 µl of reaction buffer, 10 µl of 20 mM dithiothreitol, and 10 µl of [
-32P]ATP (2.5 × 105
dpm/nmol) were added. Reaction buffer contained 25 mM MgCl2
and 2 mM EGTA dissolved in 50 ml of 100 mM imidazole (pH 6.6), and the
pH was readjusted to 6.6. The samples were incubated at 25°C for 30 min, and the reaction was terminated by addition of 0.5 ml of ice-cold
chloroform-methanol (1:2 vol/vol). The lipids were extracted by the
addition of 0.5 ml chloroform and 0.5 ml 1 M NaCl. The mixture was spun
at 14,000 g for 3 min, and the upper aqueous phase was
discarded. The lower organic phase was sequentially washed with 0.5 ml
of 1% perchloric acid, 0.3 ml chloroform-methanol (1:2 vol/vol), 0.2 ml chloroform, and 0.2 ml water. The resultant organic phase was dried
under N2 and reconstituted in 25 µl of chloroform/methanol (95:5 vol/vol). The samples were spotted onto a
Silica Gel 60 TLC plate being heat activated and developed in a solvent
mixture of chloroform-acetone-methanol-acetic acid-water (10:4:3:2:1 vol/vol/vol/vol/vol). Because DAG kinase can use
ceramide or DAG as a substrate, [32P]ceramide-phosphate
represented ceramide production and [32P]phosphatidic
acid represented DAG production (34). The TLC plates were
exposed to phosphor screens for 18 h, and
[32P]ceramide-phosphate and
[32P]phosphatidic acid were quantified using the
Molecular Dynamics System ImageQuaNT computer software. Results are
expressed as percentage of control.
Western blot analysis.
Cells were cultured in serum-free medium for 16 h and then treated
with experimental compounds for 30 min. The cells were washed twice
with ice-cold PBS and extracted with 100 µl of RIPA-L buffer [10 mM
Tris (pH 8), 1.5 mM MgCl2, 5 mM KCl, 0.5 mM DTT, 0.5 mM
phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.1% SDS, 0.5%
sodium deoxycholate, and 5 µg/ml leupeptin]. Samples were frozen on
dry ice/ethanol and stored at
80°C. Samples were sonicated 10-15 s, heated at 95°C for 5 min, centrifuged, and 40 µl/sample were loaded onto SDS-PAGE gel and electrotransferred to
nitrocellulose membrane. The membrane was blocked (1 h at room
temperature) and probed with primary antibody (1:1,000 overnight at
4°C). It was washed in TBST, incubated with horseradish peroxidase
(HRP)-conjugated secondary antibody (1:2,000) and HRP-conjugated
anti-biotin antibody (1:1,000) to detect biotinylated protein markers
(2 h at room temperature), washed, incubated with LumiGLO (1 min), and
exposed to X-ray film. Densitometric quantitation was done using the
UN-SCAN-IT program (Silk Scientific, UT), and data were normalized to control.
Statistical analysis.
The data were analyzed by repeated measure using ANOVA for each set of
experiments. The data are presented as means ± SE. of at least
three independent experiments. Differences of P < 0.05 were considered statistically significant. The percent inhibition data
was analyzed with repeated measure using ANOVA, and the data from
G
-PTX expressing clones were compared with Balb-D2S cell (wild type)
using Bonferroni multiple comparison posttest.
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RESULTS |
Apomorphine-induce DAG and ceramide production in Balb-D2S cells.
In Balb/c-3T3 cells stably transfected with the dopamine-D2S receptor
(Balb-D2S cells), DAG and ceramide production was induced by
apomorphine (dopamine receptor agonist) in a concentration-dependent manner with EC50 of 10
8 M (Fig.
1). Within 30 min, apomorphine
(10
5 M) increased DAG and ceramide compared with control
levels by 400% (500 pmol/107 cells) and 300% (430 pmol/107 cells), respectively. No response to apomorphine
was observed in Balb/c-3T3 cells (data not shown), which do not express
dopamine receptors. Exogenous SMase, used as a positive control,
hydrolyzed endogenous SM to form ceramide and also increased DAG
formation, suggesting conversion of ceramide to DAG. The bands of
radioactive DAG and ceramide comigrated with DAG and ceramide standards
and were quantified in the concentration range from 50-1,000 pmol. Apomorphine-induced DAG and ceramide production was time dependent (Fig. 2) and maximal at 1 and 6 h,
respectively. Ceramide production returned to basal at 24 h,
whereas DAG was elevated at 30 h. Apomorphine-induced DAG
paralleled and was greater than the increase in ceramide production. Pretreatment with PTX blocked DAG and ceramide formation induced by
10
6 M apomorphine (Fig. 3),
thus implicating Gi/Go proteins in D2S receptor
action. As a positive control, exogenous SMase increased the levels of
ceramide equally in cells treated or not treated with PTX (Fig.
3B), demonstrating G protein-independent hydrolysis of
endogenous SM to form ceramide.

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Fig. 1.
Apomorphine induces diacylglycerol (DAG) and ceramide
formation in a concentration-dependent manner in Balb/c-3T3 cells.
Balb-D2S cells (Balb/c-3T3 cells stably transfected with dopamine D2S
receptor cDNA) were treated with apomorphine (dopamine receptor
agonist) from 10 9 to 10 5 M for 30 min as
indicated. Lipids were extracted from cells, and
[32P]ceramide-phosphate and
[32P]phosphatidic acid were resolved by thin layer
chromatography (TLC) as a measure of DAG and ceramide content,
respectively (see MATERIALS AND METHODS). Top: a
representative image of [32P]ceramide-phosphate and
[32P]phosphatidic acid is shown here; the double bands
for ceramide represent 2 isoforms of ceramide. The amounts of DAG and
ceramide standards are as indicated. B, background (no DAG kinase); S,
cells treated with exogenous sphingomyelinase (SMase; 0.1 unit/ml for
30 min); C, control untreated cells. Bottom: the relative
levels (% of control) of DAG and ceramide were quantified by
phosphorimager scan, and the data are expressed as a function of
apomorphine concentration as means ± SE from 3 independent
experiments. *P < 0.03; **P < 0.01.
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Fig. 2.
Time course of apomorphine-induced increase in DAG and
ceramide formation. Balb-D2S cells were treated with apomorphine
(10 6 M) from 5-60 min and from 3-30 h. Lipids
were extracted from cells, and [32P]ceramide-phosphate
and [32P]phosphatidic acid were separated by TLC. A
representative image of [32P]ceramide-phosphate and
[32P]phosphatidic acid is shown. Standards for ceramide
only were used at the indicated amounts. The relative levels (% control) of DAG and ceramide were quantified and plotted as a function
of time. Data are expressed as means ± SE from 3 independent
experiments. *P < 0.05; **P < 0.03;
***P < 0.01. Cer, ceramide.
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Fig. 3.
Pertussis toxin (PTX) blocks DAG and ceramide production induced by
dopamine-D2S receptor activation. Left: Balb-D2S cells were
treated with 10 6 M apomorphine for 30 min with or without
pretreatment using 50 ng/ml PTX for 16 h. Right:
wild-type LD2S Balb/c-3T3 cells were treated with SMase (0.1 unit/ml
for 30 min) as a positive control. A representative image of
[32P]ceramide-phosphate and
[32P]phosphatidic acid is shown above. Data are
quantified and expressed as means ± SE from 3 independent
experiments. *P < 0.05. B, blank; A, apomorphine; P,
PTX; A+P, apomorphine + PTX; S, SMase; S+P, SMase + PTX; Std.,
standard.
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G
i2- and G
i3-PTX fail to rescue
apomorphine-induced DAG and ceramide formation.
Balb-D2S cells express all G
i/o-subunits, although
G
i2 and G
i3 appear to be the most
abundant based on densitometric analysis (16). To address
the G protein specificity of D2S signaling to ceramide production,
PTX-insensitive point mutants of G
i2 and
G
i3 (G
i2-PTX and G
i3-PTX)
were transfected into Balb-D2S cells to form G
i2-PTX
(BDi2-22) cells and G
i3-PTX (BDi3-3) cells. The transfectant cell lines expressed twofold more G
protein than
the corresponding endogenous G
-subunit in parental Balb-D2S (16), suggesting that approximately equal amounts of
mutant and wild-type protein were produced in the transfected cell
lines. In G
i2-PTX (BDi2-22) and
G
i3-PTX (BDi3-3) cells, apomorphine-induced DAG and
ceramide formation was blocked after pretreatment with PTX (Figs. 4 and
5),
suggesting that Gi2 or Gi3 alone do not mediate D2S-induced ceramide production. By contrast, apomorphine-induced inhibition of cAMP formation was rescued in both G
i2-
and G
i3-PTX cells (16). As a positive
control, exogenous SMase (0.1 U/ml) induced ceramide production. Hence,
neither G
i2-PTX or G
i3-PTX rescued D2S
receptor coupling to DAG and ceramide production after PTX treatment.

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Fig. 4.
G i2-PTX fails to rescue block of
apomorphine-induced DAG and ceramide production by PTX. Balb-D2S cells
expressing G i2-PTX (BDi2-22 cells), the
PTX-insensitive point mutant of G i2, were treated with
apomorphine (10 6 M) for 30 min with or without
pretreatment with 50 ng/ml PTX for 16 h. Cells were treated with
SMase (0.1 unit/ml for 30 min) as a positive control. A representative
image of [32P]ceramide-phosphate and
[32P]phosphatidic acid is presented above. At
bottom, the data are expressed as means ± SE from 3 independent experiments. *P < 0.03.
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Fig. 5.
G i3-PTX fails to rescue block of apomorphine-induced
DAG and ceramide production by PTX. BDi3-3 cells were treated with
10 6 M apomorphine for 30 min with or without pretreatment
50 ng/ml PTX for 16 h. Lipids were extracted from cells, and
[32P]ceramide-phosphate and
[32P]phosphatidic acid were separated and quantitated. At
top is shown a representative image of
[32P]ceramide-phosphate and
[32P]phosphatidic acid. At bottom, the data
are expressed as means ± SE from 3 independent experiments.
*P < 0.03.
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GRK-ct blocks apomorphine-induced DAG and ceramide formation.
We examined whether G
-subunits are involved in D2S-induced DAG
and ceramide production in stably transfection of Balb-D2S cells with
GRK-ct, a scavenger protein that binds to G
-subunits to prevent
their action. In these cells, apomorphine did not alter ceramide
production, but exogenous SMase (0.1 U/ml) did induce ceramide
production (Fig. 6). Importantly, other
D2S-induced actions, such as inhibition of forskolin-induced cAMP
accumulation, were not affected by expression of GRK-ct in these cells
(16), indicating that functional D2S receptors were
present in these clones. This result suggests a crucial role for
mobilization of G
-subunits in the D2S-mediated ceramide signal.

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Fig. 6.
Apomorphine-induced DAG and ceramide productions are
blocked in cells expressing GRK-ct. Balb-D2S cells stably transfected
with the G -blocker GRK-ct were treated with 10 6 M
apomorphine or 0.1 unit/ml SMase for 30 min. A representative image of
[32P]ceramide-phosphate and
[32P]phosphatidic acid is shown at top, and
the quantified data are expressed as means ± SE from 3 independent experiments. *P < 0.03.
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Role of phospholipase C in D2S-induced formation of DAG/ceramide.
According to the results above, we further investigated upstream of
DAG/ceramide formation by the action of apomorphine in Balb-D2S cells
(Fig. 7). Phospholipase C inhibitors
U-73122 (10
5 M, U) for phosphatidylinositol (PI)-PLC and
D609 (10
5 M, D) for PC-PLC were applied at maximal
concentrations (29, 39). U-73122 completely blocked
apomorphine-induced DAG/ceramide formation, whereas D609
partially inhibited this action by 50% or 60%, respectively. The
PI-PLC inhibitor U-73122 reduced DAG and ceramide formation in
parallel with IC50 of 1 µM (consistent with PLC
inhibitory concentration), whereas inactive analog
1-(6-[([17
]-3-methoxyestra-1,2,3-10]-trien-17yl)amino]hexyl)-2,5-pyrrolidineidione (U-73343) was without effect up to 10 µM (Fig.
8). These results are consistent with a
specific role for PLC activation in D2S-induced DAG and ceramide
formation.

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Fig. 7.
Involvement of phospholipase C in D2S-induced DAG/ceramide
generation. Balb-D2S cells were treated without (control) or with
apomorphine (10 6M), PC-PLC inhibitor D609
(10 5M; D), PI-PLC inhibitor
1-(6-[([17 ]-3-methoxyestra-1,2,3[10]-trien-17yl)amino]hexyl)-1H-pyrrole-2,5-dione
(U-73122; 10 5M; U), or SMase (0.1 U/ml) for 30 min. A
representative image of [32P]ceramide-phosphate and
[32P]phosphatidic acid is shown, and data are expressed
as means ± SE from 3 independent experiments. *P < 0.03; **P < 0.01.
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Fig. 8.
Concentration dependence of inhibition of D2S-induced
DAG/ceramide formation by U-73122 or
1-(6-[([17 ]-3-methoxyestra-1,2,3-10]-trien-17yl)amino]hexyl)-2,5-pyrrolidineidione
(U-73343). Balb-D2S cells were treated with apomorphine
(10 6 M) without or with pretreatment U-73122 or inactive
analog U-73343 for 3 h at the indicated concentration. A
representative image of [32P]ceramide-phosphate and
[32P]phosphatidic acid is shown, and at bottom
quantified data are expressed as means ± SE from 3 independent
experiments. *P < 0.03; **P < 0.01.
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Regulation of ERK1/2 by ceramide.
The possible downstream actions of ceramide were examined by comparing
the actions of apomorphine, ceramide analog C2-ceramide, and SMase on
mitogen-activated protein kinases in Balb-D2S cells (Fig.
9). Antibodies specific for
phosphorylated forms that represent activated proteins were used in
Western blot analysis. In other cell types, ceramide activates
stress-activated protein kinases, such as p38 or JNK, to mediate
apoptosis (2, 6, 41), so we examined these
pathways. Neither dopamine agonist (apomorphine), C2-ceramide, nor
SMase altered levels of phospho-p38 (Fig. 9), phospho-STAT3, or
phospho-STAT5 (not shown) compared with control as assessed by
densitometry. As previously reported (16), apomorphine induced a 2.2-fold increase in phospho-ERK1/2. In addition,
C2-ceramide and SMase also induced a 2.1- and 2.0-fold,,
respectively, increase in phospho-ERK1/2. By contrast, whereas
apomorphine induced a small (1.5-fold) increase in phospho-JNK, this
was not mimicked by C2-ceramide or SMase treatment. Thus
ceramide appears to specifically regulate ERK1/2 phosphorylation in
Balb-D2S cells, providing a novel potential mechanism for dopamine
D2S-induced ERK1/2 activation.

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Fig. 9.
Regulation of mitogen-activated protein kinases by apomorphine
and ceramide. Balb-D2S cells were treated without (control, C) or
with apomorphine (10 7 M), dopamine (10 7 M,
D), C2-ceramide (5 µM, C2), inactive analog
dihydro-C2-ceramide (5 µM,
D2-C2), or SMase (0.1 U/ml) for 30 min. After
harvesting cells, Western immunoblotting using antibodies to the
indicated phospho-protein or -actin (loading control) was carried
out. At top are shown representative blots, and at
bottom is densitometric quantitation of data (means ± SE) from 3 independent experiments expressed as %control for
phospho-JNK (left) and phospho-ERK1/2 (right).
*P < 0.05; **P < 0.01.
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DISCUSSION |
D2S-induced DAG and ceramide formation.
In Balb/c-3T3 cells expressing D2S receptors, apomorphine induced DAG
and ceramide formation in a concentration- and time-dependent manner
and was blocked by PTX, implicating Gi/Go
proteins. The D2S receptor provides a novel and interesting example of
a Gi/Go-coupled receptor that induces DAG and
ceramide formation with the same G protein specificity: these actions
were dependent on G
-subunits but were not rescued by
individual G
i2- or G
i3-PTX subunits (see model, Fig. 10). Other receptor
subtypes such as the angiotensin II type 2 (AT2) receptor
(21) or interferon-
receptor (42) mediate
PTX-sensitive ceramide production, but their G protein specificity has
not been examined. The Gi-coupled cannabinoid CB1 receptor
also induces ceramide formation to inhibit tumor growth
(14), but it is unclear whether this is a
Gi/Go-mediated action.

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Fig. 10.
D2S receptor-mediated signaling pathways in Balb/c-3T3
cells. The D2S receptor signals through PTX-sensitive
Gi/Go proteins to mobilize G i-
and G -subunits. Previous studies showed that D2S-induced
inhibition of adenylyl cyclase (AC) involved G i2- or
G i3- but not G -subunits (16).
G -subunits were essential for D2S-induced IP3-mediated calcium
mobilization (Ca2+), DAG [which activates protein kinase C
(PKC)], and ceramide formation. G -dependent phospholipase C
(PI-PLC more than PC-PLC) mediates this novel pathway as assessed by
inhibitors U-73122 and D609. Both G and, in part,
G i2 were required for D2S-induced ERK1/2 activation and
DNA synthesis (16). Ceramide induces ERK1/2 activation,
suggesting that G -dependent ceramide formation represents a novel
pathway of D2S-induced ERK1/2 activation leading to cell growth. Note
that arrows indicate stimulation, bars indicate inhibition; solid lines
denote direct effects, whereas dashed lines are indirect or multistep
pathways.
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The matching apomorphine concentration dependencies
(EC50 = 10
8 M), PLC inhibitor
concentration dependencies, G protein subunit dependencies, and the
close temporal association between D2S-induced DAG and ceramide
formation suggest that DAG formed by D2S-induced PLC activation is
converted to ceramide as shown in Fig. 10. The differential action PLC
inhibitor (U73,122 selective for PI-PLC stronger than D609 for PC-PLC)
further indicates the primary role of PI-PLC in D2S-induced DAG and
ceramide generation. Interconversion of DAG and ceramide could be
catalyzed by SM synthase, which converts ceramide and PC into SM and
DAG (22). In cells, this reaction is bidirectional and can
convert DAG into ceramide, leading to depletion of SM (25,
40). Importantly, DAG inhibits the forward reaction to favor
conversion to ceramide (18, 43). Ceramide inhibits de novo
synthesis of PC (43), again favoring the reverse reaction
to form ceramide from DAG. D609, a compound thought to be specific for
PC-PLC but that also inhibits SM synthase (25), displayed
slightly greater inhibition of ceramide than of DAG (60 vs. 50%),
suggesting at best a partial role for SMase in DAG-ceramide conversion.
Alternately, DAG can activate acidic SMase to generate ceramide
(35, 36). For example, tumor necrosis factor couples to
PC-PLC to increase production of DAG, which activates acidic SMase to
induce ceramide production. The conversion of DAG to ceramide or
DAG-induced ceramide formation would account for the identical G
and
G
dependencies of D2S-mediated DAG and ceramide formation.
G protein specificity of D2S responses.
There are striking similarities between the pattern of G protein
subunits required for D2S-induced DAG/ceramide formation and calcium
mobilization in Balb-D2S or L-D2S cells: G
-subunits were required
and no individual or pair of G
-PTX proteins reconstituted D2S-induced calcium mobilization (15, 16).
Receptor-mediated activation of PI-PLC is known to generate IP3, which
mediates calcium mobilization and DAG (3, 4), an important
second messenger that activates PKC (30). Thus the D2S
receptor mediates G
-dependent calcium mobilization and DAG
formation, possibly by activation of PI-PLC-
2 or -
3, which are
G
sensitive. D2S-induced DAG appears to be rapidly converted to
increase ceramide levels (Fig. 10). The lack of rescue of these
responses by individual (or paired) G
-PTX proteins could indicate
that activation of Gi1, Go, or multiple G
proteins is required to mobilize sufficient G
-subunits. However,
the possibility remains that PTX-insensitive G
-subunits may couple
inefficiently to this response because of the Cys-to-Ser change. By
contrast, D2S-induced inhibition of forskolin-stimulated cAMP formation
was rescued by G
i2- or G
i3-PTX,
indicating their functionality in these cells (16).
Role of D2S-induced DAG or ceramide formation in ERK1/2 activation.
As illustrated in Fig. 10, in Balb-D2S cells apomorphine-induced ERK1/2
activation and DNA synthesis are blocked by PTX and GRK-ct and are
rescued by G
i2-PTX but not G
i3- or
G
o-PTX, implicating G
i2, G
, and
ERK1/2 in cell growth (16). In the present study, D2S-induced ERK1/2 activation was mimicked by ceramide analog or
SMase-mediated ceramide generation, suggesting that D2S-induced ceramide formation mediates in part ERK1/2 phosphorylation. Although Gi2-PTX did rescue D2S-induced ERK1/2 activation and cell
growth, the rescue was only partial (16), suggesting that
Gi2-dependent and -independent pathways may contribute. One
Gi2-independent, G
-dependent pathway activated by D2S
receptors may involve ceramide formation. It is possible that
G
i1 or G
o couple D2S receptors to DAG or
ceramide formation. G
i1 appears to couple to growth regulatory signaling, because G
i1-PTX clones became
spontaneously transformed but were therefore weakly responsive to
apomorphine and were not examined. These studies illustrate the utility
of PTX-insensitive G proteins as a molecular approach to map G
protein-mediated pathways and to place novel second messengers such as
ceramide within these pathways.
 |
ACKNOWLEDGEMENTS |
The National Cancer Institute of Canada supported this research.
P. R. Albert is the Canadian Institutes of Health
Research/Novartis Michael Smith Chair in Neuroscience.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
P. R. Albert, Ottawa Health Research Institute,
Neuroscience, Univ. of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5,
Canada (E-mail: palbert{at}uottawa.ca).
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.
First published November 13, 2002;10.1152/ajpcell.00190.2002
Received 24 April 2002; accepted in final form 5 November 2002.
 |
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