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INTRODUCTION |
Phosphatidic acid phosphatase type 2 was originally identified as
a plasma membrane enzyme that catalyzes the dephosphorylation of the
putative second messenger, phosphatidic acid
(PA)1 to diacylglycerol (DG)
(1). Subsequently, multiple isoforms of phosphatidic acid phosphatase
type 2 were cloned (2-5). It was found that these enzymes
dephosphorylate a number of lipid phosphates in vitro other
than PA, including lysophosphatidic acid (LPA), sphingosine 1-phosphate
(S1P), ceramide 1-phosphate, and diacylglycerol pyrophosphate.
Therefore, they have been renamed lipid phosphate phosphatases.
Currently, there are four members of this family called LPP1, LPP1a,
LPP2, and LPP3 (6). The LPP isoforms are integral membrane proteins
with six transmembrane domains. These phosphatases contain a motif
(KXXXXXXRP(X12-54)PSGH(X31-54)SRXXXXXHXXXD), which is found in a super family of phosphatases (7). Recently, site-directed mutagenesis has been used to establish that the conserved
residues K, R, P, S, G, H, R, and H in the catalytic domain of LPP1 are
obligatory for activity (8).
There are also alternative routes through which S1P and LPA can be
dephosphorylated in cells. This involves recently identified S1P- and
LPA-specific phosphatases (9, 10). The S1P-specific phosphatase has
8-10 transmembrane domains and contains the same phosphatase motif as
the LPP isoforms, whereas the LPA-specific phosphatase has an acid
phosphatase motif
(LXXVXXVXRHGXRXP).
It has been predicted that the LPP isoforms have an extracellular
outward-facing catalytic site, raising the possibility that these
enzymes may dephosphorylate phosphorylated lipid agonists such as S1P
and LPA. Both are polar lysophospholipid metabolites that act as
extracellular mediators by binding to plasma membrane G-protein-coupled
receptors (GPCRs). To date, five closely related GPCRs of the EDG
(endothelial differentiation gene) family (EDG1, EDG3, EDG5/AGR16/H218,
EDG6, and EDG8/nrg-1) have been identified as high affinity S1P
receptors (11-16). A second group of EDG receptors (EDG2, EDG4, and
EDG7) have high affinity for LPA (35% identity to EDG1, -3, -5, -8).
The EDG receptors are coupled to multiple effectors via different
G-proteins, such as Gi1, Gi2, Gi3,
Gq, G12, and G13 (17) and Rho-GEF.
Examples include inhibition of adenylyl cyclase, stimulation of
phospholipase C, calcium mobilization, and p42/p44 MAPK activation (18,
19).
LPA may also bind to G-protein-coupled receptors that are not members
of the EDG family, such as PSP24
/
and GPR45 (20). Furthermore, PA
has been shown to stimulate cell migration via a novel
receptor-mediated mechanism in breast cancer cells (21).
Recent studies by Xu et al. (22) demonstrate that the net
association of LPA with EDG2 receptors is reduced in Rat2 fibroblasts transfected with LPP1. This leads to a decrease in the
LPA-dependent stimulation of p42/p44 MAPK, inhibition of
adenylate cyclase, calcium mobilization, phospholipase D activation,
and DNA synthesis. Such findings support the possibility that LPP1
limits bioavailability of LPA at its receptor. In contrast, Hooks
et al. (23) find that although ecto-LPP1 degrades ~90% of
the LPA in the medium over a 24-h period in HEK 293 cells, this was
insufficient to account for the decrease in LPA potency in mitogenic
assays, suggesting an alternative unidentified mechanism. One
possibility is that the LPP isoforms might dephosphorylate
intracellular PA, which is a putative second messenger molecule that
may have a role in growth factor and GPCR signaling. For instance, PA
is involved in the membrane recruitment of Raf-1 (24), which contains a specific PA binding region (amino acids 390-426) (25). Indeed, transfection of HIRcB fibroblasts with a PA binding region construct blocked subsequent insulin-stimulated p42/p44 MAPK and prevented Raf-1
translocation by sequestering PA (24). The LPP-catalyzed reduction in
PA might also increase Ras-GTPase-activating protein (26),
thereby reducing the half-life of GTP-bound Ras. Alternatively, the LPP
isoforms may modulate the membrane dynamic governing GPCR signaling.
For instance, PA and phosphatidylinositol 4,5-bisphosphate (the
formation of which is in a positive feedback loop with PA (27)) play a
role in membrane trafficking (28), where they initiate clathrin coat
assembly and facilitate vesicle formation for endocytosis (29). In this
regard, specialized areas of the plasma membrane, termed caveolae (30),
provide platforms for pre-assembled clusters of signaling molecules,
including Gi, the localization of which is essential for
GPCR signaling (31). Therefore, the LPP-catalyzed dephosphorylation of
PA may have a role in the endocytic signaling process.
Recent studies show that intracellular S1P may also function as an
intracellular second messenger. For instance, Rani et al. (32) show that platelet-derived growth factor stimulates an increase in
intracellular S1P in fibroblasts and that this may activate the p42/p44
MAPK pathway. In this regard, the LPP isoforms may dephosphorylate
intracellular S1P, thereby preventing its potential second messenger action.
In this study, we have shown that LPP1, LPP1a, and LPP2 but not LPP3
abrogate the activation of p42/p44 MAPK in response to LPA, S1P, PA,
and thrombin. Significantly, thrombin is a peptide GiPCR
agonist whose bioavailability at its receptor is not regulated by the
LPP isoforms. We have also shown that the abrogation of p42/p44 MAPK
activation by the LPP isoforms can be correlated with a reduction in
basal intracellular PA and not ecto-LPP activity. Therefore, our
findings suggest a model in which LPP1, LPP1a, and LPP2 may act on a
basal intracellular PA pool to alter the membrane dynamic governing
GPCR signaling to p42/p44 MAPK per se.
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EXPERIMENTAL PROCEDURES |
Materials--
[
-32P]ATP (3000Ci/mmol) and
[3H]palmitic acid (40-60 Ci/mmol) were from Amersham
Pharmacia Biotech. Cell culture supplies, restriction enzymes, ligases,
Taq polymerase, and LipofectAMINE Plus were supplied by Life
Technologies. pcDNA3.1 was purchased from Invitrogen (Netherlands).
Plasmid preparation kits were from Qiagen, and anti-phospho-p42/p44
MAPK and p42 MAPK antibodies were from BD Transduction Laboratories
(Oxford, UK). Reporter horseradish peroxidase anti-rabbit
antibodies were supplied by Scottish Antibody Production Unit
(Carluke, Scotland). All biochemicals, pertussis toxin, suramin, phorbol 12-myristate 13-acetate (PMA), thrombin, epidermal growth factor, LPA, PA, and sn-1,2-dioleoylglycerol were
from Sigma. Escherichia coli diacylglycerol kinase was
purchased from Calbiochem-Novabiochem, and sphingosine 1-phosphate was
from TCS Biologicals Ltd. (Botolph Claydon, UK). Antibodies were
raised to keyhole limpet hemocyanin-coupled peptides (synthesized by
Zinsser Analytical (Maidenhead, UK)) that are unique to LPP2
(ELERKPSLSLTLC) and LPP3 (KEILSPVDIIDRC).
Cell Culture--
HEK 293 cells and stable transfected cell
lines generated from them were maintained in minimum essential medium
(MEM) and 10% (v/v) fetal calf serum. Cells were maintained in MEM
supplemented with 0.1% (v/v) fetal calf serum for 24 h before experimentation.
Transfection--
Plasmid constructs were generated using
pcDNA3.1 (Zeo
) containing either gpLPP1 or gpLPP1a (5). Plasmid
constructs containing hLPP2 and hLPP3 were a generous gift from Dr.
A. J. Morris (State University of New York, Stony Brook, NY).
hLPP2 and hLPP3 were separately subcloned into pcDNA3.1 (Zeo
) at
the BamHI and HindIII sites. HEK 293 cells were
separately transfected with 1 µg of either pcDNA3.1 or
pcDNA3.1-LPP1, -LPP1a, -LPP2, and -LPP3 using LipofectAMINE Plus
and stable transfectants selected using Zeocin. Multiple
surviving colonies of each type were screened for enhanced membrane LPP
activity in an in vitro mixed micellar assay. A single colony of each type was selected and propagated for subsequent experiments.
Synthesis of 32P Lipid
Phosphates--
[32P]dioctanoyl-PA and
[32P] oleoyl-LPA were produced by the diacylglycerol
kinase-catalyzed phosphorylation of
sn-1,2-dioctanoylglycerol and
sn-1,2-dioleoylglycerol, respectively, using
[32P]ATP (1 mM, 222,000 dpm/nmol) in a mixed
micellar assay containing 0.54% v/v Triton X-100, 30 mM
imidazole (pH 7), 30 mM NaCl, 7.5 mM
MgCl2, 0.6 mM EGTA, and 0.6 mM
dithiothreitol. The phosphorylated lipid was isolated by thin layer
chromatography using silica 60 plates developed in
chloroform/methanol/acetic acid (26:6:3, v/v) and extracted using a
series of chloroform/methanol mixtures. [32P]Oleoyl-LPA
was produced by mild alkaline methanolysis of
[32P]dioleoyl-PA using NaOH (in methanol/water, 95:5,
v/v) for 15 min. [32P]Oleoyl-LPA and residual
[32P]dioleoyl-PA were separated using thin layer
chromatography (as above), and [32P]oleoyl-LPA was
similarly extracted.
[32P]S1P was prepared in a similar way by DG
kinase-catalyzed phosphorylation of
N-octanoyl-D-erythro-sphingosine
(C8-ceramide), degradation of the resulting C8-ceramide 1-phosphate by
boiling in 6 M HCl:butan-1-ol (1:1, v/v) for 60 min, and
purification of the resulting [32P]S1P by thin layer
chromatography in chloroform/methanol/acetic acid/H2O
(25:10:1:2, v/v).
Membrane LPP Activity Assay--
Membranes of the stable cell
lines were prepared by their homogenization in ice-cold buffer
(containing 50 mM Tris-maleate, 1 mM EDTA, 150 mM NaCl, and 10 mM mercaptoethanol) and
centrifugation at 30,000 × g at 4 °C for 10 min.
Pellets were resuspended in homogenization buffer (at 20-200 µg of
protein/ml) and stored at
20 °C. Membrane LPP activity was
measured as the liberation of 32Pi from
32P-labeled substrates (1000 dpm/pmol-625 dpm/nmol) in the
presence of Triton X-100 (fixed lipid:detergent ratio of 1:10), 37.5 mM Tris-maleate, 7.5 mM mercaptoethanol, and
0.2 mg/ml bovine albumin at 30 °C for 5 min. Incubations were
stopped by the addition of 5 volumes of chloroform/methanol/10
mM HCl (15:30:2, v/v). Organic and aqueous phases were
resolved by the addition of 1.25 volumes each of chloroform and 0.1 M HCl. Liberated 32Pi was measured
by counting radioactivity in the upper phase. 32Pi liberated from [32P]S1P was
measured by further processing the upper phase with an additional phase
split with equal volumes of 2 M KCl and water-saturated butanol and counting radioactivity in the lower phase. Analysis of the
reaction products by thin layer chromatography confirmed the
specificity of the assay.
Ecto-LPP Activity Assay--
Cells were rinsed twice in
phosphate-buffered saline and incubated in MEM supplemented with 0.1%
(w/v) fatty acid-free bovine albumin and 1 mM
-glycerophosphate at 37 °C for 30 min. Subsequently, [32P]S1P, [32P]dioctanoyl-PA, or
[32P]oleoyl-LPA (final concentration 50 µM,
10,000-15,000 dpm/nmol) prepared in MEM, 0.1% fatty acid-free bovine
albumin, 1 mM
-glycerophosphate was added, and the cells
were incubated for an additional 30 min. 500 µl of the medium was
extracted using chloroform, methanol, 10 mM HCl (15:30:2).
Organic and aqueous phases were resolved by the addition of chloroform
and 0.1 M HCl. 32Pi liberated from
[32P]dioctanoyl-PA or [32P]oleoyl-LPA was
measured by counting radioactivity in the upper phase.
32Pi liberated from [32P]S1P was
measured after an additional phase split as described above. The
inclusion of
-glycerophosphate was a precaution to minimize the
possibility of phospholipase A-catalyzed degradation of
32P-labeled substrates. Analysis of the reaction products
by thin layer chromatography confirmed the specificity of the assay.
Measurement of Basal PA and DG--
Lipid extracts prepared from
cells labeled with [3H]palmitate for 20 h were
halved and analyzed by thin layer chromatography on silica G150 plates
developed as above for PA or in hexane:diethyl ether:acetic acid
(60:40:1, v/v) for DG. Radioactivity comigrating with unlabeled lipid
standards was quantified by scintillation counting.
Phospholipase D Assay--
Phospholipase D activity was measured
using the transphosphatidylation assay.
Blotting--
Immunoblotting was performed as described by us
previously (33). Immunoreactive proteins were visualized using the
enhanced chemiluminescence detection kit and quantified by densitometry.
p42/p44 MAPK Assays--
The phosphorylation of p42/p44 MAPK was
detected by Western blotting using anti-phospho-p42/p44 MAPK
(extracellular signal-regulated kinase (ERK)-1/2) antibody as described
by us previously (33). Immunoreactive proteins were visualized using
enhanced chemiluminescence detection.
Fluorescence Microscopy--
HEK 293 cells stably transfected
with empty vector or expressing recombinant LPP2 and LPP3 were grown on
6-well plates, rinsed three times with phosphate-buffered saline (PBS),
and fixed using paraformaldehyde (3% w/v) in PBS. Subsequently, the
cells were washed successively in PBS, 50 mM ammonium
chloride in PBS, 0.1% (w/v) Triton-100 in PBS and incubated in
blocking buffer (0.2% (v/v) fish gelatin and 0.1% (v/v) goat serum in
PBS) before incubation with antibodies that specifically detect LPP2 or
LPP3 (1:100 dilution of each antibody in PBS, 4 h). LPP expression
was visualized using a fluorescein isothiocyanate-coupled anti-rabbit
IgG (1:400 in PBS, 1 h) on a laser-scanning confocal imaging
system and Laser Sharp software (Bio-Rad).
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RESULTS |
Lipid Phosphate Phosphatase Expression--
We set out to evaluate
whether members of the LPP family can modulate LPA- and
S1P-dependent stimulation of the p42/p44 MAPK pathway
either by limiting bioavailability of these lysophospholipid agonists
at receptors or by regulating the intracellular level of the putative
second messenger, PA. To investigate this, we generated four separate
HEK 293 stable cell lines, each overexpressing either LPP1, LPP1a,
LLP2, or LPP3.
Endogenous LPP activity of vector-transfected HEK 293 cells was very
low, making these cells ideal for transfection. The characterization of
recombinant LPP activities in the transfected cell lines is presented
in Table I. LPP1, LPP1a, LLP2, or LPP3
activity versus endogenous LPP activity in
vector-transfected cells was measured in cell membranes using 50 µM [32P]dioctanoyl-PA,
[32P]oleoyl-LPA, and [32P]S1P. The increase
in recombinant LPP activity was substantial in each transfected cell
line. The fold increases were 74-, 56-, 21-, and 6-fold
(dioctanoyl-PA), 82-, 172-, 74-, and 7-fold (oleoyl-LPA), and 128-, 41-, 271-, and 7-fold (S1P) for LPP1, LPP1a, LPP2, and LPP3,
respectively. Similar rank order activities were measured using a lower
substrate concentration (5 µM) of each substrate. The
fold increase in activities using LPA, PA, and S1P were substantially larger than any other previous report for the transfection of these
enzymes into cells. One possibility for this larger difference is that
we have used stable transfected HEK 293 cell lines, whereas other
workers have used transient transfection where expression levels of the
LPP isoforms are likely to be considerably lower.
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Table I
Expression of LPP isoforms in HEK 293 cells
HEK 293 cells were stably transfected with vector, LPP1, LPP1a, LLP2,
or LPP3, and clones were selected for Zeocin resistance. LPP activities
in cell membranes were measured at 50 µM and 5 µM [32P]dioctanoyl-PA,
[32P]oleoyl-LPA, and [32P]S1P. Activities are
expressed as the means ± S.D. for n = 3 separate
experiments.
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Kinetic estimates for Km and
Vmax for LPP1, LPP1a, LPP2, and LPP3 using these
substrates are shown in Table II. These data show that Vmax values for dioctanoyl-PA are
considerably larger for LPP1 and LPP1a compared with LPP2 and LPP3. In
contrast, LPP2 exhibits the highest Vmax for
oleoyl-LPA. The Vmax values for S1P were
considerably higher for LPP2 than LPP1 and LPP1a, which were in turn
higher than LPP3. Km values for dioctanoyl-PA are
between ~200 µM and 290 µM for all the
enzymes. Km values for oleoyl-LPA are ~ 60, 14, 190, and 250 µM for LPP1, LPP1a, LPP2, and LPP3,
respectively. Km values for S1P are between 25 and
36 µM for LPP1 and LPP3 and ~ 220 µM
for LPP1a and LPP2.
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Table II
Kinetic analysis
Comparison of the Km and Vmax for
no insert (vector alone) and LPP1-, LPP1a-, LLP2-, and LPP3-transfected
cells using [32P]dioctanoyl-PA, [32P]oleoyl-LPA and
[32P]S1P as substrates. Km and
Vmax values for each LPP isoform were calculated by
subtraction of the activities determined in vector-transfected cell
membranes from those measured in each LPP-transfected cell line. These
are the combined results of an experiment performed three times.
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We also found that LPP1, LPP2, and LPP3 exhibit low but significant
ecto activity against [32P]oleoyl-LPA (Table
III). This is in line with other studies
showing that LPP1 and LPP3 have ecto activity in transfected Rat2
fibroblasts and HEK 293 cells, respectively (34, 35). However, our
results are the first to provide evidence that LPP2 exhibits ecto
activity against oleoyl-LPA. The exception is LPP1a, which does not
display activity against this substrate. The increases in ecto activity in LPP1-, LPP1a-, LPP2-, and LPP3-transfected cells compared with vector-transfected cells were 3.6-, 0.8-, 7.7-, and 6.4-fold, respectively (Table III). These activities are similar to those reported for mouse LPP1 in Rat2 fibroblasts (34). The ecto-LPP activity
is not an artifact of uptake of [32P]oleoyl-LPA into the
cells followed by intracellular metabolism and then release of
phosphate into the extracellular medium. If this had been the case,
then the relative ecto-LPP activities between the cell types would
correlate exactly with relative activities measured in cell membranes.
This is clearly not the case since the rank order of ecto-LPP
activities (LPP2 = LPP3 > LPP1
LPP1a = vector-transfected cells) differs from that for LPP activities in membranes (LPP1a > LPP1 = LPP2
LPP3
vector-transfected cells) (see Table I).
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Table III
LPP1, LPP1a, LPP2, and LPP3 ecto activity against oleoyl-LPA,
dioctanoyl-PA, and S1P
HEK 293 cells were stably transfected with vector, LPP1, LPP1a, LPP2,
or LPP3, and clones were selected for Zeocin resistance. Ecto
activities in LPP-transfected cells were expressed as means ± S.D. for n = 3 separate experiments.
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We also found that LPP1, LPP1a, and LPP3, but not LPP2, have
significant but low ecto activity against
[32P]dioctanoyl-PA. The activity increases in LPP1-,
LPP1a-, LPP2-, and LPP3-transfected cells versus
vector-transfected cells were 4.6-, 8.3-, 1.6-, and 2.95-fold,
respectively (Table III). LPP3 also has low ecto activity against
[32P]S1P (2.14-fold increase versus
vector-transfected cells). None of the other LPP isoforms exhibited
significant ecto activity against S1P (Table III).
Additional evidence showing the expression of LPP2 and LPP3 was
obtained using Western blot analysis with isoform-specific antibodies
that recognize unique C-terminal regions in LPP2 and LPP3. Fig.
1a shows that a band with a
molecular mass of 32 kDa was specifically immunostained in
LPP2-transfected cell lysates using anti-LPP2 antibody and,
significantly, was absent from vector-transfected cells. This band was
not detected with anti-LPP3 antibodies. We can, therefore, attribute
the protein to LPP2. A band with a molecular mass of 35 kDa
corresponding to LPP3 was immunostained with anti-LPP3 antibodies in
LPP3-transfected but not vector-transfected cell lysates. This band was
not detected with anti-LPP2 antibodies. The molecular masses of these
proteins determined on SDS-polyacrylamide gel electrophoresis are in
agreement with the predicted values from the amino acid sequences of
the recombinant proteins.

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Fig. 1.
Immunological analysis of LPP2 and LPP3 in
transfected HEK 293 cells. a, Western blot of lysates
from vector-, LPP2-, and LPP3-transfected cells with anti-LPP2
(left)- and anti-LPP3 (right)-specific
antibodies. LPP2 and LPP3 have molecular masses of 32 and 35 kDa,
respectively. Molecular mass markers are also shown. b,
immunohistochemical staining of HEK 293 cells. Antibodies specific for
LPP2 (i-vi) and LPP3 (vii-xii) were used to
detect LPP isoforms by laser-scanning confocal microscopy. HEK 293 cells were transfected with vector alone (i, ii,
vii, viii), LPP2 (iii, iv,
ix, x), and LPP3 (v, vi,
xi, xii). The results show fluorescent
(i, iii, v, vii,
ix, xi) and corresponding transmission
(ii, iv, vi, viii,
x, xii) images. The bar equals 50 µm
unless otherwise indicated. These are representative results of an
experiment performed three times.
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Both antibodies are highly specific and can be used for
immunohistochemical staining of the respective transfected cells. Fig.
1b shows that the antibodies immunostained recombinant LPP2 and LPP3 at or close to the plasma membrane in transfected HEK 293 cells. We therefore propose that the LPP isoforms are located at or
close to the inner membrane. This is based upon the fact that the
ecto-LPP activities are very low and that the antibodies we have used
are raised to C-terminal regions of the LPP isoforms that are predicted
to be cytoplasmic-facing. In addition, LPP2 appears to be localized to
an undefined intracellular compartment. As controls, we also show in
Fig. 1b that the anti-LPP2 and anti-LPP3 antibodies do not
immunostain vector-transfected cells nor do they cross-react with
proteins in LPP3- and LPP2-transfected cells, respectively.
Collectively, the results clearly show that the individual LPP isoforms
are stably expressed in the four transfected cell lines and are
functionally active. Therefore, these cell lines provide an excellent
model to address whether the LPP isoforms can modulate S1P and LPA signaling.
S1P- and LPA-dependent Activation of p42/p44
MAPK--
To investigate whether the LPP isoforms can modulate S1P and
LPA signaling via G-protein-coupled receptors, we first had to establish that S1P and LPA stimulate p42/p44 MAPK via this mechanism in
HEK 293 cells. This was necessary because, in the case of S1P, the
stimulation of p42/p44 MAPK can also occur via an unidentified intracellular-dependent mechanism in certain cell types
(32).
S1P and LPA (0.5-10 µM) stimulated a
concentration-dependent activation of p42/p44 MAPK (data
not shown). This was sustained for at least 30 min and declined toward
basal thereafter (Fig. 2). Both lipid
agonists were less effective than PMA, which short circuits
G-protein-coupled and growth factor receptors to stimulate p42/p44 MAPK
via a protein kinase C-dependent mechanism (Fig. 2). Most
significant, in terms of establishing that these lipids are agonists at
receptors, was the observation that the pre-treatment of cells with the
bacterial toxin, pertussis toxin (0.1 µg/ml for 18 h), which
ADP-ribosylates the G-proteins, Gi/o, and uncouples these
G-proteins from their respective receptors, substantially reduced the
S1P- and LPA-dependent activation of p42/p44 MAPK (Fig.
3a). In addition, we found
that suramin, which can inhibit GPCR signaling blocked the S1P- and
LPA-dependent activation of p42/p44 MAPK (Fig.
3b). These findings suggest that S1P and LPA exert their
effects on p42/p44 MAPK via a GiPCR-dependent
mechanism in HEK 293 cells.

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Fig. 2.
S1P-, LPA-, and PMA-dependent
stimulation of p42/p44 MAPK in HEK 293 cells. Vector-transfected
HEK 293 cells were stimulated with S1P (5 µM), LPA (5 µM), or PMA (1 µM) for the indicated times.
Cell lysates were taken for Western blotting with antibodies that react
with the phosphorylated/activated forms of p42/p44 MAPK. Blots were
stripped and re-probed with antibodies that react with p42 MAPK to
ensure equal protein loading. These are representative results of an
experiment performed three times.
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Fig. 3.
The effect of pertussis toxin and suramin on
the activation of p42/p44 MAPK by S1P, LPA, PMA, and serum in HEK 293 cells. Vector-transfected HEK 293 cells were pre-treated with and
without pertussis toxin (0.1 µg/ml, 18 h) (a) or
suramin (50 µM, 10 min) (b) before stimulation
with S1P (5 µM, 10 min), LPA (5 µM, 10 min), PMA (1 µM, 10 min), or serum (10% (v/v), 10 min).
Cell lysates were Western-blotted with antibodies that react with the
phosphorylated/activated forms of p42/p44 MAPK. Blots were stripped and
re-probed with antibodies that react with p42 MAPK to ensure equal
protein loading. These are representative results of an experiment
performed three times.
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In general we found that the extent of p42 MAPK phosphorylation in
response to stimulation by S1P and LPA exceeded that of p44 MAPK, which
on some Western blots was barely detectable. This is accounted for by
the relative expression of p42 and p44 MAPK in these cells (data not shown).
The relatively high concentration requirement for LPA and S1P used in
this study is in agreement with other reports in various cell types
(18, 22, 36-41). Indeed, Brindley and co-workers (22) show that
decreasing extracellular calcium from 1.8 mM (concentration
in MEM) to 10 µM increased LPA binding by 20-fold, shifting the threshold for p42/p44 MAPK activation to the nanomolar range. Hence, the calcium dependence of the apparent
Kd values for ligand binding to receptors may
explain the longstanding discrepancy of why micromolar LPA is often
needed to activate p42/p44 MAPK at physiological calcium levels. These
findings may explain the similar high concentration requirement for LPA
and S1P seen in our experiments. However, this explanation remains only
a possibility and further experiments are necessary to establish its
validity. In contrast with the effect on LPA and S1P, pertussis toxin and suramin had no effect on serum- or PMA-stimulated p42/p44 MAPK activation (Fig. 3, a and b).
Lipid Phosphate Phosphatases Modulate S1P- and LPA-stimulated
Activation of p42/p44 MAPK--
We next addressed the major question
of this study as to whether the different LPP isoforms could affect the
ability of LPA and S1P to stimulate p42/p44 MAPK. Fig.
4 shows that the activation of p42/p44
MAPK by these lipid agonists was substantially reduced in LPP1-,
LPP1a-, and LPP2- but not LPP3-transfected cells. Comparison with the
ecto-LPP activities shows that there is no correlation with the
abrogation of p42/p44 MAPK activation. For instance, LPP3 exhibits ecto
activity against oleoyl-LPA and S1P that is larger than that of the
other isoforms (Table III). However, LPP3 does not abrogate the
activation of p42/p44 MAPK by LPA or S1P (Fig. 4). There was a better
correlation with membrane LPP activities against PA, LPA, and S1P
measured in Table I. Finally, the expression level of p42 MAPK was not
different between the vector- and LPP-transfected cell lines (Fig.
4).

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Fig. 4.
Lipid phosphate phosphatases attenuate the
activation of p42/p44 MAPK by S1P and LPA. HEK 293 cells stably
transfected with vector, LPP1, LPP1a, LPP2, or LPP3 were stimulated for
10 min with S1P (5 µM), LPA (5 µM), PMA (1 µM), epidermal growth factor (100 nM), or
serum (10% v/v). Cell lysates were taken for Western blotting with
antibodies that react with the phosphorylated/activated forms of
p42/p44 MAPK. Blots were stripped and re-probed with antibodies that
react with p42 MAPK to ensure equal protein loading. These are
representative results of an experiment performed three times.
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PA Stimulation of p42/p44 MAPK--
PA has been shown to stimulate
cell migration via a receptor-mediated mechanism in breast cancer cells
(22). Given that the LPP isoforms also dephosphorylate PA, we tested
first whether PA could stimulate p42/p44 MAPK via a receptor-mediated
mechanism and, second, whether the LPP isoforms might limit PA
bioavailability at this receptor. Fig.
5a shows that PA (30 nM) stimulated p42/p44 MAPK activation via a pertussis
toxin- and suramin-sensitive mechanism, suggesting that this response
is probably mediated via a Gi/o-coupled receptor. This
receptor is clearly different from those that bind LPA and S1P, as the
concentration requirement for PA was in the nM range. The
response to PA is unlikely to be mediated via EDG2 and EDG4, as PA has
a 50-fold lower binding affinity for these receptors compared with LPA
(24). Fig. 5b shows that the PA-dependent activation of p42/p44 MAPK activation was substantially reduced in
LPP1-, LPP1a-, and LPP2- but not LPP3-transfected cells. Comparison with the ecto-LPP activities against PA shows that there is no correlation with the abrogation of p42/p44 MAPK activation. For instance, LPP2 does not exhibit significant ecto activity against PA
(Table III) but does abrogate the activation of p42/p44 MAPK (Fig.
5b).

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Fig. 5.
The effect of pertussis toxin, suramin, and
lipid phosphate phosphatases on the activation of p42/p44 MAPK by PA in
HEK 293 cells. a, vector-transfected HEK 293 cells were
pre-treated with and without pertussis toxin (PTX, 0.1 µg/ml, 18 h) or suramin (50 µM, 10 min) before
stimulation with PA (30 nM, 50 min). b, HEK 293 cells, stably transfected with vector, LPP1, LPP1a, LPP2, or LPP3 were
either unstimulated (C) or stimulated with PA (30 nM, 50 min) or PMA (1 µM). Cell lysates were taken for Western
blotting with antibodies that react with the phosphorylated/activated
forms of p42/p44 MAPK. Blots were stripped and re-probed with
antibodies that react with p42 MAPK to ensure equal protein
loading. These are representative results of an experiment
performed three times.
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GPCR-mediated Stimulation of p42/p44 MAPK--
We next tested
whether the specificity of action by the LPP isoforms was restricted to
the lipid agonists used in this study. We evaluated whether the LPP
isoforms can act on other GiPCR agonists whose
bioavailability at receptors is not subject to regulation by these
enzymes. For this purpose we used the peptide agonist, thrombin, which
stimulates p42/p44 MAPK via a pertussis toxin-sensitive mechanism (Fig.
6a), thereby suggesting that
it binds to a Gi/o-coupled receptor.

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Fig. 6.
The effect of pertussis toxin and lipid
phosphate phosphatases on thrombin-dependent
activation of p42/p44 MAPK in HEK 293 cells. a,
vector-transfected HEK 293 cells were pre-treated with and without
pertussis toxin ((PTX) 0.1 µg/ml, 18 h) before
stimulation with thrombin (0.3 units/ml, 10 min). b, HEK 293 cells stably transfected with vector, LPP1, LPP1a, LPP2, or LPP3 were
stimulated with thrombin (0.3 units/ml, 10 min, upper
panel). c, vector-transfected HEK 293 cells were
pre-treated with vehicle (Me2SO (DMSO)) or
DL-threo dihydrosphingosine (DHS, 1, 5, 10 µM, 15 min) before thrombin (0.3 units/ml, 10 min).
Cell lysates were taken for Western blotting with antibodies that react
with the phosphorylated/activated forms of p42/p44 MAPK. Blots were
stripped and re-probed with antibodies that react with p42 MAPK to
ensure equal protein loading. These are representative results of an
experiment performed three times.
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Fig. 6b shows that the stimulation of p42/p44 MAPK by
thrombin was abrogated in LPP1-, LPP1a-, and LPP2- but not
LPP3-transfected cells. This correlated exactly with the effect of the
LPP isoforms on LPA, S1P, and PA stimulation of p42/p44 MAPK. Thrombin
does not cause release of S1P, PA, nor LPA from HEK 293 cells (data not
shown). In addition we have done control experiments to confirm that
thrombin does not use intracellular S1P as a second messenger to
stimulate the p42/p44 MAPK pathway. This is based upon data shown in
Fig. 6c, where pre-treatment of cells with the sphingosine kinase inhibitor DL-threo-dihydrosphingosine
(used at a concentration that has been shown to completely inhibit
platelet-derived growth factor-stimulated sphingosine kinase activity
and to block platelet-derived growth factor-stimulated p42/p44 MAPK
activation in Swiss 3T3 fibroblasts (32)) did not block the
thrombin-dependent activation of p42/p44 MAPK (Fig.
6c). These data appear to exclude the dephosphorylation of
intracellular S1P as a possible mechanism by which LPP1, LPP1a, and
LPP2 abrogate stimulation of p42/p44 MAPK by thrombin.
We also looked at other agents that do not use G-proteins to signal to
p42/p44 MAPK, such as epidermal growth factor, serum factors, and PMA.
Fig. 4 shows that none of the LPP isoforms altered p42/p44 MAPK
activation in response to serum or PMA. The response to epidermal
growth factor was only partially reduced (<20%) by LPP1, LPP1a, and LPP2.
PA Metabolism--
We next investigated whether the LPP isoforms
alter the intracellular levels of PA and DG, both of which have been
implicated as second messengers for LPA, S1P, and thrombin. Our
experiments show that there was a correlation between the ability of
the LPP isoforms to abrogate activation of p42/p44 MAPK and changes in basal intracellular PA and DG levels. Table
IV shows that basal intracellular PA
levels were decreased, whereas DG levels were increased in LPP1-,
LPP1a-, and LPP2-transfected but not LPP3-transfected cells.
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Table IV
The effect of LPP isoforms on the intracellular DG/PA ratio
HEK 293 cells were stably transfected with vector, LPP1, LPP1a, LLP2,
or LPP3, and clones were selected for Zeocin resistance. DG and PA
levels were measured according to "Experimental Procedures" and are
the means ± S.D. (per 2.5 × 105 cells) for
n = 3 separate experiments.
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Importantly, the correlation was specific with changes in basal PA/DG
levels and not agonist stimulation of PA formation. This was based upon
two pieces of evidence. First, neither S1P nor LPA increased
intracellular PA (Fig. 7). Second,
although PMA stimulates phospholipase D to generate PA in these cells
(Fig. 7), the ability of this agent to activate p42/p44 MAPK was not altered at all by expression of LPP1, LPP1a, LPP2, and LPP3 (Fig. 4 and
Fig. 5b).

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Fig. 7.
PMA stimulation of phospholipase D. Vector-transfected HEK 293 cells were stimulated with S1P (5 µM), LPA (5 µM), or PMA (1 µM) for 10 min. Phospholipase D activity was measured
using the transphosphatidylation assay. These are representative
results of an experiment performed three times. DMSO,
Me2SO; PtdBut, phosphatidylbutan-1-ol.
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DISCUSSION |
The first major finding of this study is that the stimulation of
p42/p44 MAPK by LPA, S1P, and PA is substantially reduced in LPP1-,
LPP1a-, and LPP2- but not LPP3-transfected HEK 293 cells. To date, only
LPP1 has been shown to abrogate LPA-dependent stimulation of p42/p44 MAPK and mitogenesis in Rat2 fibroblasts and HEK 293 cells,
respectively (22, 23). Therefore, the current study is not only the
first to extend this to other LPP isoforms but, significantly, is also
the first to report that the LPP isoforms can attenuate the stimulation
of p42/p44 MAPK by S1P and PA. The inability of LPP3 to abrogate
activation of p42/p44 MAPK by the lipids may be due to the fact that
its expression in HEK 293 cells is considerably less than the other LPP isoforms.
Some of the LPP isoforms exhibited Vmax values
that were considerably larger than any other previously reported for
these enzymes in transfected cells (2, 3, 5). These values simply
reflect particularly good expression of the enzymes in the
stable-transfected HEK 293 cells. The estimated Km values for dioctanoyl-PA were ~200-290 µM for all the
enzymes, whereas those for oleoyl-LPA were 14-60 µM for
LPP1/LPP1a and ~190-250 µM for LPP2/LPP3. These values
are very similar to those obtained by Hooks et al. (43), who
determined Km values for dioleoyl-PA of 98, 150, and
100 µM and for oleoyl-LPA of 170, 340, and 110 µM for LPP1, LPP2, and LPP3, respectively, in transfected HEK 293 cells. In addition, we report for the first time that LPP2 has
both intracellular and extracellular activity and have provided
evidence of its localization in transfected HEK 293 cells.
The second major finding of this study is that the abrogation of
p42/p44 MAPK activation by LPP1, LPP1a, and LPP2 is not due to
limitation of the bioavailability of the lipid agonists at their
respective receptors. This was based upon two lines of evidence. First,
there was no correlation between ecto-LPP activity against LPA, S1P,
and PA and the abrogation of p42/p44 MAPK activation. Second, LPP1,
LPP1a, and LPP2 also abrogated the stimulation of p42/p44 MAPK by
thrombin, a peptide Gi/o-coupled receptor agonist whose
bioavailability at its receptor is not subject to regulation by the LPP isoforms.
These findings contrast with studies by Xu et al. (22),
where ecto activity has been suggested to account for the effect of
LPP1 on LPA-stimulated signaling in Rat2 fibroblasts. However, it is
important to note that the study by Xu et al. (22) did not
investigate the effect of LPP1 on other GPCR agonists and so the
specificity of its action, and therefore the mechanism was not
fully defined. Our findings do agree to some extent with those of Hooks
et al. (23). These authors found that although ecto-LPP1
degrades ~90% of the LPA in the medium over a 24-h period in HEK 293 cells, this was insufficient to account for the decrease in LPA potency
in mitogenic assays, suggesting an alternative unidentified mechanism.
Moreover, these authors reported, using degradation-resistant
phosphonate analogs of LPA and stereoselective agonists of the EDG
receptors, that the mitogenic response of LPA was independent of EDG2,
-4, and -7. These authors therefore suggest that the responses to LPA
may be mediated via a distinct GPCR, possibly PSP24, an LPA receptor
that is not a member of the EDG family (20). In our studies, we found
that LPA activates p42/p44 MAPK via a GPCR that is sensitive to
pertussis toxin and suramin, although we do not know as yet whether
this receptor is a member of the EDG family. We have also found that
exogenous PA stimulates p42/p44 MAPK via a Gi/o-coupled
receptor that appears to exhibit a much higher affinity for PA compared
with LPA at its receptor. Therefore, it is possible that PA binds to a
distinct receptor from that which binds LPA.
The LPP isoforms have no effect on the stimulation of p42/p44 MAPK by
other agents that do not use G-proteins to signal, such as serum
factors and phorbol ester. Taken together, these findings represent a
major advance as they clearly show that LPP1, LPP1a, and LPP2 may
function to abrogate GPCR signaling per se.
Basal intracellular PA levels were decreased, whereas DG levels were
increased in LPP1-, LPP1a-, and LPP2-transfected but not
LPP3-transfected cells. This is in line with studies by Leung et
al. (4) using HEK 293 cells transfected with LPP1 or LPP1a. Agonist-stimulated PA has been implicated in the activation of Raf and
the inhibition of Ras-GTPase-activating protein (25, 26), both
of which are up-stream components in the p42/p44 MAPK pathway. However,
neither S1P nor LPA increase PA in HEK 293 cells. We propose that the
LPP isoforms act on a basal pool of PA, which may have a distinct
function from agonist-stimulated PA. One possibility is that basal PA
contributes to the structural integrity of lipid micro domains within
the plasma membrane, in which signal complexes are assembled.
Alternatively, PA may regulate the formation of endocytic vesicles
required for GPCR signaling.
The magnitude of the increase in DG levels in LPP1-, LPP1a-, and
LPP2-transfected cells did not equate with the reduction in PA.
Identical reciprocal changes in the amounts of DG and PA might be
expected if DG production was exclusively via the action of the LPP
isoforms. This suggests additional routes of DG synthesis in these
cells. However, it is unlikely that there is a role for increased basal
intracellular DG in abrogating p42/p44 MAPK activation, even though the
chronic increase in intracellular DG levels does raise the possibility
of down-regulation of protein kinase C, an enzyme that has been shown
to regulate p42/p44 MAPK activation. This would have the effect of
attenuating the action of agonists that use protein kinase C to
stimulate p42/p44 MAPK activation, such as LPA and S1P. However, we
suggest that functional changes in protein kinase C expression are
unlikely given that the activation of p42/p44 MAPK by acute treatment
of cells with PMA, which stimulates the same DG-sensitive protein
kinase C isoforms that would be down-regulated by chronic DG, was
unaffected by the LPP isoforms.
We speculate here that the LPP isoforms may act on a small pool of
basal intracellular PA that specifically regulates the membrane dynamic
for signaling via G-protein-coupled receptors. There are good reasons
to believe that this may be the case. First, the attenuation of p42/p44
MAPK activation by the LPP isoforms is common to several GPCR agonists
used in this study, including thrombin. Second, LPP isoforms may
localize to caveolin-enriched lipid rafts, where the enzyme could
function to maintain structural integrity by precisely regulating the
PA concentration. This is important because Gi
is also
localized in lipid rafts containing caveolin (31), which is required
for GPCR signaling. Third, the LPP isoforms may dephosphorylate a pool
of PA that plays a role in clathrin coat assembly and vesicle formation
for endocytosis (29). This is important because the endocytosis of
G-protein-coupled receptor signal complexes is required for efficient
phosphorylation of MEK-1 (mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase 1) by Ras-Raf
(45-47). Furthermore, the ratio of LPA/PA at the inner leaflet of the
plasma membrane is important for vesicular budding (48). This involves
endophilin I, an enzyme that exhibits lysophosphatidic acyltransferase
activity and generates PA, which is required for endocytic vesicle
formation. Furthermore, endophilin interacts with dynamin II (44) and
GPCRs (42).
In conclusion, the overexpression of LPP1, LPP1a, and LPP2 in cells
might deplete the intracellular pool of PA sufficiently to disrupt (i)
the recruitment of specific signaling proteins to the caveolae and/or
(ii) the endophilin I-mediated endocytic process required for
GPCR-dependent stimulation of p42/p44 MAPK. This proposal
is currently under investigation in our laboratory.