From the Departments of Biochemistry,
¶ Chemistry, and
Pharmacology, University of Virginia
Health Sciences Center, Charlotte, Virginia 22908
Received for publication, August 25, 2000, and in revised form, October 18, 2000
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ABSTRACT |
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Lysophosphatidic acid (LPA) is an extracellular
signaling mediator with a broad range of cellular responses. Three
G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as
receptors for LPA. In this study, the ectophosphatase lipid phosphate
phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated
mitogenesis. Furthermore, using degradation-resistant phosphonate
analogs of LPA and stereoselective agonists of the Edg receptors we
have demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, we found
that LPA degradation is insufficient to account for the decrease in LPA
potency in mitogenic assays, and the stereoselectivity observed at the
Edg receptors is not reflected in mitogenesis. Additionally, RH7777
cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a
mitogenic response to LPA and LPA analogs. Finally, we have determined
that the ligand selectivity of the platelet aggregation response is
consistent with that of mitogenesis, but not with Edg-2, -4, and
-7.
The structurally simple phospholipid lysophosphatidic acid
(LPA,1 1-acyl,
2-hydroxy-sn-glycerol-3-phosphate) has been shown in the
past decades to mediate an array of biological responses that is
anything but simple. LPA has been shown to signal such vital cellular
events as mitogenesis (1), platelet aggregation (2), tumor cell
invasion (3), and escape from apoptosis (4). LPA is present in serum at
low (1-20 µM) micromolar concentrations (5) and
is produced and released by stimulated platelets (6). The autocrine
activation of platelet aggregation, coupled with LPA's mitogenic
effects on smooth muscle cells (7) and fibroblasts (8), suggests a
functional role for LPA as a wound-healing hormone (see review (9)).
Furthermore, LPA accumulates to high concentrations in ovarian cancer
ascitic fluid and is mitogenic to ovarian cancer cells, implicating LPA
as a marker and mediator of ovarian cancer progression (10).
In a wide variety of tissue culture cells, LPA elicits Ca2+
mobilization with an EC50 of 0.2-10 nM (11,
12), inhibition of cAMP accumulation with an EC50 of
50-100 nM (1, 13, 14), cell rounding (15), and other
cytoskeletal rearrangements such as formation of stress fibers and
focal adhesions (16), fibrinogen binding (17), and neurite retraction
(18) also with nanomolar potency.
Recently, specific G-protein-coupled receptors for LPA have been
identified within a cluster of eight related receptors termed the Edg
(Endothelial differentiation gene)
cluster. To date, eight members of the Edg cluster have been described.
Edg-2 (15, 19), -4 (20), and -7 (21, 22) have been shown to be LPA
receptors and Edg-1 (23, 24), -3, -5 (25), -6 (26, 27), and -8 (28) are
receptors for the structurally related phospholipid sphingosine
1-phosphate. The LPA receptors differ with respect to distribution and
G-protein coupling. Edg-2 is widely expressed, with highest mRNA
levels appearing in brain (20), reflecting expression in Schwann cells
and oligodendrocytes (29, 30). Edg-4 is most highly expressed in testis
and leukocytes (20), whereas Edg-7 is very highly expressed in kidney
and prostate (21, 22). When stably transfected into the
LPA-unresponsive cell line RH7777 (Rat
Hepatoma), Edg-2 mediates inhibition of cAMP accumulation,
whereas Edg-4 and Edg-7 mediate calcium mobilization (22). Because only
the inhibition of cAMP accumulation is pertussis toxin-sensitive, this
suggests that, in this system, Edg-2 is Gi-linked,
whereas Edg-4 and -7 are Gq-linked. However, in Jurkat T
cells (31) or rat oligodendrocytes (32, 31) Edg-2 mediates calcium
mobilization via G Van Corven and colleagues first described a pertussis toxin-sensitive
mitogenic response to LPA in Rat 1 cells (8). In contrast to LPA's
effects on calcium mobilization, cAMP accumulation, and cytoskeletal
rearrangement, the mitogenic response has a half-maximal concentration
in the mid-micromolar range in fibroblasts, ovarian cancer cell lines,
and cultured vascular smooth muscle cells (7, 8, 33). This discrepancy
has been suggested to reflect degradation over the long time course of
the mitogenesis experiments (24 h or more) as compared with the assays
for higher potency responses (seconds to minutes). LPA also has
micromolar EC50 with respect to platelet aggregation,
although this response is measured in an assay of only a few minutes
duration and is therefore presumably not greatly affected by
degradation (34-36).
The degradation of LPA may be attributed to the activity of integral
membrane enzymes lipid phosphoric acid phosphatases (LPPs) (also named
type 2 phosphatidic acid phosphatases (PAP2)) (reviews: Refs. 37, 38).
The human clones LPP1 (PAP2A) and LPP3 (PAP2B) were cloned by Kanoh in
1997 (39), and our group subsequently cloned a third isoform, LPP2
(PAP2C) (40). In addition to phosphatidic acid, all three enzymes
exhibit broad substrate specificity, including the lipid-signaling
mediators sphingosine 1-phosphate and LPA (39-42). The three
isoforms of LPP act as ectophosphatases by hydrolyzing exogenous LPA
(44), suggesting a potential role in the regulation of LPA signaling by
degrading LPA at the cell surface. The Km for
LPP-mediated hydrolysis of LPA is several orders of magnitude higher
concentration than that required for most LPA-signaling responses (40)
(see Fig. 5A). Therefore, it is not clear if the activity of
the LPPs has a physiologically significant effect on the activity of
LPA at its receptors, because LPA is maximally efficacious at
concentrations well below the affinity of the phosphatases for LPA.
However, the phosphatases may significantly affect LPA receptor
activation despite their lower affinity, if the concentration of
phosphatase is much higher than the receptor concentration. Furthermore, the high Km of the phosphatases
suggests that they will exhibit first order reaction kinetics under
physiologic concentrations of LPA.
This study was initiated with the goal of determining the role of the
phosphatases in the regulation of LPA signaling; specifically, we
wanted to test the hypothesis that the markedly lower potency of LPA
with respect to mitogenesis was simply a result of LPP-mediated hydrolysis. Brindley has previously reported that in one-point assays
the mitogenicity of LPA is diminished in LPP1-overexpressing fibroblasts (44). We wanted to characterize this interaction further
and demonstrate that LPP1 lowers the potency of LPA by hydrolyzing the
phosphoester bond and thus destroying the ligand for LPA receptors. To
this end, we synthesized two LPA analogs with phosphonate head groups,
which should render them resistant to hydrolysis by the LPPs. If our
hypothesis is true, then the potency of the phosphonate compounds
relative to LPA should be greater in mitogenic assays than in assays
with a shorter incubation period. Also, increases in the activity of
LPP enzymes should diminish the activity of LPA but have no effect on
the potency of the phosphonates. In the course of testing this
hypothesis, we discovered a striking difference in the ligand
selectivity of the mitogenic response as compared with that of the
three cloned LPA receptors. The mitogenic response to LPA and LPA
analogs is consistent with the platelet aggregation response but
inconsistent with activation of Edg-2, -4, or -7.
Synthesis of Transfection and Membrane Preparation--
Plasmid DNA was
transfected into HEK 293 T cells using a calcium phosphate
transfection protocol (45). Briefly, a DNA mixture containing 25 µg
of DNA and 0.25 M CaCl was added to HEPES-buffered 2 mM Na2HPO4. Subconfluent monolayers
of HEK 293 T cells were poisoned with 25 mM chloroquine,
and the DNA precipitate was then applied to the cells. After 4 h,
the monolayers were washed with phosphate-buffered saline and refed
media (90% 1:1 Dulbecco's modified essential media (DMEM):F-12 + 10%
fetal bovine serum). The cells were harvested 48-72 h after addition
of the DNA by scraping in HME buffer (in mM: 20 HEPES, 5 MgCl2, 1 EDTA, pH 7.4) containing 10% sucrose on ice, and
disrupted using a Dounce homogenizer. After centrifugation at 800 × g, the supernatant was diluted with HME without sucrose
and centrifuged at 100,000 × g for 1 h. The resulting pellet was rehomogenized and centrifuged a second hour at
100,000 × g. This crude membrane pellet was
resuspended in HME with sucrose, aliquoted, and snap-frozen by
immersion in liquid nitrogen. The membranes were stored at Radiolabeling Lipids--
Phosphorus-32-labeled lysophosphatidic
acid was prepared by reacting monoolein (NuChek Prep) with
[ Assay for LPP (PAP2) Activity--
Assays for phosphatase
activity were carried out according to the protocol described by Kanoh
et al. (42), using a constant Triton X-100:labeled lipid
ratio of 50:1. The final specific activity of the lipids was adjusted
to 10-50 Ci/mol. Briefly, 10 µg of membrane protein was prewarmed to
37 °C in a reaction mixture containing 50 mM Tris-HCl
(pH 7.5), 1 mM EDTA, and 1 mg/ml fatty acid-free bovine
serum albumin. Reactions were started by the addition of the
radiolabeled lipid-Triton X-100 mixture, incubated at 37 °C for 5 min, and stopped by the addition of 100 µl of 0.1 N HCl
in methanol. 200 µl of CHCl3 and 200 µl of 1 M MgCl2 were then added, and the reactions were
vortexed and centrifuged briefly, and a fraction of the aqueous layer
was added to scintillation fluid for counting.
Intact Cell LPP Assays/Ectophosphatase Assays--
The
ectophosphatase activity of LPP-overexpressing cells was determined
with an assay derived from the assay described above. HEK 293 cells
were suspended (via trypsinization) in HKRB (in mM: 20 HEPES, 103 NaCl, 4.8 KCl, 0.5 CaCl2, 1.2 MgSO4,
1.2 KH2PO4, 25 NaHCO3, 15 glucose,
pH 7.4) at a concentration of 106 cells/ml. LPA was
added to each 50-µl reaction in 0.1% fatty acid free bovine serum
albumin. The reactions were allowed to proceed for 10 min at 37 °C
and stopped with the addition of acidified methanol. The reactions were
spun at 12,000 × g for 5 min to remove any cell
debris. The supernatant was extracted with chloroform, and the
radioactivity in the aqueous phase was determined with scintillation counting.
Calcium and cAMP Assays--
Calcium mobilization assays were
performed as described previously by us (13). Briefly, intercellular
calcium fluxes were measured on cell populations (2-4 × 106 cells) that had been loaded with the calcium-sensitive
fluorophore, INDO-1. Responses, which were measured in a
temperature-controlled fluorimeter (Aminco SLM 8000C, SLM Instruments,
Urbana, IL), are reported as the fraction of the maximal response
(i.e. response to 75 µM digitonin). Calcium
mobilization assays were performed in HKRB.
GTP Mitogenesis Assay--
Mitogenesis was estimated by measuring
DNA synthesis using a tritiated thymidine incorporation assay. Nearly
(70%) confluent monolayers were grown in multiwell plates (48 and 96 wells) in DMEM-F12 (HEK 293 cells), DMEM (MDA-MB231 cells), or MEM
supplemented with pyruvate and glutamine (RH7777 cells) plus 10% fetal
bovine serum. Cell lines stably transfected with LPP1 or Edg receptor were grown in the appropriate media plus 800 µg/ml G418. Cells were
deprived of serum for 30 h, and then treated with drug or vehicle
for 22 h. 1 µCi/ml of [3H]thymidine was added
4 h before harvest. Incorporated thymidine was harvested by first
fixing the cells in cold methanol, washing with phosphate-buffered
saline, and precipitating DNA with cold 10% trichloroacetic acid.
Precipitated material was either counted directly by adding scintillant
and counting in a Packard Top counter (for 96-well plates) or
solubilized with 0.2 N NaOH, 0.1% SDS, neutralized with
HCl, and added to scintillant in scintillation vials for counting (for
48-well plates).
Platelet Aggregation Assay--
Platelets were isolated from
fresh human blood from donors (47). Briefly, blood was added to
acid-citrate-dextrose to prevent coagulation, and centrifuged at low
speed to prepare platelet-rich plasma. Indomethacin and apyrase were
added to the platelet-rich plasma, and this preparation was centrifuged
at high speed to sediment platelets. Platelets were resuspended in
buffer (in mM: 140 NaCl, 0.34 Na2HPO4, 2.9 KCl, 10 HEPES, 12 NaHCO3, 5 glucose, 2 MgCl2) and their
aggregation was measured in a Lumi-aggregometer (Chrono-Log, Havertown,
PA). Prior to addition of LPA and analogs, 0.16 µM ADP
was added.
Activity of the Phosphonates at LPA Receptors--
Unfortunately,
these phosphonate compounds cannot be used in the most obvious
experiment, blocking the LPP activity with the phosphonates and
determining if there is an effect of LPA potency, because both wls-31
and wls-60 are themselves potent agonists at the LPA receptors. To
compare the potency of these compounds with that of LPA at the three
cloned LPA receptors, we used a GTP LPPs Attenuate LPA-driven Mitogenesis--
Having characterized
the activity of the phosphonates with the LPPs and the LPA receptors
Edg-2, -4, and -7, we next used the compounds to probe the interaction
between the LPPs and LPA receptors in the mitogenic response. The first
question we addressed was whether the phosphonates had a higher potency
relative to LPA in mitogenesis compared with short-term (GTP
Next, we asked if increases in LPP1 activity affect LPA-induced
mitogenesis to a greater extent than phosphonate-induced mitogenesis. If LPP enzymes down-regulate LPA signaling by hydrolyzing the phosphoester bond of LPA, then overexpression of the enzymes should lower the potency of LPA in mitogenic assays but the potency of the
phosphonates should be unaffected. We developed a stable line of HEK
293 cells overexpressing LPP1 enzymes, resulting in a severalfold increase in ectophosphatase activity, as measured with exogenous albumin-bound radiolabeled LPA as substrate (Fig.
5A). Several clonal lines of
LPP1-overexpressing HEK 293 cells were isolated, but the expression of
the gene appeared to have a negative effect on growth (i.e.
LPP1 transfected cells grew more slowly than nontransfected cells).
Additionally, the level of phosphatase activity diminished over time in
many of the lines; presumably LPP1 expression was selected against due
to the negative effects on growth. Therefore, the following experiments
were performed in a single clonal line chosen because of its stable
increase in ectophosphatase activity. Dose-response curves for
mitogenesis were determined for LPA in normal and LPP1-overexpressing
HEK 293 cells (Fig. 5B). The activity of LPA in the
phosphatase-overexpressing cells was ablated completely. Curves for the
LPP-resistant phosphonates were also determined in LPP1-overexpressing
cells, and both compounds were still of similar potency as seen in
plain HEK 293 cells (Fig. 5C). These results suggest that
the LPP1 enzymes down-regulate LPA signaling by hydrolyzing the
phosphoester bond and thereby lower the effective concentration of
ligand available for receptor binding. In contrast to the sensitivity
of LPA-induced mitogenesis to LPP overexpression, when
GTP The Ligand Selectivity of the Mitogenic Response Is Inconsistent
with That of Edg-2, -4, and -7--
As discussed above, the potency of
the phosphonates relative to LPA increased in the long term mitogenic
assay compared with the short term GTP
A possible explanation for this observation is that the mitogenic
response does not proceed through Edg-2, -4, or -7, as we had presumed.
Our laboratories have been engaged in defining the structure-activity
profiles of the three cloned LPA receptors using synthetic LPA analogs
to assign endogenous LPA responses to specific receptors. As part of
this effort, we have developed a 2-palmitoyl LPA wherein the alkyl
moiety is attached to the second carbon of the glycerol backbone by an
amide linkage, N-palmitoyl-2-methylenehydroxy ethanolamide
phosphoric acid (MHEPA) (Fig. 1). MHEPA is structurally related to
N-acyl serine phosphoric acid (NASPA) (also equivalent to
carboxy ethanolamide phosphoric acid), an LPA analog that was first
described by Hanahan's group (51) and that our group later demonstrated is a stereoselective agonist of LPA responses (14). L(s)-NASPA is a potent LPA receptor agonist,
whereas D(r)-NASPA is inactive. In MHEPA,
the chiral center of NASPA is maintained as is the stereoselectivity.
In assays of calcium mobilization, cAMP accumulation (data not shown),
and GTP
To explore further the possibility that mitogenesis in response to LPA
is distinct from Edg receptor activation, we assayed the mitogenic
response to LPA in rat hepatoma RH7777 cells. These cells are devoid of
LPA-mediated calcium mobilization and inhibition of cAMP accumulation
responses (22, 48), and do not express Edg-2, -4, and -7 mRNA as
judged by reverse transcription-polymerase chain
reaction.3 Nevertheless,
RH7777 cells show a dose-dependent incorporation of
tritiated thymidine when treated with LPA, R-MHEPA (and
S-MHEPA, data not shown), and wls-60 (Fig. 7E).
Although the curves reflect significant error due to poor adhesion of
the cells, the rank order potency of these compounds was similar to
that seen in HEK 293 and MDA MB231 cells. We also examined the
mitogenic response to LPA in clonal RH7777 cell populations that had
been transfected with Edg-2, -4, or -7 DNAs (22) and found that each of
these lines had mitogenic responses to LPA similar to those in
nontransfected cells (data not shown). Together, these data suggest
that the mitogenic response to LPA and synthetic LPA analogs is
independent of Edg-2, -4, or -7 in these three cell lines.
Platelet Aggregation Has a Ligand Selectivity Similar to the
Mitogenic Response--
Like the mitogenic response to LPA, platelet
aggregation requires micromolar concentrations (even though the assay
lasts only 1-2 min) and lacks stereoselectivity in response to
NASPA/carboxy ethanolamide phosphoric acid and related chiral LPA
analogs when tested at a single high concentration (36). We determined
platelet aggregation dose-response curves using the keto phosphonate
(wls-60), the R and S MHEPA compounds, and LPA to
determine whether the ligand selectivity correlated to that of
mitogenesis (Fig. 8). The platelet
response has a pharmacology that closely matches that of the mitogenic
response, i.e. wls-60 and LPA are nearly equipotent and both
enantiomers of MHEPA are active. The difference in the relative
potencies of LPA and wls-60 in mitogenesis versus platelet
aggregation is consistent with the amount of degradation that was
observed during the course of the mitogenic assay; because only 10% of
LPA is intact throughout the mitogenic assays, the degradation-resistant phosphonate should be one order of magnitude higher in potency relative to LPA with respect to mitogenesis than the
shorter assays of platelet aggregation. These data suggest that the
platelet aggregation response to LPA is inconsistent with the
pharmacology of Edg-2, -4, or -7, whereas it is consistent with the
mitogenic response.
The results of the study reported here are remarkable in several
aspects. First, we have described a novel pair of compounds, the
N-acyl The phosphonate compounds characterized in this study have potential to
be useful tools to study the interaction of LPPs and LPA receptors.
They are the first useful inhibitors of LPPs. It has been shown that
LPP activity is blocked by propranolol and sphingosine, but the
IC50 values are quite high. Furthermore, high dose
propranolol is nonspecific, and sphingosine is thought to inhibit by
binding substrate (49). The lack of specific inhibitors and the
ubiquity of the enzymes has made it difficult to investigate the
physiologic role of the LPPs. The ideal inhibitor for investigating the
endogenous role of the LPPs would specifically suppress the activity of
the phosphatases, but lack LPA receptor agonist activity. The compounds
reported here are not yet ideal due to their activity at the Edg
receptors and in mitogenic and platelet aggregation assays, but they
might be useful as lead structures for optimization.
Indeed, the phosphonate compounds used in this study are best described
as nonhydrolyzable or LPP-resistant LPA agonists. In GTP Our data do not support our hypothesis that LPP1-mediated degradation
of LPA is solely responsible for the lower potency of LPA in mitogenic
assays. Instead, we have shown three lines of evidence that suggest
that the mitogenic response to LPA is mediated by a pathway or
mechanism that is distinct from activation of Edg-2, -4, and -7 and has
different ligand selectivity, including an intrinsically lower affinity
for LPA. First, we have shown that the potency of the phosphonate
compounds wls-60 and wls-31 is increased in mitogenic assays to a
degree that cannot be accounted for by degradation of LPA during the
mitogenic assays. Second, R-enantiomers of compounds that
are inactive in a variety of Edg-2, -4, and -7 signaling responses
(GTP Several studies on LPA-induced platelet aggregation have revealed
inconsistencies with Edg receptor-mediated responses to LPA. The
platelet response lacks stereoselectivity (36), requires micromolar
concentrations of LPA (36, 51-53), and displays distinct ligand
selectivity with respect to 1-O-alkyl LPA (ether linkage) (34). Gueguen and colleagues (36) have proposed, based on these
observations, that there is an LPA receptor in platelets that is
pharmacologically distinct from Edg-2, -4, and -7; our data support
their idea. Furthermore, we have shown that the ligand selectivity of
the platelet response is consistent with the pharmacology of the
mitogenic response, suggesting that these responses are mediated by a
common pathway.
The data shown here do not rule out the possibility that the known LPA
receptors play a role in the mitogenic response to LPA in some cells.
It is possible that Edg receptor oligomerization or interaction with
other modulating proteins can affect the relative potency of receptors
for ligands; however, the mitogenic response in the RH7777 cells
suggests that at least in some cases the mitogenic response is
independent of these receptors. An obvious explanation is that an
additional member of the Edg receptor family remains to be identified,
but this is becoming less likely as the sequencing of the human genome
nears completion with no additional members of the Edg cluster
discovered. A more likely explanation is that a G-protein-coupled
receptor outside of the Edg cluster (presumably Gi-linked
due to pertussis toxin sensitivity) is responsible for mitogenesis. In
fact, the G-protein-coupled receptor PSP24 has been reported as a LPA
receptor and is not within the Edg cluster (54). However, the lack of
stereoselectivity and high concentration required for mitogenesis are
also consistent with the possibility that these effects are not
receptor-mediated but rather are mediated by a physical perturbation of
the membrane. Pertussis toxin sensitivity demonstrates that LPA-induced
mitogenesis is dependent on the action of a Gi-linked
protein, but this protein could be several steps downstream of LPA's
site of action. Therefore, the nature of the mediator of the mitogenic
(and platelet aggregation) response to LPA is unknown.
The data presented here do support the hypothesis that LPPs
down-regulate LPA signaling in mitogenic assays by degrading LPA, although this degradation does not account for the lower potency of LPA
in mitogenic assays. In HEK 293 cells that have severalfold overexpression of LPP1, the mitogenic response to LPA is ablated. Interestingly, we did not see an effect on the potency or efficacy of
LPA-induced GTP Brindley and colleagues (55) proposed in the same report that high and
low potency effects of LPA correlate to low and high concentrations of
calcium, respectively; however, there are many reports in the
literature of high potency LPA effects (low nanomolar EC50
levels) in the presence of high calcium ion concentrations. Hopper
et al. (56) reported calcium mobilization in response to LPA
with an EC50 of 5 nM in MDA MB-231, and
Moolenaar's group (12) has reported an LPA EC50 of 0.2 nM in A-431 cells, measuring calcium mobilization in a
buffer system containing 1 mM calcium. The potency of LPA
with respect to inhibition of cAMP accumulation has been measured and
reported in two previous papers by us to be 20 nM (13) and
52 nM (14), and these experiments were performed in 1.8 mM calcium. LPA was also shown to have a similar but
slightly lower potency (EC50 = 100 nM) in
assays of inhibition of cAMP accumulation in the absence of calcium
ions (57). Lafontan and colleagues (58) have reported LPA-induced
spreading of adipocytes with an EC50 of 4 nM in
1.8 mM calcium.
We support, however, Brindley's general proposal that the presence of
calcium greatly affects the physical state of LPA and that the physical
state of LPA can potentially affect its signaling properties. LPA has
been shown to bind calcium ions with high affinity, causing the
formation of microprecipitates of LPA at micromolar concentrations
under physiologic concentrations of calcium (11). The presence of
calcium and other LPA binding molecules such as albumin affects the
physical and signaling properties of LPA. Also potentially affecting
LPA's signaling properties is its ability to insert into the plasma
membrane bilayer. Insertion of lipid monomers into the membrane is not
expected to occur to any appreciable extent below the critical micelle
concentration; however, the critical micelle concentration of LPA
(reported to be ~1.3 mM in pure water (8)) has not been
determined in physiologic solutions in the presence of biological
membranes. Therefore, the degree to which LPA partitions into the
bilayer under physiologic conditions is not known. Much work remains to
be done to determine the existence of LPA in various physical states
(free LPA monomer, calcium aggregates, bound to albumin, or inserted in
the bilayer) under physiologic conditions and the signaling
capabilities of these different forms of LPA.
In conclusion, the data presented here suggest that the LPA receptors
that have been cloned and characterized to date are neither sufficient
nor necessary to account for the mitogenic response to LPA. We propose
a new model for LPA signaling in which at least one additional
mechanism exists that mediates LPA-induced mitogenesis and platelet
aggregation, and LPP enzymes negatively regulate this pathway by ligand
degradation (Fig. 9). The nature of this
mechanism has yet to be defined and is not necessarily a receptor. The
lack of a molecular target for this pathway is ironic in that
mitogenesis and platelet aggregation are among the first described
responses to LPA and are responsible for much of the scientific
interest in LPA signaling due to their potential involvement in
therapeutically relevant physiologic and pathologic roles for LPA
signaling in wound healing and ovarian cancer progression. This model
drastically alters our view of LPA signaling and the way in which it is
investigated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i, while Edg-4 utilizes
G
i and G
q. Thus, LPA receptor coupling
may be dependent on the system of expression. The cloning of specific
receptors for LPA represents a major advancement in the study of LPA
signaling and the physiologic significance of receptor activation;
however, endogenous cellular responses to LPA have not yet been mapped
to specific endogenous receptors due to a lack of selective ligands.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Hydroxy and
-Keto Ethanolamide Phosphonic
Acids--
The synthesis of the
-hydroxy and
-keto propanolamide
phosphonic acids will be described in detail in a manuscript currently being prepared on a series of phosphonate
compounds.2 Briefly, oleoyl
chloride was acylated using 1-aminopropanol and pyridine followed by
Dess-Martin oxidation. The resulting aldehyde was reacted with
di-t-butylphosphonate in the presence of NaOH to generate
the protected
-hydroxy derivative. Treatment with trifluoroacetic
acid provided WLS31, whereas oxidation with pyridinium chlorochromate
followed by trifluoroacetic acid deprotection provided WLS60.
70 °C.
Protein concentration was determined spectroscopically by Bradford
protein assay.
-32P]ATP in the presence of diacylglycerol kinase
(Sigma) as described by Walsh et al. (46). The method used
here differs from the published protocol in that the reaction size was
increased 10-fold, and the reaction was allowed to proceed for 18 h. The labeled products were purified by normal-phase high pressure
liquid chromatography. Using a Varian Microsorb MV, 4.6 mm x 250 mm,
5-µm silica column, lipids were eluted using the following gradient
profile: isocratic solvent A
(HCCl3/MeOH/H2O/NH4OH:
77/21.65/.85/.5) from 0 to 5 min, linear transition from solvent A to
solvent B (HCCl3/MeOH/H2O/NH4OH: 59/34.5/6/.5) from 5 to 15 min, and isocratic solvent B from 15 min
onward. The collected fractions were then washed with 0.1% HCl and
MeOH, and the organic phase was retained and dried.
S Binding Assay--
GTP
S binding experiments were
performed as described earlier (22). Ligand-mediated GTP
S binding to
G-proteins was measured in GTP binding buffer (in mM: 50 HEPES, 100 NaCl, 10 MgCl2, pH 7.5) using 25 µg of a
membrane preparation from HEK 293 T cells transiently transfected with
DNAs encoding G
i2 C351F (this mutant G-protein is
resistant to pertussis toxin inactivation), G
1, and
G
2 along with the receptor being tested. Ligand was
added to membranes in the presence of 10 µM GDP and 0.1 nM [35S]GTP
S (1200 Ci/mmol) and incubated
at 30 °C for 30 min. Bound GTP
S was separated from unbound using
the Brandel harvester (Gaithersburg, MD) and counted with a liquid
scintillation counter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Hydroxy and
-Keto Phosphonates Are LPP Inhibitors--
To
examine the effect of LPP hydrolysis on LPA's potency in mitogenesis,
we developed two LPP-resistant LPA analogs by mimicking the phosphate
group with an
-substituted phosphonate (Fig.
1). The direct carbon-to-phosphorous bond
should render phosphonates resistant to degradation by the
phosphatases. In fact, the
-hydroxy (wls-31) and
-keto (wls-60)
phosphonates were not only resistant to LPP-mediated hydrolysis, they
also acted as inhibitors of LPP activity. When added to a reaction of
LPP enzyme and LPA substrate, the phosphonates significantly blocked
the activity of each of the three LPP isoforms. Preincubation of the
phosphonates with the enzyme preparation for 30 min before addition of
substrate did not diminish the inhibitory effect, confirming that the
compounds are not substrates for the enzyme simply acting as
competitive inhibitors (Fig.
2A). The Km
values were determined for each LPP isoform using radiolabeled LPA as
substrate in the presence and absence of the phosphonates and used to
calculate Ki values (Fig. 2B). This
analysis revealed that both of the phosphonates are competitive
inhibitors of each of the three LPP isoforms with mid-micromolar
affinities (Fig. 2C). There is a significant difference in
the affinity of the phosphonates for the different LPP isoforms: LPP3 > LPP1 > LPP2. It is also noteworthy that the
Ki values for the ketone compound (wls-60) are very
nearly half the values for the hydroxy compound (wls-31) at each
isoform. This may reflect the fact that the ketone is a discrete
molecule, whereas the hydroxyl compound is racemic. If only one
enantiomer of the hydroxyl compound is active, it would be equipotent
with the ketone compound. Alternatively, the charge on the head group
of the ketone compound may be more consistent with that of LPA than the
hydroxy compound, as the pKa of the second
dissociable proton of the ketone compound is closer to that of
LPA.
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Fig. 1.
Structures of LPA and LPA analogs are shown
for comparison. LPA (1-oleoyl,
2-hydroxyl-sn-glycerol-3-phosphate); wls-31/ -hydroxy
phosphonate (N-oleoyl
-hydroxy propanolamide phosphonic
acid); wls-60/
-keto phosphonate (N-oleoyl
-keto
propanolamide phosphonic acid); MHEPA (N-palmitoyl
methylenehydroxy ethanolamide phosphoric acid).
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Fig. 2.
The phosphonates are subtype-specific
inhibitors of LPP activity. A, LPP activity in membrane
preparations from cells overexpressing each of the LPP isoforms was
assayed using 200 nM labeled LPA as substrate. 500 µM wls-31 ( -hydroxy phosphonate) was added as a 50:1
Triton X-100:phosphonate mixture, either simultaneously with the
labeled substrate or 30 min prior to the start of the reaction. Control
reactions received vehicle (Triton X-100) alone. Each data point was
measured in triplicate and is representative of at least three separate
experiments; error bars reflect standard error.
B, reaction velocities were determined for each LPP isozyme
using a range of LPA concentrations in the presence or absence of 500 µM each phosphonate inhibitor. C,
Ki values were determined from the kinetics curves
using the following equation.
S binding assay (22). Membranes
from cells overexpressing the three heterotrimeric G-protein subunits
along with a receptor are incubated with ligand, GDP, and
[35S]GTP
S. The amount of GTP
S bound to the
G-proteins is determined by filtering and liquid scintillation counting
(see "Experimental Procedures" for details). These assays measure
G-protein activation directly, and do not reflect multiple levels of
amplification, as do many measures of receptor activation. Therefore,
the dose-response curves determined by this assay are often
right-shifted with respect to assays of more downstream events
(e.g. calcium mobilization). GTP
S binding dose-response
curves show that wls-31 and wls-60 are one to two log orders less
potent than LPA at Edg-2 and Edg-4 and have negligible activity at
Edg-7 at concentrations up to 10 µM (Fig.
3). wls-60 is more potent than wls-31 at
both Edg-2 and Edg-4. The activity seen with wls-60 at Edg-7 is
consistent with the amount of activity in untransfected cells (data not
shown); however, all dose-response curves, including LPA, are strongly right-shifted at Edg-7 (21, 22), so it is possible that the phosphonates would have a similar potency relative to LPA at Edg-7 as
they do at Edg-2 and -4, if the curves were extended beyond 10 µM (21, 22). We also determined the activity of these
compounds in intact cells by measuring calcium mobilization in
nontransfected HEK 293 cells and MDA MB-231 cells. In both cell types,
dose-response curves reveal rank order potency distribution nearly
identical to that seen at Edg-2 and Edg-4 in the GTP
S assay, with
all of the curves shifted 1-2 log orders to the left reflecting
amplification (data not shown). Therefore, the relative potency of the
phosphonates with respect to LPA that we observed in the GTP
S
binding assay reflects the relative affinities of the compounds at
endogenous receptor sites in intact cells.
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Fig. 3.
wls-31 and wls-60 are LPA receptor
agonists. Receptor activation in response to LPA, wls-31, and
wls-60 was determined by measuring GTP S binding to membranes from
HEK 293 T cells transiently transfected with DNA encoding Edg-2, -4, or
-7 along with DNA-encoding G-proteins. Membranes were incubated with
drug and GTP
35S for 30 min at 30 °C and filtered to
separate bound from free radioactivity. Basal GTP
S binding has been
subtracted from the data, and the data are normalized to maximal
binding with the highest dose of LPA. Typical raw values for basal and
maximal binding are 3,500 and 6,400 dpm at Edg-2; 3,500 and
12,800 dpm at Edg-4; and 4,700 and 15,000 dpm at Edg-7, respectively.
Data points were determined in duplicate, and these curves are
representative of two separate experiments; error bars
reflect standard error.
S
binding and calcium mobilization) assays. To address this question, we
used HEK 293 cells, which respond to LPA with a pertussis
toxin-sensitive tritiated thymidine incorporation (data not shown).
These cells express Edg-2 and Edg-7 (22). Tritiated thymidine
incorporation dose-response curves for LPA and the phosphonates wls-31
and wls-60 show that indeed the trend predicted by our hypothesis was
observed (Fig. 4). The phosphonates were
3-4 log orders more potent relative to LPA in the mitogenic assay
versus the GTP
S assay.
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Fig. 4.
wls-31 and wls-60 are higher potency agonists
in mitogenesis. The mitogenicity of LPA, wls-31, and wls-60 was
determined by measuring tritiated thymidine incorporation into the
acid-insoluble fraction in serum-starved cells on treatment with drug
for 22 h. Data were normalized to the amount of thymidine incorporation
in response to treatment with 10% serum. Data were determined in
triplicate, and this curve is representative of several separate
experiments; error bars reflect standard error.
S binding curves were determined for Edg-2, -4, or -7, increases in LPP1 activity of the same magnitude (Fig.
6B) had no effect on LPA's
potency or efficacy (Fig. 6A). This may reflect a unique
sensitivity of the mitogenic response to regulation by the LPPs due to
the lower potency of LPA in mitogenesis or to the longer duration of
the assays.
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Fig. 5.
LPP1 overexpression down-regulates LPA
induced mitogenesis. A, the ectophosphatase activity of
a clonal cell line overexpressing LPP1 was determined using intact HEK
293 cells with albumin-bound exogenous radiolabeled LPA as substrate.
Reactions were incubated for 5 min, and the media were extracted and
counted as described under "Experimental Procedures" to determine
phosphatase activity. B, the nontransfected HEK 293 cells
and the LPP1-overexpressing cell line were assayed for tritiated
thymidine incorporation into the acid-insoluble fraction in response to
LPA as described under "Experimental Procedures." The response is
ablated in the LPP1-overexpressing line. Each data point was determined
in triplicate, and these curves are representative of three separate
experiments; error bars reflect standard error.
C, the LPP1-overexpressing cell line was also assayed for
wls-31- and wls-60-induced tritiated thymidine incorporation. These
compounds are high potency mitogens irrespective of increased LPP1
activity. Each data point was determined in triplicate, and these
curves are representative of two separate experiments; error
bars reflect standard error.
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Fig. 6.
LPP1 overexpression does not affect
LPA-induced Edg receptor activation. A, dose-response
curves of GTP S binding were determined for LPA at Edg-2, Edg-4, and
Edg-7 with endogenous LPP activity and overexpressed LPP1. HEK 293 T
cells were transiently transfected with DNA encoding each receptor
alone or cotransfected with DNA encoding LPP1 (and with DNA encoding
each G-protein subunit). The potency and efficacy of LPA are unchanged
by LPP1 overexpression at each receptor. Each data point was determined
in duplicate, and these curves are representative of two separate
experiments; error bars reflect standard error.
B, the amount of phosphatase activity in membranes from the
transiently transfected cells (from A) was compared with the
activity in HEK 293 cells and the stable line overexpressing LPP1 (from
Fig. 5). The membrane preparations were assayed as described under
"Experimental Procedures" using 100 µM LPA as
substrate. The degree of overexpression is similar in the transient
transfections with individual receptors as in the stable line. Each
data point was determined in triplicate; error bars reflect
standard error.
S assay at Edg-2 and Edg-4 as
predicted by our hypothesis. However, the degree to which it increased
(3-4 log orders) was much greater than predicted. If this shift is due
solely to degradation of LPA by the LPPs, then greater than 99.9% of
the LPA added at the beginning of the assay must be degraded during the
course of the incubation. To test this possibility, [32P]LPA was added to a parallel mitogenesis experiment
under identical incubation conditions. At the end of the assay period,
the reaction (including cell monolayer) was extracted with acidified
methanol and chloroform, and the radioactivity retained in the organic phase (reflecting intact LPA) was counted. These experiments revealed that at concentrations at the upper limit of the mitogenic assay and
approaching the Km of the LPPs (~150
µM), no more than 90% of the LPA is destroyed during the
24-h incubation with cells (Fig.
7A). These data suggest that
the shift of the rank order potencies of the phosphonates and LPA in
mitogenesis cannot be accounted for solely by LPP hydrolysis.
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Fig. 7.
The mitogenic response to LPA and LPA analogs
has a ligand selectivity distinct from that of Edg-2, -4, and -7. A, the increased potency of the phosphonates relative to LPA
cannot be accounted for by LPP hydrolysis; ~10% of LPA is intact
following a mitogenic assay. Phosphorous-32-labeled LPA was added to a
nearly confluent monolayer of HEK 293 cells under mitogenic assay
conditions (see "Experimental Procedures"). At the end of the assay
period, the entire reaction was extracted with acidified
methanol/chloroform, and the radioactive material remaining in the
organic phase was determined as a measure of intact LPA. Data points
were determined in triplicate; error bars reflect standard
error. B, S-MHEPA but not R-MHEPA is
active at Edg receptors. GTP S binding to membranes from HEK 293 T
cells overexpressing each subunit of the heterotrimeric G-proteins
along with Edg-2, -4, or -7 is shown in response to LPA,
R-MHEPA, and S-MHEPA (10 µM each)
(see "Experimental Procedures" for details). C, the
mitogenic response to LPA analogs lacks the stereoselectivity observed
at Edg receptors. The mitogenicity of the LPA analog MHEPA in both
R and S conformations was determined in HEK 293 cells by measuring incorporation of tritiated thymidine. Both the
R and S isomers had activities similar to that of
LPA. Data points were determined in triplicate, and these curves are
representative of two separate experiments; error bars
reflect standard error. D, the ligand selectivity of the
mitogenic response observed in HEK 293 cells is mirrored by MDA MB231
cells. Tritiated thymidine incorporation dose-response curves were
determined for LPA, wls-60, and R-MHEPA in MDA MB231 cells.
Data points were determined in quadruplicate, and these curves are
representative of two separate experiments; error bars reflect standard error. E,
RH7777 cells that do not express Edg-2, -4, and -7 exhibit a mitogenic
response to LPA and LPA analogs with the same ligand selectivity as is
seen in Edg-expressing cells. Thymidine incorporation was measured in
response to LPA, wls-60, and R-MHEPA in RH7777 cells. Data
points were determined in quadruplicate, and these curves are
representative of three separate experiments; error bars
reflect standard error.
S binding at Edg-2, -4, and -7 (Fig. 7B), MHEPA is
inactive in the R configuration but is a potent agonist in
the S-form; such ligand stereoselectivity is common and
observed in most receptor-mediated signaling. To determine whether this
stereoselectivity was echoed by the mitogenic response, we tested both
enantiomers in thymidine incorporation assays. In stark contrast to
their activity at Edg-2, -4, and -7, the R-form of MHEPA was
at least as active as the molecule in the S conformation in
mitogenic assays. Dose-response curves for these compounds revealed
that R-MHEPA is a full agonist of the mitogenic response and
is slightly less potent than LPA (Fig. 7C). We tested LPA,
both conformations of MHEPA, and wls-60 in MDA MB-231 cells for their
potency in mitogenic assays to determine whether the distinct ligand
selectivity was cell type-specific. As seen in Fig. 7D, the
rank order potencies of LPA and LPA analogs is the same in these cells
as in HEK 293 cells. The R-form of MHEPA is fully active
with respect to mitogenesis (as is the S-form, data not
shown), and the wls-60 is considerably more potent than R-MHEPA and LPA. The difference in ligand selectivity
between the mitogenic response and the response of the individual LPA receptors suggests that the activation of Edg-2, -4, and/or -7 is not
sufficient to account for the mitogenic response to LPA.
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Fig. 8.
The platelet aggregation response to LPA has
ligand selectivity that is consistent with the mitogenic response but
not with Edg-2, -4, and -7. Dose-response curves were determined
for LPA, wls-60, and both isomers of MHEPA. Aggregation was measured in
an aggregometer in response to each drug following a
subthreshold dose of ADP. Each data point was determined in triplicate
or quadruplicate; error bars reflect standard error.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxy and
-keto propanolamide phosphonic
acids, which are subtype-specific inhibitors of LPP enzymes and are
degradation-resistant agonists at Edg-2 and Edg-4. Second, we have
shown that overexpression of the LPP1 enzyme attenuates the mitogenic
response to LPA, although it does not affect LPA potency or efficacy in
GTP
S binding assays with Edg LPA receptors. Third, we present
several lines of evidence that the mitogenic response to LPA is
independent of Edg-2, -4, and -7 in three cell lines. Finally, we have
shown that the pharmacology of the platelet aggregation response
corresponds to that of the mitogenic response, but not to the known LPA
receptors. Together, these results suggest that there exists a low
affinity LPA-signaling pathway that is distinct from Edg-2, -4, and -7, which mediates mitogenesis and platelet aggregation and is regulated by
LPP enzymes.
S binding
assays, they are active at Edg-2 and -4 with potencies ~2-fold lower
than that of LPA, and these rank order potencies are reflected in
intact cells with endogenous levels of receptor and G-protein in
calcium mobilization assays, confirming the GTP
S binding assay as a
model for cellular receptor activation. Ours is not the first report of
activity of phosphonate compounds in assays of LPA receptor activation.
In 1991, Proll and Clark reported that an LPA analog with a phosphonate
head group inhibits cAMP accumulation (50), but Moolenaar's group
later showed that the same compound had little activity with respect to
calcium mobilization (12). We have synthesized and tested a similar compound in a GTP
S binding assay at each Edg/LPA receptor and found
that it is an agonist at Edg-2 but has very little activity at Edg-4 or
Edg-7.4
S binding, calcium mobilization, inhibition of adenylyl cyclase,
and calcium-induced chloride currents in Xenopus oocytes)
are full agonists in mitogenic assays. Third, RH7777 cells that do not
express Edg-2, -4, and -7 have mitogenic responses to LPA and LPA
analogs similar to those of cells that express the receptors. As
additional evidence that LPA-induced mitogenesis is Edg
receptor-independent, we have observed LNCaP cells that express
functional Edg-2 (transfected), -4, and -7 LPA receptors but lack a
mitogenic response to LPA, even though they undergo mitogenesis in
response to other stimuli such as epidermal growth factor (data not shown).
S binding with similar levels of LPP1 overexpression in membranes expressing Edg-2, -4, or -7. Several differences between
the assays for mitogenesis and GTP
S binding may be responsible for
the apparently greater sensitivity of the mitogenic response to LPP1
overexpression: hours versus minutes of incubation time, intact cells versus isolated membranes, and endogenous
versus overexpressed receptors. However, the mitogenic
response to LPA occurs over a concentration range that is much closer
to the Km values of the LPP enzymes than most Edg
receptor-mediated responses, which may allow the LPPs to specifically
regulate the lower affinity LPA pathway. Brindley and colleagues (55)
recently reported that overexpression of LPP1 attenuated LPA responses
that were suggested to be mediated by Edg-2. Because Edg-2 was the only Edg/LPA receptor expressed in their system, it was assumed to be
responsible for the responses based on the same presumption we made
entering this study: All LPA responses are mediated by one of the three
currently cloned and characterized LPA receptors. Therefore, the degree
to which LPA signaling through the Edg receptors is affected by LPP
activity is not known.
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Fig. 9.
The new model for LPA signaling derived from
the results presented here includes an Edg-receptor-independent pathway
that mediates platelet aggregation and mitogenesis by an unknown
mechanism that is down-regulated by LPPs. It is not known if the
LPPs significantly down-regulate Edg receptor-mediated LPA signaling.
Also, the physiologic cellular response to Edg receptor activation is
not known.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Erik Hewlett for the gift of pertussis toxin used in these experiments and Leman Mutlu for her input during preparation of this manuscript (Department of Pharmacology, University of Virginia).
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FOOTNOTES |
---|
* This work was supported by Grants R01-GM52722 (to K. R. L. and T. L. M.) and F31-DA05927 (to S. B. H.) from the National Institutes of Health.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.
§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Virginia Health Sciences Center, Jordan Hall, Box 440, 1300 Jefferson Park Ave., Charlottesville, VA 22908. Tel.: 804-924-2837; Fax: 804-982-3878; E-mail: sbh7d@virginia.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007782200
2 W. L. Santos, J. A. Rossi, S. B. Hooks, K. R. Lynch, T. L. MacDonald, in preparation.
3 D.-S. Im and K. R. Lynch, unpublished data.
4 W. L. Santos, S. B. Hooks, T. L. Macdonald, and K. R. Lynch, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LPA, lysophosphatidic acid (1-oleoyl, 2-hydroxyl-sn-glycerol-3-phosphate); LPP, lipid phosphoric acid phosphatase; DMEM, Dulbecco's modified Eagle's medium; GTPS, guanosine 5'-O-(thiotriphosphate); MHEPA, N-palmitoyl-2-methylenehydroxy ethanolamide phosphoric acid; NASPA, N-acyl serine phosphoric acid.
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