From the
Research into phospholipid signaling continues to flourish, as
more and more bioactive lipids are being identified and their actions
characterized. One interesting addition to the growing list of lipid
messengers is lysophosphatidic acid (LPA
It is now well
established that LPA acts on its cognate G protein-coupled receptor(s),
apparently present in many different cell types. Radioligand binding
studies have revealed the presence of specific LPA binding sites in
membranes from 3T3 cells and rat brain, with K
Despite its ubiquity and physiological importance, relatively
little is known about the physical chemistry of LPA such as its phase
behavior and cation binding properties. Having a free hydroxyl and
phosphate moiety, LPA (oleoyl) is much more water-soluble than other
long chain phospholipids
(13) . Because of its hydrophilicity LPA
does not necessarily remain membrane-associated after its formation
and, furthermore, readily escapes detection in conventional lipid
extraction procedures. Unlike other lysophospholipids, LPA is not lytic
to cells under physiological conditions because of its relatively small
headgroup. Calcium ions tend to precipitate LPA from aqueous solutions,
which can be prevented by addition of albumin
(13) . However,
where examined, the biological effects of LPA (see below) are not
critically dependent on the presence of albumin.
The list of biological responses to LPA () is
quite diverse, ranging from induction of cell
proliferation
(1, 2, 3, 4, 5, 6) to stimulation of neurite retraction
(9, 23) and even slime mold chemotaxis
(24) , and continues
to grow as more and more cellular systems are being tested for LPA
responsiveness (see also Refs. 1 and 2). Where studied, the effects of
LPA are highly specific and appear to be receptor-mediated. A caveat is
that receptor involvement has not in every case been tested thoroughly.
In general, with bioactive lipids such as LPA it is very important to
test the formal possibility that ``nonspecific''
incorporation into membranes and/or cellular uptake might somehow
influence cell behavior in a receptor-independent manner. Indeed, there
is precedent for a phospholipid mediator, notably PAF, to evoke both
receptor-dependent and receptor-independent effects
(25) . On the
other hand, receptor-independent responses to LPA have not been
documented thus far.
The mitogenic activities of LPA have been best
studied in fibroblasts. As with peptide growth factors, induction of
DNA synthesis by LPA requires its long term presence in the
medium
(1, 2, 3, 4, 26) . The
mitogenic action of LPA is almost completely inhibited by PTX,
implicating the critical involvement of one or more G
A somewhat puzzling finding
is that in certain cell types, the mitogenic action of LPA can be
reproduced by PA (but not other
glycerophospholipids
(6, 26) ), whereas PA does not
appear to act on the LPA receptor. The dose-response relationships of
PA and LPA largely overlap, implying that PA action is not mediated by
LPA impurities in the PA used
(26) . Whether this effect of PA is
receptor-mediated is still obscure. Clearly, one major challenge is now
to reconcile the mitogenic action of PA with that of LPA (see Ref. 2
for further discussion).
LPA, but not peptide growth factors, can
mimic serum in inducing invasion of carcinoma and hepatoma cells into
monolayers of mesothelial cells
(28) . The underlying mechanism
is not clear but may well involve both increased cell adhesion and
enhanced cell motility. Other lipids, including PA, have little or no
effect, but bacterial PLD does mimic LPA action
(28) . The latter
result is consistent with the finding that PLD treatment of cells may
result in activation of endogenous LPA receptors.
Like many mitogens, LPA induces rapid changes in the
actin-based cytoskeleton. In serum-deprived fibroblasts, LPA stimulates
focal adhesion assembly and stress fiber formation
(29) .
Assembly of new focal adhesions will lead to increased cell adhesion,
while stress fiber formation may contribute to the build-up of
contractile forces as occurs during wound repair. In neuroblastoma and
PC12 cells, LPA triggers acute growth cone collapse and neurite
withdrawal
(9, 23) , a process driven by contraction of
the cortical actin cytoskeleton rather than by loss of adhesion to the
substratum
(23, 30) . By stimulating neurite withdrawal,
LPA tends to inhibit and reverse the morphological differentiation of
neuroblastoma cells
(2) . The in vivo significance of
this intriguing phenomenon remains to be addressed. Given the abundance
of the putative LPA receptor in brain
(12) and the
responsiveness of neuronal cells to LPA, it is tempting to speculate
that LPA may serve a neuromodulator role, either under normal
physiological conditions or during traumatic injury of the nervous
system (discussed in Ref. 1). From a signal transduction point of view,
the early cytoskeletal and late mitogenic effects of LPA are
particularly intriguing since they cannot be explained by known second
messenger pathways, as will be outlined below.
The structural features of LPA that are important for its
biological activity are only beginning to be understood. A consistent
finding is that maximal LPA activity is observed with long acyl chains
(C
Ether-linked
LPA acts on the LPA receptor, albeit with less potency than the
corresponding ester-linked LPA, at least in A431 cells
(31) . In
platelet aggregation assays, on the other hand, ether-linked LPAs
appear to be the most potent species
(32) . One possibility to
explain this discrepancy is that platelets and carcinoma cells express
different LPA receptors. It appears that the sn-1 as well as
the sn-3 enantiomers of ether-linked LPA have about equal
activity
(31, 32) , with both isomers showing
cross-desensitization. This apparent lack of stereospecificity is
somewhat unexpected, since stereospecificity is often a critical
feature of physiological receptor ligands. The fact that ether-linked
LPAs behave as bona fide receptor agonists may be of physiological
relevance; ether-linked LPAs are potential metabolites of
1-alkyl-2-acyl-glycerophospholipids (such as PAF), which are found in
significant amounts in circulating cells such as neutrophils and
macrophages. It will now be important to find out whether ether-linked
LPA can occur extracellularly, either in physiological events or under
pathological conditions.
Thus far, structure-activity studies have
not identified specific LPA receptor antagonists (but see Ref. 33 for
potential candidates). The only known LPA antagonist is suramin; this
polyanionic compound abolishes both early and late responses to LPA in
a reversible manner (23, 26), apparently by inhibiting LPA-receptor
binding
(12) .
In summary, although many features of the LPA
structure are important for biological activity, the phosphate group is
most critical. In a simple model
(31) , the acyl chain may serve
to anchor the LPA molecule in a hydrophobic cavity of the receptor in
such a way that the glycerol-linked phosphate group is optimally
oriented for direct interaction with positively charged residues in an
extracellular region of the receptor. Such specific binding of the
phosphate moiety would then trigger a conformational change that
activates the receptor. Obviously, a direct test of this tentative
model awaits cloning of the LPA receptor cDNA. It will then be possible
to examine by site-directed mutagenesis how the LPA receptor detects
and binds its ligand.
At least four G protein-mediated signaling pathways have been
identified in the action of LPA. These are (i) stimulation of PLC and
PLD, (ii) inhibition of adenylyl cyclase, (iii) activation of Ras and
the downstream Raf/MAP kinase pathway, and (iv) tyrosine
phosphorylation of focal adhesion proteins in concert with remodeling
of the actin cytoskeleton in a Rho-dependent manner. Of these pathways,
inhibition of adenylyl cyclase and stimulation of Ras signaling are
sensitive to PTX. A simplified and tentative outline of the various
signaling cascades is given in Fig. 2and will be further
discussed below.
While the
biochemical steps between tyrosine kinase receptors and Ras activation
have been elucidated in much detail, little is still known about how
G
In other words, Rho-GTP links the
LPA receptor to cytoskeletal events and protein tyrosine
phosphorylations, but the details of the underlying signaling events
remain enigmatic, as one is hampered by not knowing the biochemical
targets of Rho. Current evidence indicates that active Rho-GTP directs
the assembly of actin-based multimolecular complexes at the plasma
membrane (such as focal adhesions; see Ref. 53 for review).
Inactivation of endogenous Rho by the Botulinum C3
ADP-ribosyltransferase inhibits not only LPA-induced cytoskeletal
effects but also tyrosine phosphorylation of FAK
(44) .
Tyrphostin 25, a tyrosine kinase inhibitor, inhibits the effects of LPA
on the actin cytoskeleton but not the effects of microinjected Rho,
suggesting that a non-receptor tyrosine kinase lies upstream of
Rho
(45) . Here is an interesting parallel with the mechanism
proposed for LPA-induced Ras activation
(37, 40) .
In
the tentative model of Fig. 2, the PTX-insensitive G
A somewhat
different picture holds for LPA-induced growth cone collapse and
neurite retraction in neuroblastoma and PC12 cells. In these cells, Rho
function appears essential for LPA-induced generation of contractile,
actomyosin-based forces in the cortical cytoskeleton. Yet, there is no
close correlation between PLC activation and Rho-dependent cytoskeletal
changes, since phosphoinositide-hydrolyzing neuropeptides fail to evoke
neurite withdrawal
(23) . LPA-induced neurite retraction is
accompanied by rapid activation of the
p60
After having long been regarded as just a metabolic
intermediate, LPA has now emerged as a multifunctional signaling
molecule. Much has already been learned about LPA as a platelet-derived
messenger, but numerous challenges remain. On the biological side,
there are questions such as what are the precise biological functions
of LPA and what cell types (other than platelets) produce and release
LPA? It seems more than likely that platelet-derived LPA stimulates
cell proliferation at sites of injury and inflammation, presumably in
synergy with other platelet-derived factors, but this needs to be
tested in vivo. At the molecular level, cDNA cloning and
characterization of a functional G protein-linked receptor for LPA is
of obvious importance; it will then be possible to address the tissue
and cell type distribution of the LPA receptor and also whether there
are multiple LPA receptor subtypes. Finally at the signal transduction
level, there is the challenge of delineating the biochemical steps that
couple the LPA receptor to activation of Ras and Rho. Ultimately,
continued study of these questions should provide a detailed picture of
the biological functions and mode of action of LPA as an intercellular
messenger.
For primary references see text and Refs. 1 and 2,
unless indicated otherwise.
I thank my colleagues in the Netherlands Cancer
Institute for helpful discussions. I have cited reviews when possible
and apologize to those whose original work could not be cited because
of space limitations.
INTRODUCTION
Biochemistry of LPA
Biological Responses to LPA
Structure-Activity Relationship
G Protein-mediated Signal Transduction
Concluding Remarks
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
;
1-acyl-glycerol-3-phosphate), the simplest of all glycerophospholipids.
While LPA has long been known as a precursor of phospholipid
biosynthesis in both eukaryotic and prokaryotic cells, only recently
has LPA emerged as an intercellular signaling molecule that is rapidly
produced and released by activated cells, notably platelets, to
influence target cells by acting on a specific cell-surface
receptor
(1) . Although its precise physiological (and
pathological) functions in vivo remain to be explored, LPA
derived from platelets has all the hallmarks of an important mediator
of wound healing and tissue regeneration. Thus, in addition to acting
as an autocrine stimulator of platelet aggregation
(1) , LPA
stimulates the growth of
fibroblasts
(1, 2, 3, 4) , vascular
smooth muscle cells
(5) , endothelial cells,
(
)
and keratinocytes
(6) ; furthermore, it promotes
cellular tension
(7) and cell-surface fibronectin
binding
(8) , which are important events in wound repair. As a
product of the blood-clotting process, LPA is a normal constituent of
serum (but not platelet-poor plasma), where it is present in an
albumin-bound form at physiologically relevant
concentrations
(9, 10) . Cellular responses to LPA in
fact show a striking overlap with those to serum, and it therefore
seems safe to conclude that albumin-bound LPA accounts for much of the
platelet-derived biological activity of serum.
values in the lower nanomolar range
(11) . Moreover,
cross-linking experiments have identified a candidate high affinity LPA
receptor with an apparent molecular mass of 38-40 kDa in
LPA-responsive cells and mammalian brain
(12) . Although to date
a functional LPA receptor has not been purified or cloned, much has
already been learned about LPA receptor signaling. The LPA receptor
couples to multiple independent effector pathways in a G
protein-dependent manner; these include not only ``classic''
second messenger pathways, such as stimulation of PLC and inhibition of
adenylyl cyclase, but also novel routes, notably activation of the
small GTP-binding proteins Ras and Rho. This minireview aims to
introduce the reader to our current understanding of LPA as an
intercellular messenger.
Production and Release
Besides being synthesized and
processed to more complex phospholipids in the endoplasmic reticulum,
LPA can be generated through the hydrolysis of pre-existing
phospholipids following cell activation. The best documented example
concerns thrombin-activated platelets, where LPA production is followed
by its extracellular release
(10) . Platelet LPA is formed, at
least in large part, through PLA-mediated deacylation of
newly generated PA
(14) , as depicted schematically in
Fig. 1
. Distinct PA-specific PLA
activity has been
identified in platelets (Ca
-dependent
(15) )
and in rat brain (Ca
-independent
(16) ), but
little is known about its mode of regulation. It remains to be examined
at what stage of the platelet activation response LPA is produced and
how it is released into the extracellular environment. Given the wide
variety of LPA-responsive cell types, LPA production and release are
unlikely to be restricted to just platelets. Indeed, there is
preliminary evidence that growth factor-stimulated fibroblasts can also
produce LPA
(17) . Furthermore, LPA may be formed and released by
injured cells, probably due to nonspecific activation of
phospholipases,
(
)
but many other cell systems
remain to be examined for LPA production.
Figure 1:
Proposed generation of LPA from newly
formed PA in activated cells, notably thrombin-stimulated platelets.
Diacylglycerol is directly formed via receptor-linked activation of PLC
and is then phosphorylated by diacylglycerol kinase to yield PA. PA may
also be directly formed through activation of PLD acting on
phosphatidylcholine and phosphatidylethanolamine (R denotes
phospholipid headgroup). In activated platelets, PA is thought to be
hydrolyzed by a specific PLA to yield bioactive LPA (see
text). The possible contribution of other pathways to generating LPA
remains to be examined.
LPA and LPA-like Lipids in Serum
In freshly
prepared mammalian serum, LPA concentrations are estimated to be in the
range of approximately 2-20 µM, with oleoyl- and
palmitoyl-LPA being the predominant species
(5, 10) . LPA
is not detectable in platelet-poor plasma, whole blood, or
cerebrospinal fluid (9, 10). In common with long chain fatty acids, LPA
binds with high affinity to serum albumin at a molar ratio of about
3:1
(18) . It is notable that serum albumin contains several
other, as yet unidentified lipids (methanol-extractable) with LPA-like
biological activity
(9) . This raises the interesting possibility
that LPA may belong to a new family of phospholipid mediators showing
overlapping biological activities and acting on distinct receptors;
conceivably, the ether-linked phospholipid platelet-activating factor
(PAF) and the mitogenic lipid sphingosine 1-phosphate (19)(
)
may also belong to this putative family.
LPA Catabolism
Besides binding to its cell-surface
receptor(s), LPA is degraded by intact cells. In fibroblasts, exogenous
LPA is rapidly hydrolyzed in part to monoacylglycerol and, to a much
lesser extent, diacylglycerol and PA
(20) . Most of the
monoacylglycerol formed remains cell-associated and is presumably
further metabolized. Ectophosphohydrolase activity toward LPA has
recently been identified in keratinocytes
(21) , and an
LPA-specific phospholipase has been purified from rat
brain
(22) . Enzymatic degradation of LPA to biologically
inactive products may serve to limit the duration and magnitude of
LPA's cellular effects.
-type
heterotrimeric G proteins. LPA also stimulates the growth of mouse
preimplantation embryos in a PTX-sensitive manner
(27) ,
suggesting a role for LPA in early mammalian development. In
keratinocytes, and possibly other cell types, LPA-induced cell
proliferation appears to be indirect as it involves the production of
transforming growth factor
in a PTX-dependent manner
(6) .
In confluent keratinocyte cultures, LPA stimulates terminal
differentiation, while its topical application to mouse skin induces
thickening of the epidermis
(6) .
(
)
to C
) and that activity decreases with
decreasing chain length; the shorter chain species, such as lauroyl-
and decanoyl-LPA, show little or no activity
(26, 31) .
Human A431 carcinoma cells are particularly convenient for
structure-activity analysis because of their high sensitivity to LPA in
terms of Ca
mobilization (EC
for
oleoyl-LPA, 0.2 nM(31) ). A recent study with these
cells has revealed the importance of the free phosphate group;
replacement of this moiety by either of three different phosphonates
results in almost complete loss of activity; similarly, the methyl and
ethyl esters of LPA are virtually inactive
(31) .
Figure 2:
Proposed scheme of signaling pathways
transduced by the LPA receptor. In this scheme, the LPA receptor
couples to both PTX-sensitive and PTX-insensitive G proteins (G and G
, respectively) to trigger various effector
pathways. The free
subunit of G
directly inhibits
adenylyl cyclase, while the
dimers are thought to mediate
LPA-induced Ras-GTP accumulation via an intermediary protein tyrosine
kinase (inhibited by genistein and staurosporine). Active Ras then
initiates the Raf/MAP kinase cascade to stimulate cell proliferation.
The G
protein is proposed to trigger PLC-mediated Rho
signaling independently of classic phosphoinositide-derived second
messengers. Perhaps, the PLC-induced loss of polyphosphoinositides may
somehow provide the signal for Rho activation. Of note, it cannot be
excluded that stimulation of PLC and Rho activation are parallel rather
than sequential events; if so, PLC and Rho might be regulated by
distinct G
subunits (cf. G
-mediated
inhibition of adenylyl cyclase and activation of Ras). A putative
protein tyrosine kinase (inhibited by tyrphostin) may act upstream of
Rho. See text for further details and references. AC, adenylyl
cyclase; IP
, inositol trisphosphate; DG,
diacylglycerol; PKC, protein kinase
C.
Phospholipase Activation
When added at nanomolar doses,
LPA activates phosphoinositide-specific PLC with consequent
Ca mobilization and protein kinase C activation via a
PTX-insensitive G protein (G
family) in a great variety of
cell types, ranging from Xenopus oocytes to mammalian neuronal
cells (Refs. 1 and 2; see Ref. 31 for an update of LPA-responsive
mammalian cell types). In fibroblasts, LPA also liberates arachidonic
acid from prelabeled lipids
(3) , but it has not yet been
established whether this results from enhanced PLA
activity
or, more indirectly, from stimulation of a diacylglycerol lipase, or
both. LPA also activates PLD to generate PA (and free choline) from
phosphatidylcholine
(34, 35) in a protein kinase
C-dependent manner, as with many PLC-activating agonists
(36) .
The signaling function, if any, of newly generated PA is still obscure.
It should be emphasized that, although enhanced phospholipid hydrolysis
mediates diverse early cellular responses to LPA, it cannot account for
LPA-induced cell proliferation
(1, 2, 3) .
G
In fibroblasts, LPA rapidly lowers cAMP levels
following forced stimulation of adenylyl cyclase
(3) . This
effect is abolished by PTX and hence attributable to
G-mediated Inhibition of Adenylyl
Cyclase
-mediated inhibition of adenylyl cyclase. By this
mechanism LPA may counteract the effects of cAMP-raising agonists,
which are often growth inhibitory. It remains to be seen whether
non-fibroblastic cell types respond similarly to LPA.
G
LPA-induced fibroblast proliferation is almost
completely inhibited by PTX and yet not attributable to known
PTX-sensitive second messenger pathways. The critical
G-mediated Ras
Signaling
-mediated mitogenic event is activation of Ras (37) and
its downstream effectors, particularly the Raf/MAP kinase
cascade
(38, 39, 40) . It seems that Ras is
necessary for both the early, transient phase and the late, sustained
phase of MAP kinase activation by LPA
(38, 41) . Other
G
-coupled receptors, including the thrombin
receptor
(37) , also couple to rapid Ras activation, at least in
a fibroblast context (reviewed in Refs. 1 and 2). It thus appears that
G
-coupled receptors and receptor protein tyrosine kinases
utilize a similar mechanism for mitogenic signaling.
mediates Ras activation. A first clue comes from COS cell
studies showing that
rather than
subunits of G
are responsible for Ras-dependent MAP kinase
activation
(41, 42) . Furthermore, in Rat-1 cells
expressing a
inhibitor polypeptide, LPA-induced Ras
signaling is significantly reduced
(43) .
dimers are
thought to couple to an as yet unidentified effector that may
stimulate, either directly or indirectly, GDP/GTP exchange on Ras, but
it is equally well possible that
acts on Ras via a
GTPase-activating protein rather than a GDP/GTP exchanger. Whatever the
precise mechanism, the G
-Ras pathway has been proposed to
comprise a non-receptor tyrosine kinase, since tyrosine kinase
inhibitors (genistein and staurosporine) selectively inhibit
LPA-induced (but not epidermal growth factor-induced) Ras-GTP
accumulation
(37, 40) . Unraveling the molecular details
of the G
-Ras connection is one of the major challenges for
future studies.
Phosphatidylinositol 3-Kinase Activation
Many
mitogens activate the p85/p110 phosphatidylinositol (PI) 3-kinase in
their target cells and so does LPA. LPA-induced PI 3-kinase activity
has been found in 3T3 cells (Ref. 44; but see ref. 45 for conflicting
data) and in megakaryoblastic cells
(46) . Whatever its precise
link to the LPA receptor and physiological function, PI 3-kinase does
not seem to be essential for mitogenic signaling, since microinjection
of a neutralizing antibody does not affect LPA-induced DNA
synthesis
(47) .
Rho Signaling: Protein Tyrosine Phosphorylation and
Cytoskeletal Changes
The least understood branch of the LPA
receptor signaling cascade is the one that involves the Ras-related Rho
protein. It accounts for LPA-induced cytoskeletal reorganizations,
actomyosin-based force generation, and tyrosine phosphorylation of
cytoskeleton-associated proteins. This pathway is somehow associated
with PLC activation. In fibroblasts, G protein-linked receptor agonists
that stimulate PLC also induce rapid tyrosine phosphorylation of focal
adhesion proteins, and LPA is no exception to the
rule
(40, 44, 48) . The major
tyrosine-phosphorylated substrates in LPA-treated fibroblasts comprise
the 125-kDa ``focal adhesion kinase'' (FAK), paxillin, and a
p130 protein
(48) , originally identified as putative v-Src
substrates. FAK is an integrin-linked tyrosine kinase that associates
with the Src and Fyn tyrosine kinases, paxillin, and adaptor proteins
and likely plays an important role in integrin-mediated signal
transduction
(49) . Immunofluorescence studies show that these
LPA-induced tyrosine phosphorylations are accompanied by the
recruitment of FAK, paxillin, talin, vinculin, as well as protein
kinase C- to focal adhesion sites, resulting in rapid reassembly
of focal adhesions and actin stress
fibers
(50, 51, 52) . Microinjected active Rho
mimics exogenous LPA in recruiting tyrosine-phosphorylated proteins to
focal adhesion sites, followed by stress fiber
formation
(29, 50) .
protein is proposed to mediate both PLC activation and Rho
signaling. In the simplest model consistent with the fibroblast data,
stimulation of PLC and Rho activation are sequential rather than
parallel events. Although LPA-induced Rho signaling appears to be
associated with PLC activation (independent of the Ras-MAP kinase
cascade), it is independent of PLC-mediated Ca
mobilization and/or protein kinase C
activation
(48, 50) . Perhaps the receptor-mediated
decrease in phosphoinositide levels somehow provides the signal for Rho
activation. Alternatively, phospholipid metabolites other than inositol
phosphates and diacylglycerol might play a role.
tyrosine kinase, but
cause-effect relationships are not clear
(23) . ADP-ribosylation
of RhoA in growing neuroblastoma and PC12 cells causes extensive cell
flattening (just opposite to what is observed in fibroblasts), probably
as a result of reduced cytoskeletal tension, followed by prominent
neurite outgrowth and growth arrest, very similar to what is observed
after long term serum starvation
(30) . These findings suggest
that, in neuronal cells, Rho acts in a LPA signal transduction cascade
that regulates actomyosin contractility in a
Ca
-independent manner and thereby directs neurite
behavior; some basal Rho activity is probably necessary to maintain
actomyosin-based tension and cell shape (30, 53). As a note of caution,
the formal possibility remains that ADP-ribosylated Rho causes subtle
modifications in the actomyosin system and thereby prevents it from
proper functioning. Microinjection of active Rho-GTP should provide
further answers.
Table:
Biological
responses to LPA
, phospholipase A
;
PLD, phospholipase D; PAF, platelet-activating factor; PTX, pertussis
toxin; MAP, mitogen-activated protein; PI, phosphatidylinositol; FAK,
focal adhesion kinase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.