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Vol. 60, Issue 6, 1161-1164, December 2001
Department of Physiology, University of Tennessee Health Sciences Center Memphis, Memphis, Tennessee
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In
this issue of Molecular Pharmacology, Heise et al. (2001)
report their results with derivatives of N-acyl ethanolamide phosphate that display subtype-selective agonist and antagonist properties for the endothelial differentiation gene (EDG) family of
lysophosphatidic acid (LPA) receptors (Heise et al., 2001). This report
follows a publication by Fischer et al. (2001) in the October issue of
Molecular Pharmacology reporting on the LPA receptor
subtype-selective antagonist properties of short-chain phosphatidic
acids. These compounds might be the prototypes of tools long awaited by
researchers attempting to unravel the physiological and
pathophysiological roles of growth factor-like phospholipids.
Lysophosphatidic acid (1-acyl-2-sn-glycero-3-phosphate) and
sphingosine-1-phosphate (S1P) have generated considerable interest among cell biologists and pharmacologists since the early 1990s because
of their ability to evoke robust Ca2+ responses
and changes in cell shape at nanomolar concentrations (for review, see
Moolenaar, 1999; Pyne and Pyne, 2000; Tigyi, 2001). This interest has
been augmented by the ability of LPA and S1P to elicit mitogenic
responses and to sustain the survival of cultured cells at micromolar
concentrations. The biological effects of these lipids are
utilized
unwittingly by mostwhen serum, which contains LPA in the 10 µM range (Baker et al., 2001) and S1P in the 100 nM range (Yatomi et
al., 1995), is added to culture media. The biological responses to LPA
and S1P are consistent with those of ligands acting through specific G
protein-coupled receptors. However, because of the hydrophobic nature
of these ligands, the application of radioligand binding assays for the detection of specific receptors has been challenging. This is one of
the factors that have made the molecular cloning of the phospholipid
growth factor receptors difficult. However, Hecht et al. (1996)
identified LPA as a ligand for a G-protein-coupled receptor they
isolated from the ventricular zone of the developing mouse brain. The
cloned and overexpressed vzg-1 gene mediated serum-induced
retraction of neurites in cortical neurons, a characteristic response
elicited by LPA application in PC12 (Tigyi and Miledi 1992) and N1E115
(Jalink et al., 1993) neuroblastoma cells noted earlier.
Vzg-1 was later named EDG2 (Contos et al., 2000) because it
was shown to be highly homologous to a family of G-protein-coupled orphan receptors. The first member of this family was cloned by Hla and
Maciag as a phorbol ester-induced early response gene in vascular
endothelial cells (Hla and Maciag 1990); hence, it was named EDG1 for
endothelial differentiation gene-1. In 1998, Lee et al. reported that
S1P was an endogenous ligand for EDG-1. After this report, several
groups have identified other members of this family, including the
genes for the LPA-specific EDG4 (An et al., 1998) and EDG7 (Aoki et
al., 2000) receptors and the S1P-specific EDG3/5 (An et al., 1997),
EDG-6 (Gräler et al., 1998), and EDG8 receptors (Im et al.,
2000). This year, the IUPHAR Nomenclature Committee proposed a new
nomenclature, based on the receptor's natural ligand and the
chronological order of its discovery, to replace the colloquial EDG
terminology (Table 1).
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Fischer et al. (1998) have shown that most cell types coexpress
multiple subtypes of the EDG family receptors. Moreover, there are
hints that the EDG family might not represent the only receptors for
LPA and S1P (Guo et al., 1996; Hooks et al., 2001). The complexity of
the pharmacology of these receptors has delayed the identification of
ligands with receptor-selective antagonist and agonist properties. Such
compounds are essential for the continued advancement of the field.
Because the coexpression of multiple receptor subtypes occurs in most
cell types, knock-out mouse models have shown minimal phenotypic
alterations (Contos et al., 2000; Ishii et al., 2001), although one of
them resulted in embryonic lethality (Liu et al., 2000). Thus, the two
recent Molecular Pharmacology publications examining
subtype-specific ligands are likely to generate additional studies that
explore the application of receptor subtype-selective agonists and
antagonists to different cell systems and animal models.
The two articles describe two different sets of compounds with
differing receptor subtype-selectivity (Table
2). The work of Fischer et al. (2001),
based on earlier structure-activity studies (Jalink et al., 1995;
Bandoh et al., 2000), has shown that truncating the length of the
sn-1 hydrocarbon chain results in a differential loss of
agonist properties toward the three LPA receptors, and that adding an
N-octyl chain to the sn-2 position of the
glycerol backbone converts the resulting dioctyl phosphatidic acid into
an antagonist. Based on computational studies and experimental validation of S1P1 and LPA1 receptor
models, our group, in collaboration with Dr. Parrill's group (Parrill
et al., 2000a,b; Sardar et al., 2001; Wang et al., 2001) has
characterized the essential requirement for ionic interactions between
LPA and two conserved positively charged residues, R3.28 (Ballesteros
and Weinstein, 1995) in the third, and K(R)7.36 in the seventh
transmembrane domains of the LPA receptors. To explore the effects of
extra charges in the polar headgroup, we have tested dioctylglycerol
pyrophosphate (DGPP), which was found to have slightly better
antagonistic properties over that of dioctyl phosphatidic acid. These
short chain phosphatidates showed preferential competitive inhibition
of the LPA3 receptor subtype
(Ki ~100 nM) over
LPA1 (Ki ~6.6 µM)
and LPA2, which is neither inhibited nor
activated by these two compounds up to 10 µM concentration. Fischer
et al. (2001) also examined a variety of LPA- and S1P-responsive cell
types that express different combinations of LPA and S1P receptors to
support a selective inhibitory action by these two compounds on the
LPA3 receptor subtype. DGPP potently inhibited
LPA-induced shape changes in human platelets and was without effect on
PAF- and thromboxane-induced response (Rother et al., 2001). One
concern with the use of DGPP is that an earlier report found that it
activated cPLA2 and ERK1/2 in P388D1, a macrophage cell line (Balboa et
al., 1999).
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The article by Heise et al. (2001) in this issue of Molecular
Pharmacology has explored a different lead compound,
N-acyl ethanolamide phosphate (NAEPA), which was originally
synthesized by Sugiura et al. (1994) in Dr. Hanahan's group. In
retrospect, these seminal observations represented a breakthrough that
was not fully appreciated by researchers in the field despite work by
our laboratory with that of Dr. Bittman's group (Bittman et al., 1996;
Liliom et al., 1996) and that of the Lynch laboratory supported by Dr.
McDonald's groups (Hooks et al., 1998, 2001; Im et al., 2000). Based
on the pioneering work of Dr. Hanahan's group (Sugiura et al., 1994),
we synthesized the enantiomers of N-palmitoyl-L(D)-serine-phosphoric
acid and
N-palmitoyl-L(D)-tyrosine-phosphoric acid, which inhibit LPA responses in Xenopus laevis oocytes
(Liliom et al., 1996; Hooks et al., 1998), platelets (Sugiura et al., 1994; Siess et al., 1999), and endothelial cells (Rizza et al., 1999;
Siess et al., 1999). However,
N-palmitoyl-L(D)-serine-phosphoric acid at micromolar concentrations was found to be a weak LPA mimetic on
LPA1 and LPA2 receptors
heterologously overexpressed in HTC4 cells, tempering further testing
of the compound (An et al., 1998). In this issue, Dr. Lynch's team
reports on the characterization of eight enantiomeric pairs of novel
NAEPA derivatives. Heise et al. (2001) identified three important
trends in the receptor selectivity of NAEPA derivatives: 1) the
receptors show a stereoselectivity, although not an absolute one, for
one enantiomer over the other; 2) compounds with short-chain
substituents on R3 (Table 2), specifically methyl (VPC12086),
methylene-amino (VPC12178), and methylene-hydroxy (VPC31143) analogs,
are agonists with a rank-order receptor preference LPA1 LPA2
LPA3; and 3) the enantiomeric pair of
benzyl-4-oxybenzyl analogs have different properties. VPC12204 is a
weak agonist, whereas VPC12249 was devoid of agonist action and showed
a selective competitive inhibitory effect with
Ki values of ~130 nM on
LPA1 and ~430 nM on the
LPA3 receptor, with no effect on
LPA2. The latter discovery of receptor subtype
selectivity has far-reaching implications for the design of
antagonists. It had been well established that LPA-elicited effects
lack stereo-specific recognition (Simon et al., 1982; Jalink et al.,
1995), because the natural (R)-LPA and unnatural
(S)-LPA are both biologically active. Heise et al. (2001),
however, have shown that NAEPA analogs, unlike LPA, are recognized in a
stereoselective manner and that this additional interaction unique to
NAEPA derivatives can provide the foundation for the design of
selective antagonists. These authors also provide data from
endogenously LPA-responsive human embryonic kidney 293T cells and from
rat arterial blood pressure measurements in support of the
pharmacological properties of their analogs.
Why should one care about novel ligands for phospholipid growth
factors? LPA receptor agonists are expected to possess antiapoptotic effects (Goetzl et al., 1999; Weiner and Chun 1999) and promote wound
healing (Balazs et al., 2001). On the other hand, LPA antagonists have
potential applications as inhibitors of inflammation (Rizza et al.,
1999), cancer invasiveness (Imamura et al., 1993), and atherogenesis
(Siess et al., 1999; Hayashi et al., 2001). Ovarian cancer cells
produce LPA-like substances that seem to act as paracrine factors to
enhance the invasiveness and chemotherapeutic resistance of this cancer
(Xu et al., 1995; Frankel and Mills 1996; Pustilnik et al., 1999; Eder
et al., 2000; Fang et al., 2000). LPA seems to cause dedifferentiation
of vascular smooth muscle cells in vitro only if it contains
unsaturated fatty acids, presumably through the
LPA3 receptor subtype; this mimics many of the
events that occur during the early stages of atheromatous plaque
formation (Hayashi et al., 2001). The inhibitors described by these two groups, some of which are now commercially available, can be tested further in a variety of in vivo model systems. The results of these two
studies should therefore provide further insights into the biology and
therapeutic applicability of phospholipid growth factor ligands.
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Acknowledgments |
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I thank Dr. Kevin Lynch for sharing his manuscript with me before its publication and Drs. Abby Parrill, Dan Baker, and Kazuaki Yokoyama for their helpful comments.
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Footnotes |
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Received October 23, 2001; Accepted October 23, 2001
Supported by USPHS grants HL9728147 and CA92160.
Dr. Gabor Tigyi, Department of Physiology, University of Tennessee Health Sciences Center Memphis, 894 Union Ave, Memphis, TN 38163. E-mail: gtigyi{at}physio1.utmem.edu
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Abbreviations |
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EDG, endothelial differentiation gene; LPA, lysophosphatidic acid; S1P, sphingosine-1-phosphate; DGPP, dioctylglycerol pyrophosphate; NAEPA, N-acyl ethanolamide phosphate.
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Article
References
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