Molecular mechanisms of lysophosphatidic acid action

Progress in Lipid Research 42 (2003) 498–526 www.elsevier.com/locate/plipres

Review

Molecular mechanisms of lysophosphatidic acid action Gabor Tigyia,*, Abby L. Parrillb a b

Department of Physiology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA Department of Chemistry and Computational Research on Materials Institute, The University of Memphis, Memphis, TN 38152-6060, USA

Contents 1. Introduction ........................................................................................................................................................... 499 2. The origins of LPA in biological fluids .................................................................................................................. 499 2.1. Generation by phospholipase hydrolysis ....................................................................................................... 499 2.2. Generation by lysophospholipase D cleavage ............................................................................................... 501 2.3. Production during oxidative modification of LDL ....................................................................................... 503 3. LPA Degradation................................................................................................................................................... 503 3.1. Degradation by phosphatases........................................................................................................................ 503 3.2. Degradation by acyltransferases.................................................................................................................... 505 3.3. Degradation by lysophospholipases .............................................................................................................. 507 4. Binding proteins in modulating the biological effect of LPA................................................................................. 507 5. LPA plasma membrane receptors .......................................................................................................................... 508 5.1. Biological targets ........................................................................................................................................... 508 5.2. Structure–activity studies............................................................................................................................... 511 6. Intracellular effects of LPA .................................................................................................................................... 515 6.1. LPA is an abundant cellular lipid ................................................................................................................. 515 6.2. The role of LPA in synaptic vesicle formation.............................................................................................. 515 6.3. The peroxisome proliferator-activated receptor-g (PPARg)—an intracellular receptor for LPA ................. 516

* Corresponding author. Tel.: +1-901-448-4793; fax: +1-901-448-7126. E-mail address: [email protected] (G. Tigyi). 0163-7827/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0163-7827(03)00035-3

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7. Pathophysiological correlates of LPA action ......................................................................................................... 517 7.1. LPA in cancer progression and metastasis .................................................................................................... 517 7.2. LPA in the pathobiology of cell survival and apoptosis ............................................................................... 518 7.3. The role of LPA in vascular pathophysiology............................................................................................... 518 8. Perspective.............................................................................................................................................................. 521 References ................................................................................................................................................................... 521

1. Introduction Lysophosphatidic acid (radyl-glycerol-phosphate, LPA) has captured the interest of lipid biochemists, cell biologists, physiologists, and physicians alike due to its myriad biological effects. Since it was first identified in 1957 as the active ingredient of Darmstoff (smooth muscle-stimulating substance) [1,2], our knowledge of LPA biology has steadily increased. In spite of hundreds of publications, we still lack a clear understanding of the role of LPA in disease or even its physiological role. Since the last comprehensive review on LPA in this journal by Tokumura in 1995 [3] major advances have occurred with regard to the production and breakdown, as well as the identification of plasma membrane and intracellular receptors for LPA. The present review is focused on these two aspects of LPA biology with a strong commitment to the physiological and pathophysiological context of these fundamental discoveries.

2. The origins of LPA in biological fluids LPA has long been known as a product of the lipid synthetic pathways. LPA is generated from glycerol-3-phosphate and acyl-CoA by glycerophosphate acyl transferase [4] and from monoacylglycerol by monoacylglyerol kinase [5] in mitochondria and microsomes. Three other mechanisms have been identified (Fig. 1) that can lead to LPA production in a stimulus-coupled manner. These pathways in their order of discovery include: (1) PLA1- or PLA2-mediated hydrolysis of PA, (2) Lyso-PLD-mediated hydrolysis of lysophospholipids, and (3) Oxidative modification of LDL. Despite recent advances identifying the enzymes involved in the various pathways; their regulation still remains obscure. 2.1. Generation by phospholipase hydrolysis Hydrolysis of fatty acids at the sn-1 position by PLA1 or at the sn-2 position by PLA2 enzymes generates LPA from phosphatidic acid (PA). In blood, platelets and to a smaller degree red blood cells, have been identified as sources of LPA [6,7]. Mauco and colleagues first reported the synthesis of PA and LPA in phospholipase C-treated platelets with identical specific activities and a sequential time course, suggesting that their synthetic pathways were coupled [8]. PA was found to be rapidly generated in thrombin-stimulated platelets and its conversion to LPA was proposed

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through PLA enzymes in the platelet [9–11]. PA-specific PLA1 and PLA2 have been reported in human [12–14], pig [15], and horse [16] platelets. Based on several independent reports, it is now accepted that generation of LPA from PA contributes only a minor proportion, estimated to be 10% [13], of LPA detected in serum [6,13,17] LPA generated through this mechanism appears within 15 min following thrombin stimulation of platelets. This is not to be confused with the contribution of platelets to LPA production in plasma. Aoki and colleagues, [6] using antibody depletion of platelets in rat blood, found a 50% reduction in LPA production. Platelets release PLA1 and PLA2 enzymes [13] that generate a de novo pool of lysophospholipids, primarily lysophosphatidylcholine (LPC) in plasma, which is further metabolized by lysophospholipase D. Thus, the rate-limiting step in the generation of LPA in blood is not the constitutively present plasma enzyme lyso-PLD but the release of PLA1 and PLA2 enzymes and the de novo generation of a new pool of lysophospholipids. The rank order of molecular species of LPA in thrombin stimulated platelets is 16:0 >18:0 > >20:4> >16:1>18:1> 18:2, whereas the rank order of PA molecular species after inhibition of PLA2 with U10029A is 18:0>20:4> >18:1>18:2> 16:0>18:2>16:1 [18]. This mismatch between the molecular species of LPA and PA suggests that the U10029Asensitive PLA2-mediated production of LPA is not the only mechanism that contributes to LPA production in the platelet. Secretory or type II PLA2 has been implicated in LPA production from microvesicles shed by activated inflammatory cells. These vesicles have altered membrane asymmetry that leads to PA accumulation in the outer leaflet of the plasma membrane [19]. Fourcade and colleagues [19] suggested that secretory PLA2 released from cells stimulated with interleukin-1b or tumor

Fig. 1. Biochemical pathways of LPA synthesis and degradation. PA, phosphatidic acid, LPX, lysophospholipid representing LPE, LPS or LPC, MAG, monoacylglycerol, PG, phosphatidylglycerol, PA–PLA1&2, PA-specific PLA1&2, Lyso-PLD, lysophospholipase D, LPP, lipid phosphate phosphatase, LPAAT, LPA acyltransferase, LPA– LPL, LPA-lysophospholipsae, GPAT, glycerophosphate acyltransferase.

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necrosis factor-a could participate in releasing arachidonate and LPA from the outer leaflet of microvesicles. Type II PLA2 is nearly inactive against phosphocholine but hydrolyzes aminophospholipids [17]. Thus, phosphatidylethanolamine and phosphatidylserine hydrolysis via sPLA2 can generate the corresponding lysophospholipid substrates of lyso-PLD cleavage. These products are substrates of lyso-PLD Such a mechanism has been proposed for LPA production in adipocytes [20]. 2.2. Generation by lysophospholipase D cleavage LPA is generated in aged serum [21] and plasma prepared from heparinized blood [22] indicating that these biological fluids contain the enzymes necessary to generate the lipid mediator. However, in these cases several hours elapse prior to a substantial increase in LPA concentrations indicating a very low rate of LPA production. There are characteristic differences in the LPA acyl species composition in human plasma and serum. In plasma the five major species show a rank order of 18:2 (50%) > 18:1 (15%) > 18:0 (13%) > 16:0 (12%) > 20:4 (11%), whereas after a 24 h incubation of serum the rank order is 20:4 (39%)518:2 (38%) > 16:0 (10%)518:1 (9%) > 18:0 (4%) [23]. There is consensus that the plasma concentration of LPA is low [22], approximately 130 nM [13], and slowly increases even in the presence of EDTA to reach a concentration of 680 nM after 1 h and 950 nM by 24 h [23]. In whole blood ex vivo, LPA levels increase rapidly to
1.2 mM within 1 h and to 5.2 mM after 24 h of incubation at 25  C [23]. Interestingly, LPA concentration in serum from healthy human female donors is slightly higher than in males, 5.6 mM versus 4.9 mM, respectively. The concept of lyso-PLD-mediated production of LPA in rat plasma [22] was first proposed in the 1980s. This pathway was subseqently confirmed in several other species including rabbits [24] and humans [13,25]. However, identification of the enzyme remained elusive until recently. Two groups independently reported purifying lyso-PLD [26,27], which unexpectedly is identical to autotaxin (ATX), a 125 kDa glycoprotein that stimulates tumor cell motility. With this breakthrough, many issues became clear. ATX promotes tumor cell motility, progression, metastasis, and angiogenesis via a pertussis toxin-sensitive mechanism [28–30]. LPA has been reported to have similar activities [31,32]. ATX/lyso-PLD is a member of the nucleotide pyrophosphatase/ phosphodiesterase (NPP) family, which includes PC-1/NPP-1 [33] and gp130RB13-6/NPP3 [34]. ATX/lyso-PLD and the other NPP-family members are capable of hydrolyzing ATP and ADP generating nucleoside 50 -monophosphates and are distinct from the lipase superfamily of proteins. While ATX/lyso-PLD can hydrolyze ATP with a Km estimated in the millimolar range, the Km for ATX/lyso-LPD hydrolysis of lyso-PC and sphingosylphosphorylcholine (J. Aoki, personal communication) is in the micromolar range. The ATX/lyso-PLD enzyme is comprised of a short N-terminal tail, a transmembrane domain, two cysteine-rich somatomedin B-like domains and a C-terminal catalytic domain. A soluble form of ATX is cleaved near the transmembrane domain [30] and is nearly ubiquitously present in biological fluids. Lyso-PLD activity in serum has been found to increase steadily during pregnancy from 66.7 nmol choline/ml per 24 h in nonpregnant healthy volunteers to 280 nmol choline/ml per 24 h at the end of the third trimester [35]. Interestingly, in patients with threatened preterm delivery lyso-PLD activity was significantly elevated to
220 nmol choline/ml per 24 h [35]. This increase in lyso-PLD activity is not accompanied by a concomitant rise in plasma LPA, which was attributed to increased metabolism and uptake of

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the lipid [35]. Whether this increase in enzymatic activity is due to an increase in enzyme expression remains to be demonstrated. Nonetheless, bFGF [29], BMP-2 [29], retinoic acid [36], as well as activation of Wnt-1 signaling [36] have been reported to upregulate ATX/lyso-PLD expression. Interestingly, in the plasma of pregnant women, 16:0 LPA accumulates to a greater degree followed by 18:0, whereas the unsaturated species remain low [35]. This is in sharp contrast to that seen in serum from non-pregnant donors [13], where the unsaturated species dominate. These profiles indicate different mechanisms and/or pools of lysophospholipids likely contribute to LPA generation in these distinct populations. Lyso-PLD activity does not seem to be regulated during blood clotting as the activity of serum and plasma appear to be the same [6]. The concentration of LPA rises rapidly during blood coagulation [6,13]. If ATX/lyso-PLD activity is not regulated, what explains the rapid generation of LPA? Platelets do not generate significant amounts of LPA, however they do secrete several phospholipases that in turn generate a pool of lysophospholipids de novo, primarily LPC and lysophosphatidylserine (LPS). Sano et al. [13] used activated plasma, isolated within 2 min after blood was drawn, to show that LPA generation increases 20-fold during the first hour. Several authors have shown the release of PLA1 [37] and PLA2 [13] activities from thrombin-stimulated human platelets that are capable of generating LPC, LPS, and lysophosphatidylethanolamine (LPE) from exogenously supplied PC, PS, and PE. Plasma LPC is derived from two main sources: liver PLA1 and lecithin-cholesterol acyltransferase (LCAT) [38]. Tokumura and colleagues proposed that LCAT is an important source of LPC that is accessible to lyso-PLD in plasma. This notion has been strengthened by the recent demonstration of reduced LPA production in plasma isolated from LCAT-deficient donors [6]. These authors showed that LPA concentration does not rise above
4 mM in LCAT deficient plasma. Unfortunately the acyl composition of LPA generated in LCAT deficient plasma is not yet known. This would be key information in assessing the metabolic pathway supplying LPC to lyso-PLD since there is a close correlation between the acyl-composition of plasma LPC and LPA [22]. LCAT deficient patients have abundant LPC in their plasma [6], thus there are multiple mechanisms generating this LPA precursor. The plasma concentration of LPC is 125–150 mM, making it the most abundant lysophospholipid in plasma [39,40]. As much as 41% of plasma LPC is the 1-lyso-2-acyl isomer. The origin of the sn-1-lyso-PC, which is highly enriched in polyunsaturated fatty acids, has been attributed to the action of liver PLA1 and is recognized as an important mechanism for delivering polyunsaturated fatty acids to tissues [41]. Because linolenoyl and arachidonoyl LPA together constitute the majority of LPA in activated plasma and serum [13,23], the role of PLA1 in supplying LPC to lyso-PLD should not be overlooked. Alkyl-GP is a naturally occurring analog of LPA. It has been found to be more potent in activating platelet aggregation than its acyl counterpart [42,43]. Tokumura and colleagues [42] have shown that human donors display differential sensitivity to alkyl-GP (16:0) and LPA (20:5) in that platelets from some donors do not respond to alkyl-GP despite responses to LPA. There was no apparent difference in the expression of LPA receptor transcripts found in alkyl-GP responsive and non-responsive platelets, both expressed LPA2 receptor transcripts. Alkyl-GP activates all three EDG-family LPA receptors albeit with a lower potency than LPA [44]. Tokumura and colleagues proposed that platelets of some donors express a distinct and as yet unidentified receptor selective for alkyl-GP [45]. The origin of alkyl-GP in biological fluids remains unknown. One possible mechanism includes transacetylases, which can transfer acetate from PAF [46] to lysophospholipid acceptors followed by lyso-PLD cleavage of the choline headgroup. Alkyl-LPA

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is abundant in brain tissue and is also found in egg yolk and saliva [47–49]. Alkyl-LPA may exert unique, non-receptor-mediated signaling effects [50]. Bacterial PLD has been shown to generate LPA from LPC if the precursor is present in the outer leaflet of the plasma membrane [51]. However the pathological significance of this reaction as well as the role of the LPA produced remains unknown. LPA is enriched in tumor cell effusions and ascites from ovarian cancer cells [52–54]. ATX/lysoPLD production seems to be underlying this phenomenon [35,55]. 2.3. Production during oxidative modification of LDL Siess and colleagues [56] investigated the lipid constituents formed during oxidation of low density lipoprotein (LDL) responsible for induction of shape change in platelets and endothelial cells. Mild oxidation of LDL (moxLDL) catalyzed by Cu2+ changes the biological properties of LDL to that of pro-thrombotic and pro-atherogenic. These authors showed that LPA-like biological activity is generated by this non-enzymatic oxidation that co-migrates with an authentic LPA standard by TLC. The pro-thrombotic effects of mox-LDL were inhibited with NP-tyrosine and NP-serine phosphoric acids, inhibitors of LPA-induced platelet shape change. These inhibitors of LPA receptors also blocked the shape change induced by LPA and mox-LDL in human umbilical cord vein endothelial cells. LPA and mox-LDL but not PAF or ADP induce cross desensitization of the shape change response in platelets. The CD36 scavenger receptor has been shown to mediate the uptake of LDL into cells. In this context, the finding of Siess et al. [56] that LPA accumulates in human atherosclerotic plaques is significant, particularly because of recent evidence that LPA activates the peroxisome proliferator activator receptor g (PPARg), which in turn regulates CD36 expression [57]. Neither the acyl/alkyl species composition nor the precursor of LPA formed in LDL is presently known. An alternative to the oxidative mechanism generating LPA directly, is that oxidation could also lead to the activation of ATX if present in LDL. The mechanism responsible for LPA generation in moxLDL will have to be addressed in future studies.

3. LPA degradation LPA degradation includes three major pathways (Fig. 1). The first is phosphate removal to form monoacylglycerol by phosphatase or phosphohydrolase enzymes. The second is conversion to PA by acyltransferases. The third is removal of the sn-1 acyl chain to form glycerol phosphate by LPA-specific lysophospholipases. 3.1. Degradation by phosphatases LPA is dephosphorylated to form monoacylglycerol by the action of membrane spanning phosphatase enzymes in the phosphatidate phosphatase type 2 (PAP-2, lipid phosphate phosphohydrolase, LPP) family [58] as well as by an as yet unknown nuclear lysophosphatidic acid phosphohydrolase [59]. Four isoforms of LPP have been cloned and characterized in mammals, LPP1/ PAP-2a/PAP-2a1 [60], LPP1a/PAP-2a2 [61], LPP2/PAP-2c/PAP-2g [62] and LPP3/PAP-2b/PAP-2b

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[60]. While these isoforms are all capable of dephosphorylating LPA and other glycerolipid and sphingolipid phosphates, they do demonstrate different selectivities for phospholipid substrates in vitro. LPP1 most efficiently catalyzes the dephosphorylation of LPA, followed by phosphatidic acid, sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) [62,63]. LPP2 is selective for PA, followed by C1P, LPA and finally S1P [62]. LPP3 shows similar activity toward PA and LPA, and less efficiently dephosphorylates C1P and S1P [62]. Substrate recognition by the LPP1 isoform is not stereoselective nor is it selective for acyl versus alkyl LPA analogs [64]. The topology of LPP enzymes is well-understood based on insertion studies on a rat homolog of LPP3, Dri 42, which demonstrated that the six hydrophobic segments span the membrane and that both termini are cytosolic (Fig. 2) [65]. All four isoforms are expected to exhibit this topology due to the high sequence identity ( >50%) and functional homology. Two PRG isoforms are also expected to share this topology, but differ in the presence of a hydrophilic extension at the Cterminus comprising half the length of the overall protein sequence [66]. Table 1 demonstrates that the percent of identical amino acid residues in the LPP isoforms is significantly lower from

Fig. 2. Topology of LPP enzymes. Table 1 Percent amino acid identity (homology) among human LPP sequences aligned using the default parameters in the Moe [217] program

LPP1 LPP1a LPP2 LPP3

LPP1 (AB000888, AF014402, AF017116)

LPP1a (AF014403)

LPP2 (AF035959, AF056083, AF044760)

LPP3 (AB000889, AF043329, AF017786)

100/100/100 88.4/56.2/99.5 58.1/46.6/62.1 51.1/43.9/53.6

88.1/55.4/99.5 100/100/100 55.8/37.8/62.1 48.1/32.4/53.6

57.3/47.9/60.4 55.2/39.4/60.4 100/100/100 45.8/36.6/48.8

46.6/31.4/54.1 41.1/23.5/54.1 42.4/25.5/50.7 100/100/100

The first, second and third numbers represent overall homology, homology from the amino terminus through the end of the second hydrophobic domain (residue 73 in LPP1), and homology in the remainder of the sequence, respectively. GenBank protein accession numbers are shown in parentheses for each isoform.

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the amino-terminus through the end of the second transmembrane domain than it is for the remainder of the sequence. This trend is particularly evident on comparing LPP1 and LPP1a, which have 88% homology overall, with only 56% homology through the end of the second hydrophobic segment and greater than 99% homology after that point. This suggests that functional similarities among the LPP isoforms such as substrate recognition and catalysis may depend on amino acid residues after the second hydrophobic domain. On the other hand, functional differences such as substrate selectivity may arise from structural features through the end of the second transmembrane domain. The second and third extracellular loops of the LPP enzymes contain motifs in three regions conserved across the phosphatase superfamily [58,67]. These conserved regions are also present in vanadate chloroperoxidase which, like the LPP isoforms, exhibits a vanadate-inhibited phosphatase activity [68]. The X-ray structure of this chloroperoxidase [69] demonstrates that many of the residues in these conserved regions hydrogen bond with the vanadate ion, which is thought to occupy a position analogous to the phosphate group [68]. Conservative mutations of the corresponding residues, K120, R127, P128, S169, H171, R217 and H223, in mouse LPP1 result in a 95% loss of specific activity relative to the wild type enzyme [70]. Mutation of the conserved G170 resulted in a 60% loss of specific activity whereas mutation of nonconserved residues both within and outside of these regions had more modest effects [70]. Thus, the second and third extracellular loops of the LPP enzymes comprise the catalytic site. Mutation of H252 in PRG-1, which is analogous to H171 of LPP1, similarly reduced ecto-phosphatase activity by 95% [66]. The phosphate moiety of LPA is likely to bind to the amino acid residues identified by Zhang et al. [70] with the hydrophobic tail within the bundle formed by the six transmembrane domains of LPP. The LPP isoforms exhibit characteristic expression patterns that suggest potential physiological roles. The LPP3 isoform is ubiquitously expressed [60] whereas the LPP1 isoform presents varying expression levels in different tissues [60,71,72]. Northern blot and dot-blot analysis indicates that LPP1 is most abundant in prostate with reduced expression levels in heart, bladder, uterus and other tissues. LPP1 expression in the LNCaP human prostate adenocarcinoma cell line was found to be induced by androgens in a dose- and time-dependent manner [72]. The induction was not inhibited by cycloheximide and is thus independent of other androgen-induced proteins. The important role of androgens in prostate development, growth, function [73] and pathology implicates LPP1 in prostate cancer. Studies of LPP isoform expression in adipose tissue suggest another physiological role for LPPs [74]. Amounts of LPP1 and LPP3 mRNAs exceeded that of LPP2 mRNA levels detected in preadipocytes. Differentiation into adipocytes reduced both phosphatase activity and LPP expression. These results suggest LPA phosphatase activity as a potential target to control adipose tissue development. Finally, PRG-1 has been shown to be expressed in the brain in a spatially and developmentallyregulated fashion correlated with axon outgrowth [66]. Transfection of PRG-1 into N1E-115 cells, which retract neurites upon LPA treatment, demonstrated that PRG-1 attenuates this retraction. Thus PRG-1 appears to play an important role in the development of the brain. 3.2. Degradation by acyltransferases The second LPA degradation pathway involves the action of 1-acylglycerol 3-phosphate acyltransferase (AGPAT) enzymes, also called lysophosphatidic acid acyltransferases (LPAAT).

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These enzymes catalyze the transfer of an acyl group from acyl-CoA to LPA to form PA. Proteins with LPAAT activity include a transmembrane family of enzymes [75,76], and membraneassociated proteins involved in membrane fission such as endophilin [77–79] and CtBP/BARS [80]. Members of the LPAAT family have been proposed to mediate the majority of activity in most cells [76]. Five members of the LPAAT family have been sequenced, LPAATa/1-AGPAT 1 [75,81], LPAATb/1-AGPAT 2 [75], LPAATg/1-AGPAT 3(GenBank accession AF156774), LPAATd/ 1-AGPAT 4(GenBank accession AF156776) and LPAATe/1-AGPAT 5(GenBank accession AF375789). LPAATa and LPAATb appear responsible for most LPAAT activity in cells due to their higher catalytic activity in relation to the other family members [76]. LPAATa shows a marked preference for LPA over other acyl acceptors [82], including LPC, LPE, LPI and lysoPAF. Only an alkyl-LPA species was able to serve as an alternative acyl acceptor, although only half the acyltransferase activity was observed in comparison with the acyl-LPA. Both LPAATa and LPAATb show less discrimination with regards to the acyl donor [75,82,83]. Acyltransferase activity was observed with acyl-CoA chain lengths varying from 14 to 20, including both saturated and unsaturated chains. hLPAATb preferred arachidonic acid over palmitic acid whereas the opposite selectivity by hLPAATa was observed, although differences in the assay conditions were noted [75]. Hydropathy analysis of LPAATa [76] and LPAATb [83] identified several hydrophobic segments. Further consideration of cleavage at a signal site after residue 58 and the alignment of several sequences resulted in a suggested topology involving only two transmembrane domains [75]. This topology places the termini in the lumen of the endoplasmic reticulum (ER) and one conserved region on the cytosolic side [75]. The conserved region includes the motif PEGTR found at residues 177–181 of human LPAATa. This is only one of two highly conserved motifs, the other having a consensus sequence NHQSxxD found at residue 103 of human LPAATa [76]. The topology proposed by Aguado et al. places these two conserved regions on opposite sides of the membrane. Another two transmembrane domain topology involving the first two hydrophobic segments and no signal cleavage site has been proposed that places these two segments on the cytoplasmic side of the ER [76]. This second topology is more consistent with mutagenesis studies on the related sn-glycerol-3-phosphate acyltransferase which found significantly diminished Vmax values for mutations within both of these conserved motifs [84]. The two major LPAAT isoforms show distinctly different expression patterns [76]. LPAATa has been detected uniformly in most tissues examined, whereas the b isoform is differentially expressed. LPAATb was also found to be elevated in several tumor tissues relative to matched normal tissues, [61,76] implicating the overproduction of PA in several types of cancer. In several cases, expression changes in LPAATb and PAP-2a were inversely related indicating additional likelihood of elevated PA levels due to both increased production from LPA and decreased degradation to diacylglycerol [61,76]. Congenital generalized lipodystrophy, which characteristically involves low body fat percentage, insulin resistance and early onset diabetes, has been linked to mutations in LPAAT enzyme AGPAT2/LPAATb [85]. The observed mutations may cause lipodystrophy due to impaired triglyceride synthesis, or through LPA accumulation in tissues. LPA accumulation may contribute to lipodystrophy due to its influence on adipocyte function [74]. LPAATa/AGPAT1 is encoded by a gene found within the Class III region of the major histocompatibility complex (MHC) [75].

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The abundance of diseases associated with MHC gene products suggests that dysfunction of LPAATa may also be tied to human disease, although further research is necessary to identify relevant diseases. 3.3. Degradation by lysophospholipases The third pathway involved in LPA degradation involves the hydrolysis of the acyl group from the phosphoglycerol headgroup by the action of lysophospholipase enzymes. The majority of characterized lysophospholipase enzymes act on LPC [86]. However, LPA has been identified as a competitive inhibitor of LPC lysophospholipase activity in rabbit neuronal nuclei [59] and as the substrate of a distinct LPA lysophospholipase activity that is not substantially enriched in the nuclear fraction over the microsomal fraction [59]. An 80 kDa protein with LPA lysophospholipase activity has been purified and characterized from rat brain [87]. This enzyme catalyzes hydrolysis of 1-oleoyl and 1-stereoyl LPA most readily, followed by the 1-palmitoyl and 1-myristoyl LPA species. The purified lysophospholipase did not hydrolyze LPE, LPI or LPS as substrates. Characterization of the rat brain enzyme involved mostly elucidation of substrate selectivity [87], whereas evaluation of the LPA phospholipase activity in rabbit neuronal nuclei focused on effects of inhibitors, phospholipids, CoA, and MgATP [88], thus it is impossible to determine if they represent the same protein.

4. Binding proteins in modulating the biological effect of LPA Due to its hydrophobic character, LPA associates with other lipids and proteins both in biological fluids and within cells. These interactions modify the biological properties of LPA. Association of LPA with binding proteins has been suggested to be responsible for the disparity between the LPA concentration in plasma (
100 nM), which is far in excess of the nanomolar Kd of LPA receptors, and the lack of LPA-like biological activity of plasma [13]. LPA receptors are differentially activated with LPA carried by different proteins [89,90]. While LPA1 and LPA2 receptors show little sensitivity to the presence of blood plasma or albumin, activation of LPA3 is fully blocked by 104 dilution of blood plasma or 1% bovine serum albumin. Interestingly, seminal plasma diluted 103-fold completely blocks the activation of LPA1 and LPA2 receptors, whereas it does not affect the activation of LPA3 receptors [89,90]. Albumin has been identified as a carrier of LPA in blood plasma [91–95]. Albumin can bind up to 3 mol LPA/mol of protein at the long-chain fatty acid binding sites with a nanomolar affinity, comparable to that of oleate [96]. Interestingly, LPC and LPE both bind to sites distinct from those for LPA, which appear to be identical to that for bilirubin and medium-chain fatty acids [96,97]. Although albumin is almost always applied as a carrier for LPA in experiments, caution must be exercised since it interferes with the binding of LPA-like compounds including alkylacetyl-PA and PAF to platelets [90]. Liver fatty acid binding protein (LFABP) has been recognized as an intracellular carrier of LPA [98]. LFABP has 2 LPA binding sites per molecule and binds LPA, LPC, LPE and LPG with affinities in the micromolar range [99]. The intracellular concentration of LFABP is estimated to be as high as 0.2–0.4 mM [100], which would suggest a stoichiometric binding of these

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lipids to the carrier. Interestingly, besides the liver and the intestine, LFABP is expressed in the proximal tubules of the kidney [101] where it could play a role in the reabsorption of lysophospholipids. LFABP is hypothesized to play a role in extracting LPA from the membrane and buffering high lysophospholipid concentrations generated by phospholipases in the hepatocyte. Whether LFABP binding of LPA-like lipids affects the activation of the peroxisome proliferators activating receptor-g (PPARg) remains to be investigated, but if it does, it could provide a physiological mechanism of attenuation. Gelsolin, discovered in 1979 as an intracellular actin binding protein involved in the remodeling of cellular actin filaments associated with cell shape changes and movement [102], has a secreted isoform as well. The secretory gelsolin isoform, called plasma gelsolin, circulates in human and rodent blood at concentrations of 25050 mg/l. Gelsolin binds both monomeric and filamentous actin, although it prefers the latter. This binding requires micromolar calcium concentrations and is of high affinity with a nM dissociation constant. Gelsolin also binds LPA [103] with nanomolar affinity [104]. Lind and colleagues proposed a novel hypothesis for the role of plasma gelsolin in inflammatory homeostasis [105]. In local injury, activated platelets and leukocytes generate LPA and at the same time cell lysis releases actin, which binds to plasma gelsolin. This local gelsolin depletion allows LPA to exert effects on defense and repair. In catastrophic injury, a more drastic gelsolin depletion permits LPA and possibly other mediators to impact distant organs, especially the lung. These authors have shown [105] that plasma gelsolin levels fell to about one third of normal in the adult respiratory distress syndrome (ARDS) which is associated with massive tissue damage and presumed actin release.

5. LPA plasma membrane receptors 5.1. Biological targets Extracellular LPA evokes a variety of biological responses that are mediated through a subfamily of G protein-coupled receptors (GPCR). Three members of this family have been identified in mammals as LPA receptors. The first reported LPA receptor, LPA1/EDG2/vzg-1 was cloned from sheep [106] and was identified as highly homologous to the orphan GPCR, endothelial differentiation gene 1 (EDG1), which was later identified as an S1P receptor and renamed S1P1. LPA1 was later cloned from mouse [107–109] and human and identified as a receptor for LPA [110–112]. The human protein sequence of LPA1 can be accessed in GenBank using accession numbers JC5293, NP476500, NP001392 and Q92633. Northern blot analysis demonstrated LPA1 mRNA is most abundant in brain, followed by heart with less abundance in tissues of the gastrointestinal tract and reproductive system [110,112]. The second described LPA receptor, LPA2/EDG4, was identified from a database of expressed sequence tags on the basis of its similarity to LPA1 [110]. LPA2, unlike LPA1, is expressed abundantly in leukocytes and testis [110]. The human protein sequence of LPA2 is available from GenBank under accession numbers Q9HBW0, NP004711, AAG28521, AAF43409 and AAC27728. The first four entries are identical in sequence whereas the last entry, commonly thought to be a sequencing error, replaces the four residues at the C-terminus with 33.

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LPA3/EDG7 was subsequently identified as an LPA receptor by two research groups [113,114]. Northern blot analysis detected LPA3 mRNA in human heart and prostate [113,114], rat kidney and testis [114], and human testis and pancreas [113]. The human LPA3 sequence is deposited in GenBank with protein accession numbers NP036284, AAF91291 and AAD56311. The human sequences of all three LPA receptors are shown in Fig. 3. Molecular models of both the active [115,116] and inactive [115] conformations of the three LPA receptors have been developed by homology to a validated S1P1 receptor model [117]. These models were used in docking studies to identify the interactions of the LPA receptors with LPA [115,116]. These modeling and subsequent site-directed mutagenesis studies identify three key interactions between the receptors and the polar headgroup of LPA, one of which confers specificity for LPA over S1P [116]. In particular, cationic residues from the third and seventh transmembrane domains (TM) were found to interact with the phosphate group of LPA and a glutamine residue in the third TM was found to hydrogen bond with the sn-2 hydroxyl group. Table 2 summarizes the positions of these residues. S1P recognition similarly involved two cationic residues from the third and seventh TM, but differed in that an anionic glutamate residue in the same position as the glutamine residue in the LPA receptors formed an ion pair with the S1P ammonium moiety [117,118]. Docking studies involving these receptor models were also used to explore binding of the LPA3-selective antagonist, dioctylglycerol pyrophosphate [115]. The studies with DGPP were the first to propose a ligand-binding site involving the extracellular loops (EL) of the LPA receptors. Although this binding site involved the EL, the pyrophosphate group

Fig. 3. Alignment of human LPA receptor sequences. Transmembrane domains (TM) are enclosed in boxes and labeled. Ovals enclose amino acid residues discussed in the text.

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of DGPP interacted with the same cationic residues within TM3 and TM7 that interact with LPA. Additionally, these studies identified a residue in the first extracellular loop that may be involved in the observed selective DGPP antagonism of LPA3 with a Ki of 106 nM, compared with a Ki of 6600 nM at LPA1 and the lack of antagonism at LPA2 [115,119]. The amino acid K95 present in LPA3 can ion pair with the pyrophosphate group of DGPP. The R114 residue found in LPA1 can also ion pair, but is not as well positioned. The LPA2 receptor has an alanine at the corresponding position, which is unable to participate in ion pairing. These amino acid residues are highlighted in Fig. 3. Modeling studies have also been used to rationalize the unusual pharmacology of a series of fatty alcohol phosphate (FAP) structures [120]. FAP structures with saturated alkyl chains ranging from 10 to 14 carbons in length were specific partial agonists of the LPA2 receptor (700 nM EC50 for FAP-12), while those having saturated alkyl chains of 10 and 12 carbons were antagonists of LPA3 (90 nM Ki for FAP-12) and weak antagonists of LPA1. Modeling studies demonstrated that binding of FAP to LPA2 was similar to LPA binding, involving phosphate interactions with cationic residues in the third and seventh TM domains and a hydrophobic tail position in the TM bundle. Modeling of FAP-12 interactions with LPA3 and LPA1 gave results similar to studies of DGPP binding in that FAP-12 occupied a binding site involving the extracellular loops and the same residue in the first extracellular loop seems to play an important role in selectivity (unpublished data). These modeling studies indicate that phosphate groups of both agonists and antagonists interact with common residues, but the agonist and antagonist binding pockets are otherwise distinct. Elucidation of the events downstream of LPA receptor activation utilize heterologous overexpression of individual LPA receptors in LPA-unresponsive cells. These studies have been recently reviewed [121–126]. More recently, characterization of genetically engineered mice lacking LPA receptors has provided insight into the role of the LPA receptors in intact organisms. Targeted deletion of the gene coding for LPA1 in mice resulted in impaired suckling behavior leading to 50% neonatal lethality, craniofacial dysmorphism, and increased apoptosis of sciatic nerve Schwann cells [127]. Subsequent characterization of LPA2 knockout mice demonstrated no phenotypic abnormalities [128]. The only significant abnormality in double knockouts lacking the genes for both LPA1 and LPA2 was an increased incidence of perinatal frontal hematoma compared with the LPA1 knockout mice [128]. Studies on cell lines derived from these mice indicate that these receptors have both overlapping and distinct responses. A likely explanation of these Table 2 Predicted interactions between the polar headgroup of LPA and the LPA receptors based on molecular modeling [115,116] Receptor

LPA1 LPA2 LPA3

Residues ion-pairing with phosphate

Residue hydrogen bonding with hydroxyl (3.29)

(3.28)

(7.35)

(7.36)

R124 R107 R105

– – K275

K294 K278 R276

Q125 Q108 Q106

The prediction validated by site-directed mutagenesis [116] is shown in bold. Numbers in parentheses indicate the helix in which the residue appears and the position of that residue relative to the most conserved residue in the GPCR family which is numbered 50 in each helix [218].

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results is that there are additional LPA receptors unrelated to the EDG family. At the time of writing this review there is preliminary evidence of at least one such mammalian receptor, termed LPA4 [219]. 5.2. Structure–activity studies LPA and structural analogs can all be described as having a polar headgroup, a linker, and a hydrophobic tail (Fig. 4). Of these three motifs, modifications to the polar headgroup are least well tolerated. Serum response element-driven luciferase expression assays of LPA1 and LPA2 activation demonstrated that serine, choline, or ethanolamine derivatives of LPA, LPS, LPC and LPE, respectively, were not active [110]. Competition binding assays in Sf9 cells [113] as well as [gS35]-GTP binding to HEK293T cell membranes [114] demonstrated that recombinant LPA3 does not bind or respond to LPS, LPC, LPE or lysophosphatidylinositol (LPI). Only modifications of the phosphate headgroup that retain negative charge under physiological conditions have been demonstrated to retain receptor activation. Two different structures with phosphonate headgroups (tail¼oleoyl, R1, R2¼H, X¼CHOH or C¼O and headgroup=PO3H2 in linker E in Fig. 5) stimulate [gS35]-GTP binding to HEK293T cell membranes expressing either LPA1 or LPA2 (about two-fold less potently than LPA) but not LPA3 [129]. One other agonist with a modified headgroup has been reported [130]. This structure, 1-oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), contains a sulfur substitution for one of the phosphate oxygen atoms as well as a methoxy group in the linker region in place of the hydroxyl found in LPA. OMPT potently activates the LPA3 receptor, with an EC50 of 276 nM for [gS35]-GTP binding to HEK293T cell membranes, a number similar to the 196 nM EC50 observed for LPA in the same assay. Activation of LPA2 was observed at high OMPT concentrations, but no LPA1 activation was noted even at 1 mM. This selectivity can be rationalized on the basis of the second pKa values of phosphorothioic acid and phosphoric acid, 5.4 and 7.2, respectively [131]. This difference of nearly two orders of magnitude in Ka indicates that in neutral solution, nearly all OMPT molecules will be present with a 2 charge whereas LPA molecules with 1 and 2 charges will be present in approximately equal concentration. Molecular modeling of the LPA receptors has identified the presence of an additional cationic residue interacting with the LPA phosphate in the LPA3 complex relative to the LPA1 and LPA2 complexes (Table 3) [115]. This additional charge may be necessary for interaction with a dianionic molecule such as OMPT. One other modification of the headgroup retains receptor recognition, but not receptor activation. Dioctylglycerol pyrophosphate (DGPP) selectively antagonizes LPA action at the LPA3, and to a lesser extent the LPA1 receptor [119]. Modeling studies examining DGPP interactions with the LPA receptors indicate that the presence of two alkyl chains in DGPP rather than the single chain found in LPA results in binding at a distinct, but overlapping site [115]. The LPA receptors tolerate changes in the linker region better than changes in the headgroup. Fig. 5 contains the range of linker structures that have been tested in recombinant receptor-

Fig. 4. Structural regions of LPA and analogs.

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Fig. 5. Linker structures present in LPA analogues tested in recombinant systems. Linkers below the dashed line are not found in any active compounds.

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expressing systems, with linkers found in active compounds above the dashed line and those found only in inactive compounds below the line. One feature of the linker SAR is a lack of stereoselective recognition of glycerol-linked structures. This effect was demonstrated using an enantiomeric pair of ether-linked phospholipids, 1-O-hexadecyl-sn-glycero-3-phosphate (1-C16GP, linker A in Fig. 5) and 3-O-hexadecyl-sn-glycero-1-phosphate (3-C16-GP, linker B in Fig. 5) [64]. Both 1-C16-GP and 3-C16-GP mobilized intracellular calcium in RH7777 cells stably expressing LPA1 and LPA3, and RH7777 cells transiently expressing LPA2. Responses to 1 mM lipid in stably-transfected cells and to 5 mM lipid in transiently-transfected cells were equivalent. A second linker SAR feature is the lack of regioselectivity for hydrophobic tail attachment observed for the LPA1 and LPA2 receptors, in contrast to the significant preference of the LPA3 receptor for 2-O-linked structures (Linker C in Fig. 5) [44,113]. An 8–10-fold difference in EC50 values for intracellular calcium mobilization was observed in Sf9 cells expressing LPA3 upon stimulation with a series of 1-acyl (linker A) and 2-acyl (linker C) compounds [44]. In addition to minor variations of glycerol as a linker, compounds containing ethanolamine have also been studied. Three important observations have resulted from the use of ethanolamine and its derivatives as linkers (E in Fig. 5) [114,132]. First, the parent compound, N-oleoyl ethanolamine phosphoric acid (NAEPA 18:1, R1, R2=H, X=O in linker E in Fig. 5), exhibits activity similar to LPA 18:1 in stimulating [g35S]-GTP binding to HEK293T cell membranes of LPA1 and LPA2expressing cells. In contrast, NAEPA 18:1 was much less active against LPA3 [114]. Second, the LPA receptors demonstrate stereoselectivity for the ethanolamine-linked analogues with R1 substitutions being more active than R2 substitutions [132] in contrast to the non-stereoselective recognition of the glycerol-linked analogs. The fold-difference in EC50 between enantiomeric pairs acting at the LPA1 receptor ranged from 44 for ethyl substitution to greater than 1000 for methylene amino substitution. Differences at the LPA2 receptor ranged from 26-fold for ethyl substitution to greater than 273-fold for methyl substitution, disregarding pairs in which neither isomer was particularly active. While each compound was less active at the LPA3 receptor, the fold-difference between enantiomers when measurable was much smaller as evidenced by the 4and 13-fold differences in EC50 for the ethyl- and methylene hydroxyl-substituted isomers, respectively. Third, the benzyl-4-oxybenzyl derivatives were competitive antagonists of LPA action at the LPA1 and LPA3 receptors [132]. These glycerol and enthanolamine-based derivatives demonstrate that significant changes to the linker region are tolerated by the LPA receptors. However, the fluorine-substituted glycerol backbone [57,133] and the sphingosine backbone [110,113,114] have not yet produced active compounds. A notable discrepancy between observed biological effects and results of assays using recombinant systems involves two structures with amino acid linker regions (Fig. 6). NPSerPA and N-palmitoyl-l-tyrosine phosphoric acid (NPTyrPA) were initially reported to antagonize the effects of LPA on platelets [134], endothelial cells [56] and in Xenopus oocytes [135]. Subsequent assays of these two structures against recombinant Tag-Jurkat cells expressing LPA1 or LPA2 showed that 1 mM NPSerPA produces a very weak increase in aequorin luminescence relative to 100 nM LPA whereas NPTyrPA was inactive [136]. NPTyrPA did not antagonize LPA action at either of these receptors. These results may indicate that the observed antagonism is mediated through the LPA3 receptor, which is specific for several compounds featuring dianionic headgroups as these two structures have. Alternatively, currently unknown plasma membrane receptors may be the targets of these structures.

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Several structure–activity trends are evident from studies on LPA species with differing hydrophobic tails. An extensive comparison of intracellular calcium mobilization in LPA-receptor overexpressing Sf9 cells by LPA species with different hydrophobic groups highlights several of these trends [44]. The first trend, decreasing activity upon decreased chain length, was consistent with early SAR studies of LPA-induced calcium mobilization in A431 cells [137] and mitogenesis in fibroblasts [138]. Second, the LPA3 receptor shows a marked preference for cis-
9-unsaturated hydrophobic tails as demonstrated by the data in Table 3. Note in particular the >650-fold increase in EC50 over 1-oleoyl LPA for the saturated 1-stearoyl LPA, the unnatural trans stereoisomer 1-elaidoyl LPA and the
12 positional isomer 1-petroselinoyl LPA. Neither LPA1 nor LPA2 showed greater than a 10-fold difference among any of these analogues. This preference for an unsaturated hydrophobic tail is parallel to the structure–activity data collected for LPAinduced cardiovascular effects in rats [139]. Separate investigations of ether-linked (alkyl) LPA analogs have focused on 16:0 (hexadecyl) [64] and 18:1 (cis-
9 hexadecenyl) [44] hydrophobic tail groups. Both research groups used FURA-2 assays of intracellular calcium mobilization. Full

Fig. 6. LPA analogs with amino acid linker regions.

Table 3 LPA acyl group activity mobilizing intracellular calcium in Sf9 cells overexpressing LPA3 Acyl group

Length/unsaturation

1-Oleoyl 1-Linoleoyl 1-Linolenoyl 1-Arachidonoyl 1-Elaidoyl 1-Palmitoleoyl 1-Petroselinoyl 1-Stearoyl

18:1 18:2 18:3 20:4 18:1 16:1 18:1 18:0

(
9) (
9,
12) (
9,
12,
15) (
5,
8,
11,
14) (trans
9) (
9) (
12)

EC50 (nM) [44]

Fold-increase over 1-oleoyl


75
80
80
1000 > 50,000 > 25,000 > 50,000 >50,000

1 1 1 13 > 650 > 300 > 650 > 650

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dose–response curves were generated for the unsaturated analog acting on LPA receptor-expressing Sf9 cells, giving EC50 values 10-, 100- and 80-fold higher than for 1-oleoyl LPA against LPA1, LPA2 and LPA3, respectively. The saturated analog was compared with 1-oleoyl LPA at 1 mM concentrations in stably-transfected RH7777 cells expressing LPA1 and LPA3, and at 5 mM concentrations in transiently-transfected RH7777 cells expressing LPA2. At these concentrations, the alkyl- and acyl-LPA species showed similar effect against LPA1 and LPA2, with the alkyl-LPA species showing decreased effect at LPA3. As LPA3 shows a significant preference for unsaturated hydrophobic tails, this difference in maximal effect may be due to the saturation, rather than the ether linkage.

6. Intracellular effects of LPA 6.1. LPA is an abundant cellular lipid Due to the interest and advances in identifying LPA receptors and receptor-mediated signaling, intracellular roles of LPA have largely been overlooked. The tissue concentrations of LPA are rather high, 58 nmol/g in rat liver, 53 nmol/g in kidney and 92 nmol/g in the brain [140]. The acyl chain composition of LPA (total concentration 143 nmol/g) in guinea pig liver is dominated by stearoyl-LPA (65.9 mol%) followed by linolenoyl (14.3 mol%) and palmitoyl (9.2%). StearoylCoA comprises 72% of liver saturated fatty acyl CoAs [141] suggesting that acylation of glyero-3phosphate in mitochondria and dihydroxyacetone phosphate in microsomes could be responsible for the relative abundance of this species. While these LPA concentrations are surprisingly high the compartmental distribution (plasma membrane, Golgi membrane, mitochondrial, nuclear envelope, cytoplasm) of LPA is not yet known. 6.2. The role of LPA in synaptic vesicle formation The shape of membrane lipids determines membrane curvature [142]. Inverted cone-shaped lipids abundant in the outer leaflet of a membrane bud promote positive curvature, whereas coneshaped lipids favor negative curvature and are found in the inner leaflet of a bud. LPA is an inverted cone-shaped lipid, whereas PA is a cone-shaped lipid [142]. Thus membrane curvature can be influenced by the accumulation and local changes in the ratio of LPA to PA. The seminal findings of Schmidt and colleagues [143] have provided evidence that endophilin I, an SH3 domain-containing membrane associated protein that binds to dynamin in a GTP-dependent manner is a lysophosphatidic acid acyltransferase (LPAAT). LPAATs catalyze the reaction of LPA with acyl-CoA, primarily arachidonoyl-CoA. LPAATs come in two types, transmembrane LPAATs have apparent molecular masses of
29 to 31 kDa and are involved in modulating cytokine responses [83], and membrane-associated LPAATs of the endophilin family with molecular masses of
40 kDa [144]. Increase in the activity of endophilin is regulated by direct phosphorylation by a cyclic-AMP dependent protein kinase or Ca2+/calmodulin-dependent protein kinase II [145,146]. Endophilin I has a high specificity for LPA over other lysophospholipids including LPC, LPS, LPI and LPE and can use both arachidonoyl-CoA an effective membrane-bending substrate and palmitoyl-CoA an inhibitor of membrane fusion [143]. Exogenously added LPA also inhibits membrane fusion, which suggests that it flip-flops to the

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inner leaflet and increases the substrate pool of inverted cone-shaped lipids. Inhibition of cytosolic-Ca2+-dependent and independent PLA2 potentiated synaptic-like vesicle formation presumably via the regulation of the LPA pool available to endophilin I. The catalytic activity of endophilin I is not sufficient to promote membrane fission, as a deletion mutant in the SH3 domain was inactive. Endophilin I binds to dynamin [144] and synaptojanin [147] through its SH3 domain. Schmidt et al. [143] proposed a model in which dynamin and synaptojanin recruit endophilin to the membrane, where its catalytic activity is regulated by phosphorylation. The local accumulation of PA not only changes membrane curvature but can help the docking of proteins with pleckstrin-homology domains including phosphatidylinositol-4-phosphate 5-kinase, which in turn can generate inositol polyphosphates that are recognized by a variety of signaling molecules [148]. It appears that endophilin catalyzes a reaction generating two products that promote the formation of synaptic vesicles by consuming LPA and by generating sn-2arachidonoy PA. Plasma membrane receptors for LPA activate Ca2+ transients and cause membrane depolarization [149]. Both of these effects are likely to contribute to LPA-induced neurotransmitter release from rat cerebral cortex [150] and PC12 cells [151]. 6.3. The peroxisome proliferator-activated receptor-g (PPARg)—an intracellular receptor for LPA The effects of LPA on cell growth and differentiation are hard to reconcile with the three LPAx receptors of the EDG gene family and for this reason it has been suggested that additional GPCR and other non-GPCR LPA receptors likely exist. McIntyre and colleagues [57] provided the first evidence that the transcription factor PPARg can function as an intracellular LPA receptor. These authors showed that oleoyl–LPA, palmitoyl–LPA and hexadecyl–GP competitively displace rosiglitazone and hexadecyl azelaic phosphatidylcholine, previously identified ligands of PPARg. These authors provided conclusive evidence that LPA activates transcription of genes containing peroxisomal proliferator-responsive element (PPRE) including the scavenger receptor CD36. Several lines of evidence support that exogenously applied LPA in the low micromolar range (45 mM), a concentration equivalent to the serum concentration of LPA, is capable of activating PPARg-mediated gene transcription. LPA was selective for PPARg since neither PPARa nor PPARbd expression enhanced the transcription of an acyl-CoA oxidase PPRE reporter construct. Enhanced access of LPA to the intracellular compartment using translocase-3 enhanced PPARg activation, whereas ectopic over-expression of LPAAT inhibited it. sn-1 dihydrodifluoro and sn-2 dehydroxyfluoro analogs (Fig. 5) of LPA that do not activate EDG-family LPAx GPCR below 105 M were potent activators of PPARg at a 5 mM concentration. Decisive evidence for the role of the LPA-mediated activation of the PPARg-retinoic acid receptor (RXR)–PPRE transcription factor axis was obtained by transfection of these signaling molecules into S. cerevisiae that lack nuclear hormone receptors and contain only the mating pheromone GPCR. In this artificial system both LPA and rosiglitazone activated transcription of a PPREreporter gene whereas no response was detected if any of the three components was omitted. Lastly, these authors showed that the supernatant of activated platelets, which is enriched in LPA but not thrombin, was capable of activating PPARg-mediated gene transcription of CD36 only if the CD36 promoter included an intact PPRE. Thus, PPARg responds to LPA but its specificity is

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distinct from that of LPAx as it accommodates fluorine substituted LPA and other lipids that do not activate LPAx. What might be the physiological significance of LPA’s action on PPARg? LPA has been shown to promote adipocyte differentiation [152–154] and PPARg regulates genes that control energy metabolism [155]. Interestingly, it has been reported that LPA induced cell proliferation in RH7777 cells that do not express LPAx receptors and perhaps PPARg might be a mediator of this effect. In a pathophysiological context, LPA has been implicated in atherogenesis as LPA accumulates in the lipid-rich atherosclerotic plaques and is generated in oxidized LDL [56]. A role for PPARg has been proposed in the development of atherogenesis via the regulation of CD36 expression which mediates the cellular accumulation of lipids [156]. It is easy to predict that this area of research will yield many important new discoveries in the near future.

7. Pathophysiological correlates of LPA action LPA GPCR receptor knockout mice for LPA1, LPA2 and LPA1LPA2 have been generated and the animals are without lethal phenotypic alterations [128,157]. Many cells co-express multiple subtypes of LPA receptors, therefore the lack of major physiological defects in the LPA1LPA2 double knockouts is a surprising finding. This results indicates it is likely that more LPA receptors remain to be described This section describes three disease-related conditions where corollary evidence suggests an important role for LPA, including: (1) cancer progression and metastasis, (2) pathobiology of cell survival and apoptosis, and (3) vascular pathophysiology. 7.1. LPA in cancer progression and metastasis The first reports implicating LPA in cancer metastasis by Imamura et al. [31,158] originated from the long-standing observation that serum is required for invasion of certain tumor cells across cellular monolayers and that one of the active components responsible for this effect was LPA. These early reports suggested a connection between invasiveness and the production of PLD-type LPA-producing enzymes by tumor cells [158]. However, it was not until last year that the elusive LPA-producing enzyme lyso-PLD was purified and identified as ATX [26,27], a previously identified phosphodiesterase secreted by invading cancer cells [159, 160]. Xu and colleagues made the observation [53] that ovarian cancer cells generated substantial amounts of a lipid factor, which showed biochemical properties similar to that of LPA. These authors have found elevated plasma LPA concentrations in patients with ovarian and other gynecological cancers [54] (mean, 8.6 mmol/l; range, 1.0–43.1 mmol/l) compared with the healthy control group (mean, 0.6 mmol/l; range, <0.1–6.3 mmol/l). However, other groups were unable to reproduce these findings casting doubt on the diagnostic usefulness of plasma LPA levels in the early detection of ovarian cancer [23,161]. Nevertheless, there is a clear consensus between the different groups using distinct methodologies that ovarian cancer ascites and other malignant tumor effusates contain elevated concentrations of LPA [23,162,163]. Baker et al. [164] have also showed that the acyl species composition of plasma- and ascites-derived LPA differ, indicating a lack of exchange between the two biological fluids. Another interesting connection between LPA and ovarian cancer has been suggested at the level of LPA2 receptor mRNA expression [165,166]. Ovarian cancer cells but not

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nonmalignant ovarian surface epithelial cells express this receptor subtype. LPA has also been shown to increase therapeutic resistance to chemo- and radiation therapy-induced apoptosis [167,168]. Taken together, LPA receptor antagonists may offer therapeutic potential in supplementing current cancer therapy by blocking the growth-supporting and anti-apoptotic effects of LPA generated by cancer cells. The mechanisms of enhanced tumor cell invasion by LPA include two important molecular machineries. On one hand, LPA receptor mediated activation of the Rho and Rac GTPase pathways are essential for the regulation of the actin cytoskeleton and cell motility [32,169–172]. On the other hand, LPA has been shown to regulate the activity of matrix metalloproteinases, which are also intricately involved in metastasis as well as in the LPAinduced transphosphorylation of the EGF receptor [173–175]. In summary, autotaxin-mediated production of LPA and the expression of LPA receptors, in particular the LPA2 subtype, appear to be associated with malignant transformation. The regulation of these genes during transformation is predictably going to be a topic of intense future investigation. 7.2. LPA in the pathobiology of cell survival and apoptosis Serum is used to provide trophic support to cultured clonal, as well as primary cells. The assumption by many has been that polypeptide growth factors present in serum are solely responsible for sustaining cell survival and promoting cell proliferation. In light of the fact that the serum concentration of LPA is in the high micromolar range [23,161,176], a concentration sufficient to elicit mitogenesis and prevent apoptosis in every LPA-responsive cell line examined, its role as an ubiquitous survival factor cannot be underestimated. The capacity of LPA to enhance cell survival through the inhibition ofapoptosis has been shown in a variety of cell lines. These include promoting Schwann cell survival [127], suppressing apoptosis induced during ischemia and reperfusion in cardiomyocytes [177–180] and by serum withdrawal in fibroblasts [181] and renal tubular cells [182], preventing tumor necrosis factor (TNF-a) induced apoptosis in hepatocytes [183] and ceramide induced apoptosis in T cells [177] and cardiac myocytes [184]. The anti-apoptotic effect of LPA offers at least two potential clinical applications. As outlined above, LPA receptor antagonists [119] could be applied to supplement current cancer chemotherapy. On the other hand, because LPA prevents radiation and chemotherapy-induced apoptosis [168] in the gut and promotes epithelial wound healing [185,186], LPA analogs resistant to intestinal lipases could be effective in reducing the unwanted side effects of cancer treatment [120]. LPA is capable of inhibiting the apoptotic pathway activated either though death receptors [183,187] or by DNA damage [168] by attenuating activation of the mitochondrial pathway [168]. The precise interactions of LPA signal transduction with the apoptotic signaling pathway remains unclear but the ERK1/2 and Akt kinases appear to play a crucial role [168,183,187,188]. The simple chemical structure of LPA together with its potent and ubiquitous effects enhancing cell survival make it an ideal drug candidate for the prevention of cell death in degenerative diseases. 7.3. The role of LPA in vascular pathophysiology Atherosclerosis is the leading cause of death in the United States and is responsible for more than half of all mortality in developed countries [156]. Neointima formation is the initial step in atherogenesis [189,190]. Several cellular mechanisms are involved in neointima formation

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including platelet activation and thrombus formation; endothelial cell activation and injury; infiltration by inflammatory cells; and activation, migration, phenotypic modulation, and proliferation of vascular smooth muscle cells [189,190]. Neointima proliferation ultimately leads to neointimal-plaque formation, lipid accumulation and calcification. Rupture of the inflamed atheromatous-plaque can trigger acute thrombembolic events, which are direct causes of heart attack and stroke. Cellular responses elicited by LPA include proliferation, migration, de-differentiation and antiapoptotic effects [191]—responses that are all involved in neointima formation. Production of LPA is tied to platelet activation and involves two enzymatic steps: First, activated platelets release phospholipase A1 and A2 enzymes, which hydrolyze plasma and membrane phospholipids and generate a variety of lysophospholipids [6,13]. Subsequently, the de novo generated lysophospholipids become substrates for lysophospholipase D, which produces LPA through cleavage of phosphate head groups. LPA generated by this metabolic pathway is enriched in the 18:2 and 20:4 polyunsaturated fatty acyl forms [6,13]. Platelet activation is the trigger for LPA formation in blood. The concentration of LPA in plasma is nanomolar, whereas in serum as a result of platelet activation it increases 100-fold to as high as 10 mM. In addition to the platelet-linked mechanism, minimally oxidized LDL (mox-LDL) also contains LPA-like activity [56,192]. Likewise, the lipid-rich atheromatous plaques, which accumulate oxidized lipids including mox-LDL [56,192], contain several acyl and alkyl species of LPA. LPA is a potent activator of platelet aggregation [21,43,193,194]. By studying the interaction of oxidatively modified LDL with platelets, it was shown that LPA produced during mild oxidation of LDL is one of the key mediators responsible for platelet activation induced by mox-LDL [56,192]. In the event of plaque rupture, circulating platelets come into contact with this highly thrombogenic material [195]. Indeed, oxidatively modified LDL and lipid extracts of human atherosclerotic plaques have been shown to stimulate platelets [56,192]. Platelet activation elicited by LPA, moxLDL and plaque lipids can be blocked by diacylglycerol pyrophosphate (DGPP), an LPA1&3 selective antagonist [192]. Oxidized LDL is also present in circulating blood and may be responsible for enhanced aggregation of platelets and the prothrombotic state often observed in patients with cardiovascular disease [196]. LPA and mox-LDL are capable of activating endothelial cells [56,197], macrophages [198] and modulating endothelial/leukocyte interactions [199]. Our group and others have shown that DGPP and another LPA receptor antagonist, N-palmitoyl-serine phosphoric acid (NPSerPA), inhibit the effects of LPA on platelets and endothelial cells [119,135]. LPA has been identified as a potent mitogen [200,201] and motility factor for VSMC [202]. In a recent study, Hayashi and colleagues reported that LPA induced vascular SMC dedifferentiation in vitro [203]. These authors noted that only LPA containing unsaturated fatty acids (18:1, 18:2 and 20:4) and not LPA containing saturated fatty acids (16:0, 18:0) are capable of inducing VSMC dedifferentiation [203]. This selectivity for unsaturated LPA species does not match the selectivity of LPA, and LPA4 receptors expressed in the arterial wall, as these receptors are activated by both saturated and unsaturated LPA analogs [44]. McIntyre et al. reported that PPARg was an intracellular receptor for LPA [57]. Peroxisome proliferator-activated receptors (PPARs) are transcription factors of the nuclear receptor superfamily. The PPAR family consist of three isoforms: PPARa, PPARd, and PPARg. Binding of PPAR ligands leads to activation and heterodimerization with retinoic acid X receptor (RXR),

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the receptor for 9-cis-retinoic acid. PPAR/RXR heterodimers bind to specific peroxisome proliferator response elements, or PPRE, located upstream of responsive genes [204]. Although these three isoforms belong to the same superfamily, their biological actions are distinct. PPARs can be activated by a vast number of compounds including synthetic drugs such as Rosiglitazone and Troglitazone, oxidized phospholipids, fatty acids, eicosanoids, and oxidized LDL. The actions of PPARs were originally thought to be limited to the control of lipid metabolism and homeostasis. Recent studies, however, have shown that PPAR activation regulates inflammatory responses, cellular proliferation, differentiation, and apoptosis [205–208]. Interestingly, unsaturated 18:1 LPA but not saturated 18:0 binds to PPAR-g and transactivates PPRE responsive elements [57]. PPARg is expressed in monocytes/macrophages, vascular smooth muscle cells, endothelial cells, and is highly expressed in atherosclerotic lesions and hypertensive vascular wall [206,209–212]. The role of PPARg in vascular diseases remains unclear. Based on these studies we propose a novel hypothesis that assigns a pivotal role to LPA in atherogenesis (Fig. 7). It has been proposed that local activation of platelets at hemodynamically compromised sites leads to release of platelet-borne mediators [156,211]. Activated platelets release enzymatic activities involved in LPA biosynthesis [13]. Locally produced LPA activates the expression of adhesion molecules including E-selectin and VCAM [199], which in turn leads to increased platelet adhesion. Because LPA also activates platelets, this positive feedback mechanism likely recruits more platelets, as well as stimulating the generation of LPA enriched in unsaturated fatty acid species. Although many lipids can activate PPARg, LPA stands out as its

Fig. 7. Mechanism potentially involved in the atherogenic effect of LPA. LPA generated from activated platelets induces expression of adhesion molecules on endothelial cells through LPAx receptors. Platelet aggregates adhering to the endothelium generate LPA in loco. A portion of this LPA is taken up by cells and combines with PPARg. PPARg among other genes regulates the expression of CD36, which is responsible for the uptake of moxLDL rich in LPA that contributes to further and sustained activation of PPARg.

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concentration can rise to 10 mM in serum [6,23,164] and concentrations near the vessel wall are likely to be even higher. Locally generated LPA, due to its lipophilic character, is able to cross the plasma membrane and combine with PPARg in the cytoplasm. The LPA–PPARg complex translocates to the nucleus and transactivates genes with PPRE elements in their promoters. Of these genes the scavenger receptor gene, CD36, is perhaps the most interesting, as it is involved in lipid import into the cell [213,214]. Increased expression of CD36 at the site of atheromatous plaques is well documented [214] and it leads to increased mox-LDL uptake into the cell, also providing a sustained source of LPA and other activating lipids of PPARg. LPA has been found to upregulate CD36 transcription [57]. This novel hypothesis is supported by several experimental observations [215] including the neointima-inducing effect of unsaturated LPA species and moxLDL in vivo that is fully blocked by the PPARg antagonist GW9662 [216].

8. Perspective The LPA field is still a relatively new area of research with many more questions than answers. Since the molecular cloning of some of the LPA synthesizing and degrading enzymes as well as the LPA receptors, many details of the biochemical pathways and signaling events have been characterized. However, this is still the tip of the iceberg. It is clear that several key synthetic enzymes (e.g. serum PLA1), LPA ectophosphatases, and LPA lysophospholipases remain to be identified. Similarly, LPA receptors, including the cyclic-PA receptor are yet to be identified. Identification of PPARg as an intracellular receptor will foster new investigation into the mechanism of LPA transport and delivery to the intracellular receptor. Identification of plasma factors, both proteins and lipids that attenuate the biological effects of LPA, can lead to novel inhibitors of LPA action. Although in the last 2 years the first LPA receptor antagonists have been identified, these compounds will have to be reassessed for their effect on the new LPA receptors including PPARg. Since the original submission of this manuscript the report describing the identification of the LPA4 receptor has appeared [219], and dioleoyl-PA and S1P have been identified as ligands of GPR63 [220], a mammalian homolog of the Xenopus LPA receptor PSP24, reinforcing the high pace of discovery in the field. Genomics-based approaches to the LPA receptor and metabolic enzyme genes are likely to identify important correlations with certain diseases and types of cancer. In conclusion, a better understanding of the biology of LPA has the potential to provide us with novel strategies for the treatment of the two major killers in Western societies: cancer and atherosclerosis. References [1] [2] [3] [4] [5] [6] [7]

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