Current progress in non-Edg family LPA receptor research

Biochimica et Biophysica Acta 1831 (2013) 33–41

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Review

Current progress in non-Edg family LPA receptor research☆ Keisuke Yanagida a,⁎, Yoshitaka Kurikawa a, Takao Shimizu a, Satoshi Ishii b a b

Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan Department of Immunology, Graduate School of Medicine, Akita University, Akita City, Akita 010-8543, Japan

a r t i c l e

i n f o

Article history: Received 29 May 2012 Received in revised form 1 August 2012 Accepted 2 August 2012 Available online 11 August 2012 Keywords: LPA4 LPA5 LPA6 GPR23/p2y9 GPR92/GPR93 p2y5

a b s t r a c t Lysophosphatidic acid (LPA) is the simplest phospholipid yet possesses myriad biological functions. Until 2003, the functions of LPA were thought to be elicited exclusively by three subtypes of the endothelial differentiation gene (Edg) family of G protein-coupled receptors — LPA1, LPA2, and LPA3. However, several biological functions of LPA could not be assigned to any of these receptors indicating the existence of one or more additional LPA receptor(s). More recently, the discovery of a second cluster of LPA receptors which includes LPA4, LPA5, and LPA6 has paved the way for new avenues of LPA research. Analyses of these non-Edg family LPA receptors have begun to fill in gaps to understand biological functions of LPA such as platelet aggregation and vascular development that could not be ascribed to classical Edg family LPA receptors and are also unveiling new biological functions. Here we review recent progress in the non-Edg family LPA receptor research, with special emphasis on the pharmacology, signaling, and physiological roles of this family of receptors. This article is part of a Special Issue entitled Advances in Lysophospholipid Research. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Lysophosphatidic acid (LPA) is a pluripotent lipid mediator with various biological functions including cell growth, differentiation, survival, motility, and cytoskeletal morphology [1]. LPA is known to be a major growth factor in serum [2], and significant amounts of LPA can be detected in other body fluids as well such as saliva [3], seminal plasma [4], and bronchoalveolar lavage fluid [5]. Recent genetargeting studies in mice have elucidated at least two major pathways of extracellular LPA production which occur in vivo [6]. Autotaxin (ATX) converts lysophosphatidylcholine and other lysophospholipids to LPA by its lysophospholipase D activity, and this is a primary mechanism of LPA production in plasma/serum as best demonstrated by the observation that ATX heterozygous mice had a plasma LPA level half that of wild-type mice [7,8]. Another mechanism of LPA production occurs in hair follicles, where LPA with acyl chains at the sn-2 position is produced by membrane-bound phosphatidic acidselective phospholipase A1α (mPA-PLA1α) [9]. LPA exerts its many biological functions through binding and activation of multiple G protein-coupled receptors (GPCRs) localized in the plasma membrane of target cells [10]. Originally, the functions

☆ This article is part of a Special Issue entitled Advances in Lysophospholipid Research. ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: + 81 3 5802 2925; fax: + 81 3 3813 8732. E-mail address: [email protected] (K. Yanagida). 1388-1981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2012.08.003

of LPA were thought to be elicited exclusively by three subtypes of the endothelial differentiation gene (Edg) family of GPCRs — LPA1, LPA2 and LPA3 [11]. These receptors do mediate some biological effects of LPA including chemotactic [12,13] and mitotic activities [12,14,15], but several biological activities of LPA could not be assigned to the Edg family LPA receptors [16–18] reflecting the existence of additional, non-Edg LPA receptor(s). In 2003, we identified the first phylogenetically distant, non-Edg LPA receptor, LPA4 [19], and subsequently two additional LPA receptors (LPA5 and LPA6) were discovered [20–22]. These three receptors share 35–55% amino acid identity with each other and constitute the non-Edg family LPA receptors. This novel cluster of LPA receptors can account for several of the biological functions of LPA that could not previously be assigned to any of the Edg family LPA receptors, and has also revealed new and unexpected functions of LPA. Here we extend our previous reviews [23,24] and discuss recent progress in the non-Edg family LPA receptor research, with special emphasis on the pharmacology, signaling, and physiological roles of this family of receptors.

2. Nomenclature and classification of LPA receptors Chun and his colleagues discovered the first LPA receptor (LPA1, formerly known as vzg-1 or Edg2) in 1996 [25]. This was followed by the identification of two additional structurally related LPA receptors, LPA2 [26] and LPA3 [27] (formerly known as Edg4 and Edg7, respectively). These LPA receptors share 50–57% amino acid identity with each other and along with five sphingosine-1-phosphate receptors, S1P1–5, comprise the Edg family of GPCRs [11].

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During the course of searching for the natural ligands of several orphan GPCRs, our research group found that LPA is a ligand for GPR23/p2y9 [19]. This fourth LPA receptor, now referred to as LPA4, does not share high homology with Edg family LPA receptors but is more closely related to the receptor for platelet-activating factor [19]. Identification of LPA4 expanded the repertoire of known LPA receptors beyond the Edg family GPCRs. Subsequently, two orphan GPCRs structurally related to LPA4 were also found to respond to LPA, GPR92/93 [28,29] and p2y5 [20–22], now known as LPA5 and LPA6, respectively. Together these three LPA receptors comprise the nonEdg family of LPA receptors. Several other orphan GPCRs have also been proposed to function as LPA receptors including GPR87 [30], p2y10 [31], and GPR35 [32], but await confirmation by further research. Thus, a final consensus has not been reached yet on whether each of these orphan GPCRs actually mediate biological effects of LPA [10]. 3. Pharmacological characterization of LPA receptor ligands on non-Edg family LPA receptors LPA is not a single molecular entity; there are many LPA species that differ in the chemistry of their hydrocarbon chains and linkages to the glycerol backbone [33]. Importantly, each LPA receptor has distinct ligand selectivities for the various LPA species. For example, LPA3 is more potently activated by LPA with an acyl chain at the sn-2 position (2-acyl-LPA) rather than the sn-1 position (1-acyl-LPA) [34]. In this section, we discuss ligand selectivities of non-Edg family LPA receptors and also summarize the agonist/antagonist effects of commercially available compounds. 3.1. A “platelet-type” LPA receptor: LPA5 The first reported biological activity of LPA was contraction of smooth muscle [35]. Ensuing studies in the 1970s revealed that polyunsaturated acyl-LPA has greater potency to induce smooth muscle contraction than saturated acyl-LPA or alkyl ether-linked LPA (alkyl-LPA) species [33,36]. However, in 1982, Simon et al. found that human platelets aggregate more potently in response to alkyl-LPA than to acyl-LPA [16]. This difference in LPA-species preference indicated the existence of at least two distinct LPA receptor subtypes, a “smooth muscle-type” LPA receptor and a “platelet-type” LPA receptor [33]. However, all Edg family LPA receptors are more potently stimulated by acyl- rather than alkyl-LPA species, consistent with the “smooth muscle-type” rather than “platelet-type” LPA receptor activities [34]. Thus, “platelet-type” LPA receptor(s) remained to be identified even after identification of the Edg family LPA receptors LPA1–3 [18]. We examined ligand selectivities of non-Edg family LPA receptors, and found that the ligand selectivity of LPA5 coincides with that of the “platelet-type” LPA receptor, with a preference for alkyl- rather than acyl-LPA (K.Y., S.I. and T.S., FASEB Summer Research Conference 2007). The ligand selectivity was determined by Ca 2 + mobilization assay, and 1-hexadecyl-LPA (1-alkyl-LPA 16:0) was more potent than 1-oleoyl-LPA (1-acyl-LPA 18:1) in RH7777 cells stably expressing LPA5 but not in cells stably expressing other LPA receptors. It is notable that LPA5 is one of the most highly expressed GPCRs in human platelets as revealed by a gene expression profiling study [37]. Recent studies with LPA5-selective agonists or siRNA against LPA5 are also consistent with the role of LPA5 as a “platelet-type” LPA receptor [38–40]. Khandoga et al. used many LPA or phosphatidic acid structure-based compounds, and found that the structure–activity relationships are similar between LPA5 activation potency and human platelet shape change potency [38]. Utilizing an in silico screening strategy, Williams et al. identified non-lipid LPA5 antagonists that also suppress LPA-induced human platelet activation [39]. Furthermore, by using RNAi, Khandoga et al. successfully showed that LPA-induced

shape changes of the megakaryocytic cell lines Dami and Meg-01 cells were mediated by LPA5 [40]. Taken together, these studies indicate that LPA5 is at least one of the elusive “platelet-type” LPA receptors which had been postulated for the past several decades. 3.2. LPA6: a preference for 2-acyl-LPA species Many missense mutations have been reported in LPAR6 or mPAPLA1α gene of patients with autosomal recessive wooly hair/ hypotrichosis [41]. Since mPA-PLA1α is a membrane bound PLA1 that produces 2-acyl-LPA species [42], there is a possibility that 2acyl-LPA species produced by this enzyme act as endogenous ligands for LPA6 in hair follicles [6]. We examined the ligand selectivity of LPA6 in heterologous expression systems and found that the 1-acylLPA species with unsaturated fatty acids were more potent for LPA6 than those with saturated fatty acids, with a rank order of activity 1-linoleoyl > 1-oleoyl > 1-arachidonoyl = 1-palmitoyl ≫ 1-stearoyl = 1-myristoyl-LPA. Furthermore, among LPA species with unsaturated fatty acid tails, 2-acyl-LPA was a more potent agonist than 1-acyl-LPA [21]. Inoue et al. later obtained a similar ligand structure–activity relationship for LPA species using a different assay system and mouse rather than human LPA6 [9]. The biological relevance of the LPA species‐ligand selectivity of LPA6 will be further discussed in Section 5.6. 3.3. VPC31143(R) and VPC31144(S): distinctive ligand selectivities of LPA4 and LPA5 The chemical compound N-acyl ethanolamide phosphate (NAEPA) evokes human platelet aggregation as potently as LPA [43]. Many 2substituted derivatives of NAEPA have been reported, some of which are commercially available. VPC31143(R) and VPC31144(S) are enantiomers of a 2-methylene hydroxy-containing analog of NAEPA [44] (Fig. 1). It was reported that VPC31143(R) displays much higher activity than VPC31144(S) for all Edg family LPA receptors [44]. In Ca2 + mobilization assays utilizing RH7777 cells stably expressing each of three Edg family LPA receptors, we consistently observed that all the Edg family LPA receptors are more potently stimulated by VPC31143(R) than by VPC31144(S) (Fig. 2A–C). We examined whether non-Edg family LPA receptors have similar stereoselectivities for VPC31143(R). Ligand selectivity of LPA6 was similar to that of the Edg family LPA receptors (Fig. 2F) as measured by Gs/13-mediated cAMP production (discussed further in Section 4.3), while LPA4 and LPA5 exhibited a clear preference for VPC31144(S) over VPC31143(R) in Ca2 + mobilization assays (Fig. 2D and E). In human erythroleukemia (HEL) cells, which express mRNA of LPA5 but not other LPA receptors (data not shown), alkyl-LPA showed higher activity than acyl-LPA in stimulating intracellular Ca2 + mobilization (Fig. 2G). Of note, VPC31144(S) showed much higher activity than VPC31143(R) in the Ca 2 + mobilization of these cells (Fig. 2H). This stereoselectivity suggests that VPC31143(R) and VPC31144(S) may serve as useful tools to distinguish activities elicited by LPA4 and LPA5 from those elicited by other LPA receptors (Table 1). 3.4. Other ligands for LPA receptors In addition to VPC31143(R) and VPC31144(S), there are other commercially available compounds with the NAEPA-based structure. VPC12249(S) and VPC32183(S) are known antagonists of LPA1/LPA3, while VPC32179(R) has LPA3 antagonist activity and LPA1 partial agonist activity [44–47]. Although there is no report on their efficacies on non-Edg family LPA receptors, we found that VPC32183(S) showed agonist or partial agonist activity against all of the non-Edg family LPA receptors, LPA4–6. Notably, VPC32183(S) had a similar affinity to LPA4 as LPA1 as measured by displacement of [ 3H]-1-oleoyl-LPA (K.Y., S.I. and T.S., unpublished observation). Furthermore, pretreatment with VPC32183(S) abolished LPA-induced

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Fig. 1. Chemical structures of LPA and commercially available LPA analogs.

Ca2 + responses in RH7777 cells stably expressing LPA4 (K.Y., S.I. and T.S., unpublished observation). Thus, when utilizing of these compounds as LPA1 or LPA3 antagonists, their activities on non-Edg family LPA receptors should be considered. 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT) is a synthetic LPA analog which contains a thiophosphate rather than phosphate headgroup [48,49]. OMPT and an alkyl-chain-substituted OMPT analog (alkyl-OMPT) [50] have been used in many studies because of their selectivity for LPA3 among the Edg family LPA receptors [13,51–57]. In the course of our studies, we found that alkyl-OMPT is also a potent agonist for LPA6 [21]. Inoue et al. also observed potent agonistic activity of OMPT at mouse LPA6 [9]. AlkylOMPT is protected from degradation by both phosphatases and phospholipases, and this compound may be especially useful in future studies to elucidate the function of the LPA6 receptor in vivo. Diacylglycerol pyrophosphate and dioctyl-phosphatidic acid 8:0 are antagonists of LPA1 and LPA3, with higher potency for LPA3 [58]. Although their efficacies on non-Edg family LPA receptors have not yet been evaluated, some analogs of dioctyl-phosphatidic acid 8:0 [59] were shown to exhibit agonist activities against LPA5. The data obtained using these analogs supports the idea that LPA5 is a “platelet-type” LPA receptor [38] as discussed in Section 3.1. Farnesyl monophosphate (FMP) and farnesyl pyrophosphate (FPP) were reported as endogenous antagonists for LPA2 and LPA3, with preferences for LPA3 [60]. Williams et al. showed that FMP and FPP are also antagonists for LPA4 [39]. However, these compounds show agonist activity against LPA5 receptor. Oh et al. reported that FPP activates LPA5 with higher potency than LPA [61]. Williams et al. also identified FPP as an agonist of LPA5 but found that 1-oleoyl-LPA was more potent than FPP in activating this receptor [39]. Our independent experiments with RH7777 cells stably expressing LPA5 also demonstrated that 1-oleoyl-LPA is a more potent agonist of LPA5 than FPP (K.Y., S.I. and T.S., unpublished observation). Lee et al. showed that LPA6 is also activated by FPP as well as geranylgeranyl pyrophosphate, although the signaling cascades induced by these agonists differed from those induced by LPA [22]. Ki16425 is the most commonly used antagonist for LPA receptors [12]. This compound exerts antagonistic effects on all three Edg family LPA receptors although it is a somewhat weak antagonist of LPA2. Ki16425 and its related analog Ki16198 [62] show no antagonist activities at any of the three subtypes of non-Edg family LPA receptors

LPA4–6 [21,28,62,63]. These compounds have non-lipid-like structures, suggesting that their selectivities in inhibiting Edg but not nonEdg family LPA receptors may reflect different modes of ligand recognition between these two families of receptors. AM966 is an orally active LPA1 receptor antagonist with a non-lipid-like structure similar to Ki16425 [64]. This compound is 100-fold more potent for the LPA1 receptor over other LPA receptors including the non-Edg family LPA receptor LPA4 and LPA5 [64]. Its activity on LPA6 has not been determined yet and awaits characterization. The combined use of the LPA1- or Edg family LPA receptor-selective antagonists with VPC31143(R)/VPC31144(S) which display unique selectivities for LPA4 and LPA5 may be useful for determination of which LPA receptor subtypes mediate various LPA-induced biological responses. 4. Non-Edg family LPA receptor signaling G protein coupling of Edg family LPA receptors has been well characterized; LPA1 and LPA2 couple to Gi, Gq and G12/13 proteins, and LPA3 couples to Gi and Gq proteins [65]. On the other hand, there is some controversy as to the coupling specificities of non-Edg family LPA receptors. In this section we will summarize areas of consensus and controversy in the G protein coupling selectivities of non-Edg family LPA receptors (Table 2). 4.1. G protein coupling of LPA4 Multiple independent studies demonstrated that LPA4 activates Rho-signaling pathways through G12/13 proteins in cell lines exogenously expressing LPA4 [63,66]. LPA-induced Rho activation was also observed in mouse embryonic fibroblasts (MEFs), which endogenously express LPA4 [67], but this activation was severely attenuated in MEFs isolated from LPA4 knockout embryos. As discussed later in more detail, some LPA4 knockout embryos exhibited vascular defects [68] similar to those observed in G13 knockout embryos [69–71], suggesting an important role for LPA4/G13 signaling in vasculogenesis in vivo [68]. LPA increased intracellular cAMP levels in Chinese hamster ovary (CHO) cells [19], B103 cells [63] and RH7777 cells (K.Y., S.I. and T.S., unpublished observations) exogenously expressing LPA4. Involvement of Gs protein in the cAMP production was demonstrated in B103 cells exogenously expressing LPA4 (B103-LPA4 cells) by blocking this response

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Fig. 2. Distinctive ligand selectivity of LPA4 and LPA5. (A–E) RH7777 cells stably expressing LPA1 (A), LPA2 (B), LPA3 (C), LPA4 (D), or LPA5 (E) were loaded with Fluo-3 AM, and intracellular Ca2 + mobilization in response to the indicated ligands was measured (n = 1). (F) Ligand selectivity of LPA6 was examined using B103 cells stably expressing LPA6 and Gs/13 in the presence of PTX (n = 5, mean ± S.D.). (G) and (H) HEL cells were loaded with Fura-2 AM, and intracellular Ca2 + mobilization in response to the indicated ligands was measured. All data are representative of three independent experiments with similar results.

using a Gs minigene [63]. Coupling of endogenous LPA4 to Gs protein is also proposed to occur in some cells. In human fibrosarcoma HT1080 cells, LPA induced LPA4-dependent invadopodia production, which can be induced by increased intracellular cAMP [72]. Furthermore, LPA increased intracellular cAMP levels in human mesenchymal stem cells undergoing osteogenic differentiation in an Edg family LPA receptorindependent manner [73]. It is notable that these cells highly express LPA4 but not LPA5, and knockdown of LPA4 enhanced osteogenic differentiation of these cells. However, our data failed to support the LPA4 coupling to Gs protein; LPA did not increase intracellular cAMP in B103-LPA4 cells under our experimental conditions [66]. In addition, when we applied LPA to MEFs, little or no increase in intracellular cAMP was observed even after inhibition of Gi signaling with pertussis toxin (PTX) to unmask any potential LPA-stimulated cAMP production (K.Y., S.I. and T.S., unpublished observations). Thus, the coupling efficiency of LPA4 to Gs protein might depend on the cell type, the culture conditions, or the receptor expression levels. Molecularly, differences in adenylyl cyclase subtypes or Gs/Gi switching as observed in other GPCRs [74,75] might affect the coupling efficiencies.

Previous studies reported that LPA4 couples to Gi and Gq proteins in cells exogenously transfected with LPA4 [19,63]. However, the coupling of Gi and Gq proteins with endogenously expressed LPA4 has not been reported. Indeed, we observed little or no differences in LPAinduced adenylyl cyclase inhibition and Ca 2 + mobilization between MEFs derived from wild-type and LPA4 knockout embryos (K.Y., S.I. and T.S., unpublished observations). Thus, the coupling of endogenous LPA4 to Gi and Gq proteins in physiological settings is still unclear and warrants further investigation. 4.2. G protein coupling of LPA5 Exogenous or endogenous LPA5 has been shown to mediate LPAinduced intracellular cAMP production in several different cell lines [28,29,61]. We have consistently observed that exogenous expression of LPA5 in RH7777 cells imparts responsiveness to LPA as measured by increases in intracellular cAMP levels (K.Y., S.I. and T.S., unpublished observations). Notably, two independent studies suggest that LPA5mediated cAMP production may occur independently of Gs signaling.

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Table 1 EC50 values (nM) of VPC31143(R) and VPC31144(S) for each LPA receptor.

1-Oleoyl-LPA VPC31143(R) VPC31144(S) (S) selectivityc

LPA1a

LPA2a

LPA3a

LPA4a

LPA5a

LPA6b

64 ± 6.3 59 ± 12 461 ± 69 0.128

9.1 ± 1.7 16 ± 5.2 2592 ± 313 0.00617

321 ± 7.8 130 ± 4.4 7123 ± 2743 0.0182

26 ± 3.1 341 ± 66.9 18 ± 0.7 18.9

11 ± 1.6 126 ± 24 16 ± 2.5 7.88

1495 ± 148 1484 ± 115 4835 ± 577 0.307

Data represents means ± SEM of three independent experiments. a Agonist activities on LPA1–5 were determined by measuring intracellular Ca2 + mobilization in response to increasing concentrations of the indicated ligands as shown in Fig. 2. b Agonist activities on LPA6 were determined by measuring cAMP increases as shown in Fig. 2. c Selectivity for VPC31144(S) over VPC31143(R) was calculated by dividing mean EC50 values for VPC31143(R) by those for VPC31144(S).

Lee et al. reported that a Gs minigene did not inhibit LPA-induced cAMP production in B103 cells exogenously expressing LPA5 [28]. In another study, Jongsma et al. analyzed LPA5-dependent intracellular cAMP increases in detail using a fluorescence resonance energy transfer-based cAMP sensor in B16 melanoma cells, which endogenously express LPA5 [76]. Increase of intracellular cAMP by LPA5 in these cells was transient and clearly differed from the response to α-melanocyte-stimulating hormone, which causes a more sustained increase in intracellular cAMP through a Gs-dependent mechanism. The coupling of exogenously expressed LPA5 to Gq protein and/or Ca 2 + signaling has been observed in several cell lines [28,29,61,77]. Although coupling of endogenous LPA5 to Gq protein requires further confirmation, LPA5 knockdown experiments demonstrated the involvement of LPA5 in LPA-induced Ca 2 + mobilization in primary rat dorsal root ganglion neurons [61] and human LAD2 mast cells [78]. As discussed earlier in Section 3.3, LPA-induced Ca 2 + mobilization in HEL cells might also be mediated by LPA5. LPA5-dependent G12/13/Rho/Rho-associated kinase (ROCK) activation has been observed in B103 cells and RH7777 cells exogenously expressing LPA5 [28]. In addition, LPA-induced and ROCK-dependent morphological changes in two megakaryocytic cell lines, Dami and Meg-01, were blocked by knockdown of endogenous LPA5 [40]. Endogenous LPA5 in human platelets may also activate the Rho/ROCK pathway to induce shape change; however, the involvement of G12/13 in this response requires further investigation. 4.3. G protein coupling of LPA6 Studies by three independent groups including ours have established a consensus that LPA6 couples to G12/13/Rho/ROCK activation pathways [9,21,22]. We found that LPA induced ROCK-dependent cytoskeletal changes in both B103 and RH7777 cells exogenously expressing LPA6 (B103-LPA6 cells or RH7777-LPA6 cells, respectively) [21]. In B103-LPA6 cells, G13 coupling was demonstrated using a Gs/13 chimeric protein which allows G13-coupled receptors to stimulate Gsmediated adenylyl cyclase activation. In RH7777 cells, G13 coupling was demonstrated by immunoprecipitation of [ 35S]GTPγS-labeled Gα13 protein in membrane fractions from LPA6-transfected but not vector-

transfected cells following LPA treatment. Coupling of endogenous LPA6 to the Rho activation pathway was demonstrated by LPA6 knockdown: LPA-induced cytoskeletal shape change of human umbilical vein endothelial cells was suppressed following transfection of LPA6 siRNA [21]. Independent experiments by Lee et al. also demonstrated that LPA increases serum response element-linked luciferase reporter activity via the G12/13/Rho/ROCK pathway in hBRIE 380 cells exogenously expressing LPA6 [22]. In their experiments, the LPA-induced activation could be blocked by either a G12/13 inhibitor (the regulator of G protein signaling domain of p115RhoGEF), a Rho inhibitor (C3 exoenzyme), or a ROCK inhibitor (Y-27632). Additionally, a recent study by Inoue et al. showed that LPA induced TGFα release in HEK cells exogenously expressing LPA6 in a ROCK-dependent manner, further demonstrating that LPA6 couples to G12/13/Rho/ROCK activation pathways [9]. Lee et al. reported that LPA inhibited forskolin-triggered elevation of intracellular cAMP levels in CHO cells transfected with LPA6 [22]; however, we could not elicit similar responses in B103-LPA6 or RH7777-LPA6 cells [21]. It is notable that the Gs/13 chimeric proteindependent cAMP increase in B103-LPA6 cells was observed only when the cells were pretreated with PTX, suggesting that LPA6 may activate some Gi-mediated responses that mask cellular responses mediated by other Gα subclasses [21]. It has also been reported that LPA increased intracellular cAMP levels in CHO cells transfected with LPA6 [20]; however, the LPA6-mediated cAMP production has yet to be confirmed by other independent studies. Further investigation may be required to establish a consensus on the coupling specificities of LPA6 to the Gi and Gs subclasses of proteins. 5. Roles of non-Edg family LPA receptors in physiology and disease 5.1. Vascular development and LPA4 In 2006, two groups reported that ATX knockout embryos died with severe vascular defects [7,8], and these phenotypes have subsequently been confirmed by additional independent research groups [79,80]. Although ATX also has non-enzymatic activities, catalytically inactive ATX (T210A mutant) knock-in mice showed similar phenotypes to the

Table 2 G protein coupling of non-Edg family LPA receptors. Receptor subtype

Pathways activated in heterologous systems

Cell types in which endogenous coupling has been demonstrated

LPA4

G12/13 (+) Gs (+) Gq (+) Gi (+) cAMP↑ (+)a Gq (+) G12/13 (+) G12/13 (+) Gi (+/−) Gs (+/−)

Mouse embryonic fibroblasts [67] HT1080 cells [72], human mesenchymal stem cells [73] – – B16 cells [76] Rat dorsal ganglion neurons [61], LAD2 cells [78] Dami cells [40], Meg-01 cells [40] Human umbilical vein endothelial cells [21], mouse keratinocytes [9], HaCaT cells [9], HEK293 cells [9] – –

LPA5

LPA6

(+), Supported by multiple independent reports (+/−), Controversial a Noncanonical, Gs-independent pathway is suggested.

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ATX knockout mice, indicating that the vascular defects result from loss of ATX-dependent LPA production [81]. G13 knockout mice also have impaired vascular development [69–71], suggesting the existence of a G13-coupled LPA receptor(s) that is essential for vascular development. Among Edg family LPA receptors, LPA1 and LPA2 have been known to activate G12/13 protein [65]; however, LPA1, LPA2, and LPA3 single and multiple receptor knockout mice do not display vascular defects at all [14,15,82,83]. We found that some LPA4 knockout embryos (approximately 30%) did not survive gestation and displayed hemorrhages in many organs [68]. In defective embryos, the blood vessels were dilated, and recruitment of smooth muscle cells and pericytes was impaired. Thus, LPA4 is probably essential for normal blood vessel development and may be a missing link connecting ATX to G13-dependent vasculature development. However, there are still some unsolved questions. First, two other research groups have reported that LPA4 knockout mice are born with the expected Mendelian ratio and no obvious physiological abnormalities [67,73]. The apparent discrepancy between these and our studies might be caused by strain differences (mixed versus pure C57BL/6 genetic backgrounds) or different environmental conditions. Second, the phenotype observed in LPA4 knockout mice was only partially penetrant, showing a sharp contrast with ATX knockout mice. Thus, it is possible that another G13-coupled LPA receptor(s) participates in vascular development in a cooperative or redundant manner with LPA4. Recently, Yukiura et al. reported that ATX morphant zebrafish embryos showed severe vascular dysfunction, indicating that the role of ATX in vascular development is widely conserved among vertebrate species [84]. More interestingly, although single LPA1 or LPA4 morpholino antisense oligonucleotides (MOs) did not affect vascular development in zebrafish, treatment with LPA1/LPA4 double MOs or treatment with LPA4 MOs in conjunction with the LPA1 inhibitor Ki16425 phenocopied the ATX morphants. This elegant study further supports the key role of LPA4 in vascular development and also the redundant roles of LPA1 and LPA4 in this process. Some of the LPA4 knockout embryos generated in our laboratory suffered from edema as well as bleeding [68]. In these embryos, lymphatic vessels were dilated, indicating a role for LPA4 in proper formation of lymphatic vessels. So far, defects of lymphatic vessels in ATX knockout embryos have not been reported, most likely because ATX knockout embryos die at a stage that precedes formation of lymphatics. Notably, it was reported that LPA1-knockdown by MOs in zebrafish resulted in defective lymphatic vessel formation [85]. Thus, similar to their roles in blood vessel formation, LPA1 and LPA4 might have redundant or cooperative roles in lymphatic vessel formation. 5.2. Bone and LPA4 LPA4 mRNA is highly expressed in bone. Liu et al. reported that LPA4 knockout mice have increased bone density compared to wild-type mice [73]. LPA4 activation seems to inhibit osteoblast differentiation, probably through Gs protein activation and intracellular cAMP increase. However, LPA1 knockout mice showed an opposite phenotype, with abnormal facial bone development [14] and reduced bone formation [86]. Thus, in contrast to the redundant or cooperative functions of LPA1 and LPA4 in vascular development, LPA1 and LPA4 may have opposing functions in bone formation. The ability of LPA4 signaling to either cooperate or oppose LPA1 signaling may reflect the coupling of LPA4 to different classes of G proteins such as G12/13 and Gs under different physiological settings. Since LPA1 couples to Gi protein and LPA4 may couple to Gs protein in mesenchymal stem cells or osteoblasts, differential regulation of intracellular cAMP levels might explain the opposite bone phenotypes observed in LPA1 versus LPA4 knockout mice [87]. In vitro studies also revealed reciprocal roles of LPA1 and LPA4 in B103 cells exogenously expressing LPA1 or LPA4 [66] and MEFs [67]. These studies suggested that

LPA1 and LPA4 mediate intracellular signaling through Gi/Rac and G12/13/ Rho pathways, respectively, to mediate opposing roles in cytoskeletal morphology and chemotaxis. Thus, the differential regulation of Rho and Rac pathways might explain the opposite bone phenotypes of LPA1 and LPA4 knockout mice. 5.3. Mast cells and LPA5 Several studies have suggested that LPA is involved in mast cell functions [52,88,89]. LPA induces histamine release from mast cells [90] and provokes itch–scratch responses in mice [89–91]. Lundequist and Boyce reported that human LAD2 mast cells and human primary mast cells abundantly express mRNA and protein of LPA5 [78]. LPA induced rapid Ca 2 + mobilization and release of macrophage inflammation protein-1β in LAD2 cells, and these responses were abolished following siRNA-mediated LPA5 knockdown. These results strongly suggest that LPA5 is a dominant LPA receptor subtype mediating biological responses in human mast cells. ATX is reported to be abundant in mast cells of the human gastrointestinal tract [92], and LPA5 mRNA expression is also reported to be high in the gastrointestinal tract [28,29,61,77]. Since mast cells are considered to play important roles in gastrointestinal diseases such as allergic enteritis and inflammatory bowel disease, LPA5 signaling might contribute to these diseases via ATX-dependent LPA production. Future studies with LPA5 knockout mice will be useful to address the role of LPA5 in gastrointestinal diseases. 5.4. Cancer and LPA5 ATX was first isolated from the conditioned media of cancer cell cultures as a highly potent factor which stimulates cancer cell motility [93]. LPA, a primary product of ATX, functions as a potent chemoattractant for many cancer cell lines [94,95]. The LPA1 (or LPA2)/Gi protein pathway is thought to mediate the chemotactic activity of LPA and to play a major role in cancer invasion and metastasis [13,96]. In contrast to the general notion that LPA is a chemoattractant for malignant cancer cells, Jongsma et al. recently reported that LPA is a chemorepellent for B16 melanoma cells [76]. They found that LPA and ATX strongly inhibited migration of B16 cells, which endogenously express LPA5 and LPA6. Knockdown of LPA5 but not LPA6 blocked the chemorepellent activity of LPA. Furthermore, alkyl-LPA was more potent as a chemorepellent than acyl-LPA, which is consistent with a LPA5-mediated response based on the ligand structure–activity relationship of this receptor subtype. Because LPA is known to enhance cell growth and cell motility, ATX and LPA receptors are regarded as attractive targets to develop inhibitors/antagonists that may have efficacy as anti-cancer drugs [97]. However, the study by Jongsma et al. raised the possibility that LPA5 “agonists” might have therapeutic potential, whereas ATX inhibitors might promote rather than inhibit tumor cell migration and invasion in some types of cancer such as melanoma where LPA5 plays a dominant role. Carba analogs of cyclic phosphatidic acid are proposed to function as ATX and LPA receptor inhibitors and have been shown to block growth and metastasis of B16 melanoma cell tumors in mouse models [98,99]. The possibility that these compounds have agonistic activity on the LPA5 receptor should be examined because this would also be consistent with their anti-cancer effects. The activity on the LPA5 receptor and possible therapeutic impacts should be considered in the further development of anti-cancer drugs which target ATX and LPA receptors [100,101]. 5.5. Neuropathic pain and LPA5 LPA functions in the initiation of neuropathic pain [102], and the roles of ATX [103], LPA1 [104], and LPA3 [105] in this process have been investigated in studies utilizing knockout mice. Following nerve

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injury, LPA produced by ATX activates the LPA1/Rho axis in Schwann cells, thereby contributing to dorsal root demyelination and mechanical allodynia. LPA3 in microglia also plays a role in neuropathic pain by amplifying ATX-dependent LPA biosynthesis. Very recently, Lin et al. revealed that LPA5 is also involved in the development of neuropathic pain [106]. They found that LPA5 knockout mice were protected from neuropathic pain induced by the partial sciatic nerve ligation. Importantly, the mechanism underlying the protection in LPA5 knockout mice is clearly different from that in LPA1 knockout mice. Contrary to the role of LPA1 in neuropathic pain, LPA5 signaling is unlikely to contribute to demyelination, because severe demyelination occurred in LPA5 knockout mice to the same extent as in wild-type mice. In LPA5 but not LPA1 knockout mice, spinal cord dorsal horn neurons had diminished levels of phosphorylated CREB, suggesting that LPA5-mediated cAMP/CREB signaling enhances synaptic activity in the dorsal horn, leading to the central sensitization.

5.6. Hair development and LPA6 The first description of the effect of LPA on hair growth was published in 2003 when Takahashi et al. found that phosphatidic acid and LPA promoted the growth of hair epithelial cells and epidermal keratinocytes [107]. They showed that phosphatidic acid enhanced hair growth in an in vivo murine model and that LPA also induced hair growth but with much lower potency. In 2006, Kazantseva et al. reported that the gene which encodes mPA-PLA1α, a membrane bound PLA1 that produces 2-acyl-LPA, is a causative gene for autosomal recessive hypotrichosis [108]. Subsequently, it was reported by two independent research groups that several mutations in the p2y5 (now known as LPAR6) gene are causative factors that underlie autosomal recessive hypotrichosis/woolly hair [20,109]. mRNAs for both mPA-PLA1α and LPA6 are expressed in the hair follicle inner root sheath and the epidermis in humans [41], suggesting that mPA-PLA1α-derived 2-acyl-LPA may activate LPA6 in an autocrine/paracrine manner to stimulate hair growth [6]. It is noteworthy that LPA6 shows a ligand preference for 2-acyl-LPA over 1-acyl-LPA in heterologous expression experiments as discussed earlier (Section 3.2). Recently, a study by Inoue et al. utilizing mPA-PLA1α knockout mice gave great insight into the role of the mPA-PLA1α/LPA6 axis and precise mechanisms by which LPA regulates hair growth [9]. mPAPLA1α knockout mice showed wavy hairs, which is consistent with previous genetic studies showing the importance of mPA-PLA1α and LPA6 in normal human hair growth. They examined relative abundance of many LPA species in mouse hair follicles, and found that LPA species with unsaturated fatty acids were dramatically decreased in mPA-PLA1α knockout mice. Furthermore, they confirmed the reduction of 2-acyl-LPA (> 90%) but not of 1-acyl-LPA in hair follicles of mPA-PLA1α knockout mice. Their results showed that unsaturated 2-acyl-LPAs are clearly the most abundant LPA species in mouse hair follicles and dominantly produced by mPA-PLA1α. In vitro experiments utilizing primary keratinocytes, HaCaT cells, and HEK293 cells supported the role of the 2-acyl-LPA/LPA6/G12/13/ROCK axis in promoting the TNFα converting enzyme/TGFα/epidermal growth factor receptor (EGFR) signaling pathway, which is an important regulator of hair follicle development. This was also supported by data acquired in vivo — the amount of TGFα in hair follicles and phosphorylation of EGFR in dorsal skins were reduced in mPA-PLA1α knockout mice compared to wild-type mice. However, the involvement of G12/13 in hair development in vivo was not examined. This point should be addressed in future studies. Of note, the gene which encodes G12 protein is located on chromosome 7p21.3-p22.3a in a region determined by genetic mapping to be a disease locus for the autosomal recessive transmission of hereditary hypotrichosis in a Pakistani family [110].

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6. Conclusion and future prospects Because of its low homology with Edg family LPA receptors, the first non-Edg LPA receptor, LPA4, represented a new paradigm in LPA signaling that took some time to be widely accepted. However, following independent confirmation by other research groups and the discovery of additional non-Edg LPA receptors, these receptors are now recognized as major mediators of LPA-stimulated biological responses. Here we have summarized recent reports on the ligand selectivities, signaling and physiological roles of non-Edg family LPA receptors. These reports are filling in gaps in the LPA field that could not be accounted for by the classical Edg family LPA receptors such as platelet aggregation and vascular development. Furthermore, the study and analysis of non-Edg family LPA receptors are now unveiling new biological functions and deepening our understanding of LPA signaling. Elucidation of the cellular signaling downstream of non-Edg family LPA receptors requires further investigation. Compared to Edg family LPA receptors, these signaling pathways are less well characterized, particularly G protein coupling specificities which may vary in different cell lines and biological contexts. Unfortunately, there are currently very few pharmacological agents that selectively target non-Edg family LPA receptors, and the search for specific agonists and antagonists remains an extremely pressing issue. Analyses of LPA signaling by knockout of non-Edg family LPA receptor genes in mice will also yield critical information. These pharmacological and genetic approaches will not only shed light on the function of non-Edg family LPA receptors but also illuminate new opportunities where targeting LPA receptors may provide therapeutic benefits. Acknowledgements The authors thank Dr. William J. Valentine (Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo) for critical reading of the manuscript and useful suggestions. This work was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan (to K.Y., S.I. and T.S.), the Health and Labour Sciences Research Grants for Research on Allergic Disease and Immunology from the Ministry of Health, Labour and Welfare of Japan (to S.I.), a grant to the Respiratory Failure Research Group from the Ministry of Health, Labour, and Welfare of Japan (to S.I.) and grants from the Naito Foundation and the Uehara Memorial Foundation (to S.I.). References [1] J.W. Choi, D.R. Herr, K. Noguchi, Y.C. Yung, C.W. Lee, T. Mutoh, M.E. Lin, S.T. Teo, K.E. Park, A.N. Mosley, J. Chun, LPA receptors: subtypes and biological actions, Annu. Rev. Pharmacol. Toxicol. 50 (2010) 157–186. [2] T. Sano, D. Baker, T. Virag, A. Wada, Y. Yatomi, T. Kobayashi, Y. Igarashi, G. Tigyi, Multiple mechanisms linked to platelet activation result in lysophosphatidic acid and sphingosine 1-phosphate generation in blood, J. Biol. Chem. 277 (2002) 21197–21206. [3] T. Sugiura, S. Nakane, S. Kishimoto, K. Waku, Y. Yoshioka, A. Tokumura, Lysophosphatidic acid, a growth factor-like lipid, in the saliva, J. Lipid Res. 43 (2002) 2049–2055. [4] K. Hama, K. Bandoh, Y. Kakehi, J. Aoki, H. Arai, Lysophosphatidic acid (LPA) receptors are activated differentially by biological fluids: possible role of LPA-binding proteins in activation of LPA receptors, FEBS Lett. 523 (2002) 187–192. [5] A.M. Tager, P. LaCamera, B.S. Shea, G.S. Campanella, M. Selman, Z. Zhao, V. Polosukhin, J. Wain, B.A. Karimi-Shah, N.D. Kim, W.K. Hart, A. Pardo, T.S. Blackwell, Y. Xu, J. Chun, A.D. Luster, The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, Nat. Med. 14 (2008) 45–54. [6] J. Aoki, A. Inoue, S. Okudaira, Two pathways for lysophosphatidic acid production, Biochim. Biophys. Acta 1781 (2008) 513–518. [7] L.A. van Meeteren, P. Ruurs, C. Stortelers, P. Bouwman, M.A. van Rooijen, J.P. Pradere, T.R. Pettit, M.J. Wakelam, J.S. Saulnier-Blache, C.L. Mummery, W.H.

40

K. Yanagida et al. / Biochimica et Biophysica Acta 1831 (2013) 33–41

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24] [25]

[26] [27]

[28]

[29]

[30]

[31]

[32] [33]

Moolenaar, J. Jonkers, Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26 (2006) 5015–5022. M. Tanaka, S. Okudaira, Y. Kishi, R. Ohkawa, S. Iseki, M. Ota, S. Noji, Y. Yatomi, J. Aoki, H. Arai, Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281 (2006) 25822–25830. A. Inoue, N. Arima, J. Ishiguro, G.D. Prestwich, H. Arai, J. Aoki, LPA-producing enzyme PA-PLAalpha regulates hair follicle development by modulating EGFR signalling, EMBO J. 30 (2011) 4248–4260. J. Chun, T. Hla, K.R. Lynch, S. Spiegel, W.H. Moolenaar, International Union of Basic and Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature, Pharmacol. Rev. 62 (2010) 579–587. J. Chun, E.J. Goetzl, T. Hla, Y. Igarashi, K.R. Lynch, W. Moolenaar, S. Pyne, G. Tigyi, International Union of Pharmacology. XXXIV. Lysophospholipid receptor nomenclature, Pharmacol. Rev. 54 (2002) 265–269. H. Ohta, K. Sato, N. Murata, A. Damirin, E. Malchinkhuu, J. Kon, T. Kimura, M. Tobo, Y. Yamazaki, T. Watanabe, M. Yagi, M. Sato, R. Suzuki, H. Murooka, T. Sakai, T. Nishitoba, D.S. Im, H. Nochi, K. Tamoto, H. Tomura, F. Okajima, Ki16425, a subtype-selective antagonist for EDG-family lysophosphatidic acid receptors, Mol. Pharmacol. 64 (2003) 994–1005. K. Hama, J. Aoki, M. Fukaya, Y. Kishi, T. Sakai, R. Suzuki, H. Ohta, T. Yamori, M. Watanabe, J. Chun, H. Arai, Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1, J. Biol. Chem. 279 (2004) 17634–17639. J.J. Contos, N. Fukushima, J.A. Weiner, D. Kaushal, J. Chun, Requirement for the lpA1 lysophosphatidic acid receptor gene in normal suckling behavior, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13384–13389. J.J. Contos, I. Ishii, N. Fukushima, M.A. Kingsbury, X. Ye, S. Kawamura, J.H. Brown, J. Chun, Characterization of lpa(2) (Edg4) and lpa(1)/lpa(2) (Edg2/Edg4) lysophosphatidic acid receptor knockout mice: signaling deficits without obvious phenotypic abnormality attributable to lpa(2), Mol. Cell. Biol. 22 (2002) 6921–6929. M.F. Simon, H. Chap, L. Douste-Blazy, Human platelet aggregation induced by 1-alkyl-lysophosphatidic acid and its analogs: a new group of phospholipid mediators? Biochem. Biophys. Res. Commun. 108 (1982) 1743–1750. G. Gueguen, B. Gaige, J.M. Grevy, P. Rogalle, J. Bellan, M. Wilson, A. Klaebe, F. Pont, M.F. Simon, H. Chap, Structure–activity analysis of the effects of lysophosphatidic acid on platelet aggregation, Biochemistry 38 (1999) 8440–8450. A. Tokumura, J. Sinomiya, S. Kishimoto, T. Tanaka, K. Kogure, T. Sugiura, K. Satouchi, K. Waku, K. Fukuzawa, Human platelets respond differentially to lysophosphatidic acids having a highly unsaturated fatty acyl group and alkyl ether-linked lysophosphatidic acids, Biochem. J. 365 (2002) 617–628. K. Noguchi, S. Ishii, T. Shimizu, Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid, structurally distant from the Edg family, J. Biol. Chem. 278 (2003) 25600–25606. S.M. Pasternack, I. von Kugelgen, K.A. Aboud, Y.A. Lee, F. Ruschendorf, K. Voss, A.M. Hillmer, G.J. Molderings, T. Franz, A. Ramirez, P. Nurnberg, M.M. Nothen, R.C. Betz, G protein-coupled receptor P2Y5 and its ligand LPA are involved in maintenance of human hair growth, Nat. Genet. 40 (2008) 329–334. K. Yanagida, K. Masago, H. Nakanishi, Y. Kihara, F. Hamano, Y. Tajima, R. Taguchi, T. Shimizu, S. Ishii, Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6, J. Biol. Chem. 284 (2009) 17731–17741. M. Lee, S. Choi, G. Hallden, S.J. Yo, D. Schichnes, G.W. Aponte, P2Y5 is a G(alpha)i, G(alpha)12/13 G protein-coupled receptor activated by lysophosphatidic acid that reduces intestinal cell adhesion, Am. J. Physiol. Gastrointest. Liver Physiol. 297 (2009) G641–G654. S. Ishii, K. Noguchi, K. Yanagida, Non-Edg family lysophosphatidic acid (LPA) receptors, Prostaglandins Other Lipid Mediat. 89 (2009) 57–65. K. Yanagida, S. Ishii, Non-Edg family LPA receptors: the cutting edge of LPA research, J. Biochem. 150 (2011) 223–232. J.H. Hecht, J.A. Weiner, S.R. Post, J. Chun, Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex, J. Cell Biol. 135 (1996) 1071–1083. R.M. Sullivan, S.G. Lucas, Fossil squamata from the San-Jose formation, early eocene, San-Juan Basin, New-Mexico, J. Paleontol. 62 (1998) 631. K. Bandoh, J. Aoki, H. Hosono, S. Kobayashi, T. Kobayashi, K. MurakamiMurofushi, M. Tsujimoto, H. Arai, K. Inoue, Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid, J. Biol. Chem. 274 (1999) 27776–27785. C.W. Lee, R. Rivera, S. Gardell, A.E. Dubin, J. Chun, GPR92 as a new G12/13- and Gq-coupled lysophosphatidic acid receptor that increases cAMP, LPA5, J. Biol. Chem. 281 (2006) 23589–23597. K. Kotarsky, A. Boketoft, J. Bristulf, N.E. Nilsson, A. Norberg, S. Hansson, C. Owman, R. Sillard, L.M. Leeb-Lundberg, B. Olde, Lysophosphatidic acid binds to and activates GPR92, a G protein-coupled receptor highly expressed in gastrointestinal lymphocytes, J. Pharmacol. Exp. Ther. 318 (2006) 619–628. K. Tabata, K. Baba, A. Shiraishi, M. Ito, N. Fujita, The orphan GPCR GPR87 was deorphanized and shown to be a lysophosphatidic acid receptor, Biochem. Biophys. Res. Commun. 363 (2007) 861–866. M. Murakami, A. Shiraishi, K. Tabata, N. Fujita, Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor, Biochem. Biophys. Res. Commun. 371 (2008) 707–712. S. Oka, R. Ota, M. Shima, A. Yamashita, T. Sugiura, GPR35 is a novel lysophosphatidic acid receptor, Biochem. Biophys. Res. Commun. 395 (2010) 232–237. A. Tokumura, A family of phospholipid autacoids: occurrence, metabolism and bioactions, Prog. Lipid Res. 34 (1995) 151–184.

[34] K. Bandoh, J. Aoki, A. Taira, M. Tsujimoto, H. Arai, K. Inoue, Lysophosphatidic acid (LPA) receptors of the EDG family are differentially activated by LPA species. Structure– activity relationship of cloned LPA receptors, FEBS Lett. 478 (2000) 159–165. [35] W. Vogt, Intestine-stimulating activity of various phosphatides and glycolipids, Naunyn Schmiedebergs Arch. Exp. Pathol. Pharmakol. 240 (1960) 134–139. [36] A. Tokumura, K. Fukuzawa, H. Tsukatani, Effects of synthetic and natural lysophosphatidic acids on the arterial blood pressure of different animal species, Lipids 13 (1978) 572–574. [37] S. Amisten, O.O. Braun, A. Bengtsson, D. Erlinge, Gene expression profiling for the identification of G-protein coupled receptors in human platelets, Thromb. Res. 122 (2008) 47–57. [38] A.L. Khandoga, Y. Fujiwara, P. Goyal, D. Pandey, R. Tsukahara, A. Bolen, H. Guo, N. Wilke, J. Liu, W.J. Valentine, G.G. Durgam, D.D. Miller, G. Jiang, G.D. Prestwich, G. Tigyi, W. Siess, Lysophosphatidic acid-induced platelet shape change revealed through LPA(1–5) receptor-selective probes and albumin, Platelets 19 (2008) 415–427. [39] J.R. Williams, A.L. Khandoga, P. Goyal, J.I. Fells, D.H. Perygin, W. Siess, A.L. Parrill, G. Tigyi, Y. Fujiwara, Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate receptor indicates role in human platelet activation, J. Biol. Chem. 284 (2009) 17304–17319. [40] A.L. Khandoga, D. Pandey, U. Welsch, R. Brandl, W. Siess, GPR92/LPA5 lysophosphatidate receptor mediates megakaryocytic cell shape change induced by human atherosclerotic plaques, Cardiovasc. Res. 90 (2011) 157–164. [41] Y. Shimomura, Congenital hair loss disorders: rare, but not too rare, J. Dermatol. 39 (2012) 3–10. [42] H. Sonoda, J. Aoki, T. Hiramatsu, M. Ishida, K. Bandoh, Y. Nagai, R. Taguchi, K. Inoue, H. Arai, A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid, J. Biol. Chem. 277 (2002) 34254–34263. [43] T. Sugiura, A. Tokumura, L. Gregory, T. Nouchi, S.T. Weintraub, D.J. Hanahan, Biochemical characterization of the interaction of lipid phosphoric acids with human platelets: comparison with platelet activating factor, Arch. Biochem. Biophys. 311 (1994) 358–368. [44] C.E. Heise, W.L. Santos, A.M. Schreihofer, B.H. Heasley, Y.V. Mukhin, T.L. Macdonald, K.R. Lynch, Activity of 2-substituted lysophosphatidic acid (LPA) analogs at LPA receptors: discovery of a LPA1/LPA3 receptor antagonist, Mol. Pharmacol. 60 (2001) 1173–1180. [45] B.H. Heasley, R. Jarosz, K.R. Lynch, T.L. Macdonald, Initial structure–activity relationships of lysophosphatidic acid receptor antagonists: discovery of a high-affinity LPA1/LPA3 receptor antagonist, Bioorg. Med. Chem. Lett. 14 (2004) 2735–2740. [46] W.L. Santos, B.H. Heasley, R. Jarosz, K.M. Carter, K.R. Lynch, T.L. Macdonald, Synthesis and biological evaluation of phosphonic and thiophosphoric acid derivatives of lysophosphatidic acid, Bioorg. Med. Chem. Lett. 14 (2004) 3473–3476. [47] B.H. Heasley, R. Jarosz, K.M. Carter, S.J. Van, K.R. Lynch, T.L. Macdonald, A novel series of 2-pyridyl-containing compounds as lysophosphatidic acid receptor antagonists: development of a nonhydrolyzable LPA3 receptor-selective antagonist, Bioorg. Med. Chem. Lett. 14 (2004) 4069–4074. [48] L. Qian, Y. Xu, Y. Hasegawa, J. Aoki, G.B. Mills, G.D. Prestwich, Enantioselective responses to a phosphorothioate analogue of lysophosphatidic acid with LPA3 receptor-selective agonist activity, J. Med. Chem. 46 (2003) 5575–5578. [49] Y. Hasegawa, J.R. Erickson, G.J. Goddard, S. Yu, S. Liu, K.W. Cheng, A. Eder, K. Bandoh, J. Aoki, R. Jarosz, A.D. Schrier, K.R. Lynch, G.B. Mills, X. Fang, Identification of a phosphothionate analogue of lysophosphatidic acid (LPA) as a selective agonist of the LPA3 receptor, J. Biol. Chem. 278 (2003) 11962–11969. [50] L. Qian, Y. Xu, T. Simper, G. Jiang, J. Aoki, M. Umezu-Goto, H. Arai, S. Yu, G.B. Mills, R. Tsukahara, N. Makarova, Y. Fujiwara, G. Tigyi, G.D. Prestwich, Phosphorothioate analogues of alkyl lysophosphatidic acid as LPA3 receptor-selective agonists, ChemMedChem 1 (2006) 376–383. [51] M.D. Okusa, H. Ye, L. Huang, L. Sigismund, T. Macdonald, K.R. Lynch, Selective blockade of lysophosphatidic acid LPA3 receptors reduces murine renal ischemia– reperfusion injury, Am. J. Physiol. Renal Physiol. 285 (2003) F565–F574. [52] D.A. Lin, J.A. Boyce, IL-4 regulates MEK expression required for lysophosphatidic acid-mediated chemokine generation by human mast cells, J. Immunol. 175 (2005) 5430–5438. [53] M.M. Murph, J. Hurst-Kennedy, V. Newton, D.N. Brindley, H. Radhakrishna, Lysophosphatidic acid decreases the nuclear localization and cellular abundance of the p53 tumor suppressor in A549 lung carcinoma cells, Mol. Cancer Res. 5 (2007) 1201–1211. [54] C. Zhao, M.J. Fernandes, G.D. Prestwich, M. Turgeon, J. Di Battista, T. Clair, P.E. Poubelle, S.G. Bourgoin, Regulation of lysophosphatidic acid receptor expression and function in human synoviocytes: implications for rheumatoid arthritis? Mol. Pharmacol. 73 (2008) 587–600. [55] Z. Zhou, J. Niu, Z. Zhang, The role of lysophosphatidic acid receptors in phenotypic modulation of vascular smooth muscle cells, Mol. Biol. Rep. 37 (2010) 2675–2686. [56] J.P. Mansell, M. Barbour, C. Moore, M. Nowghani, M. Pabbruwe, T. Sjostrom, A.W. Blom, The synergistic effects of lysophosphatidic acid receptor agonists and calcitriol on MG63 osteoblast maturation at titanium and hydroxyapatite surfaces, Biomaterials 31 (2010) 199–206. [57] J.W. Lee, C.H. Kim, Y.Y. Wang, X.M. Yan, U.D. Sohn, Lysophosphatidic acid presynaptically blocks NO uptake during electric field stimulation-induced relaxation via LPA(1) receptor in cat lower esophageal sphincter, Arch. Pharm. Res. 34 (2011) 169–176. [58] D.J. Fischer, N. Nusser, T. Virag, K. Yokoyama, D. Wang, D.L. Baker, D. Bautista, A.L. Parrill, G. Tigyi, Short-chain phosphatidates are subtype-selective antagonists of lysophosphatidic acid receptors, Mol. Pharmacol. 60 (2001) 776–784.

K. Yanagida et al. / Biochimica et Biophysica Acta 1831 (2013) 33–41 [59] G.G. Durgam, R. Tsukahara, N. Makarova, M.D. Walker, Y. Fujiwara, K.R. Pigg, D.L. Baker, V.M. Sardar, A.L. Parrill, G. Tigyi, D.D. Miller, Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands, Bioorg. Med. Chem. Lett. 16 (2006) 633–640. [60] K. Liliom, T. Tsukahara, R. Tsukahara, M. Zelman-Femiak, E. Swiezewska, G. Tigyi, Farnesyl phosphates are endogenous ligands of lysophosphatidic acid receptors: inhibition of LPA GPCR and activation of PPARs, Biochim. Biophys. Acta 1761 (2006) 1506–1514. [61] D.Y. Oh, J.M. Yoon, M.J. Moon, J.I. Hwang, H. Choe, J.Y. Lee, J.I. Kim, S. Kim, H. Rhim, D.K. O'Dell, J.M. Walker, H.S. Na, M.G. Lee, H.B. Kwon, K. Kim, J.Y. Seong, Identification of farnesyl pyrophosphate and N-arachidonylglycine as endogenous ligands for GPR92, J. Biol. Chem. 283 (2008) 21054–21064. [62] M. Komachi, K. Sato, M. Tobo, C. Mogi, T. Yamada, H. Ohta, H. Tomura, T. Kimura, D.S. Im, K. Yanagida, S. Ishii, I. Takeyoshi, F. Okajima, Orally active lysophosphatidic acid receptor antagonist attenuates pancreatic cancer invasion and metastasis in vivo, Cancer Sci. 103 (2012) 1099–1104. [63] C.W. Lee, R. Rivera, A.E. Dubin, J. Chun, LPA(4)/GPR23 is a lysophosphatidic acid (LPA) receptor utilizing G(s)-, G(q)/G(i)-mediated calcium signaling and G(12/13)-mediated Rho activation, J. Biol. Chem. 282 (2007) 4310–4317. [64] J.S. Swaney, C. Chapman, L.D. Correa, K.J. Stebbins, R.A. Bundey, P.C. Prodanovich, P. Fagan, C.S. Baccei, A.M. Santini, J.H. Hutchinson, T.J. Seiders, T.A. Parr, P. Prasit, J.F. Evans, D.S. Lorrain, A novel, orally active LPA(1) receptor antagonist inhibits lung fibrosis in the mouse bleomycin model, Br. J. Pharmacol. 160 (2010) 1699–1713. [65] Y. Takuwa, N. Takuwa, N. Sugimoto, The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities, J. Biochem. 131 (2002) 767–771. [66] K. Yanagida, S. Ishii, F. Hamano, K. Noguchi, T. Shimizu, LPA4/p2y9/GPR23 mediates rho-dependent morphological changes in a rat neuronal cell line, J. Biol. Chem. 282 (2007) 5814–5824. [67] Z. Lee, C.T. Cheng, H. Zhang, M.A. Subler, J. Wu, A. Mukherjee, J.J. Windle, C.K. Chen, X. Fang, Role of LPA4/p2y9/GPR23 in negative regulation of cell motility, Mol. Biol. Cell 19 (2008) 5435–5445. [68] H. Sumida, K. Noguchi, Y. Kihara, M. Abe, K. Yanagida, F. Hamano, S. Sato, K. Tamaki, Y. Morishita, M.R. Kano, C. Iwata, K. Miyazono, K. Sakimura, T. Shimizu, S. Ishii, LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis, Blood 116 (2010) 5060–5070. [69] S. Offermanns, V. Mancino, J.P. Revel, M.I. Simon, Vascular system defects and impaired cell chemokinesis as a result of Galpha13 deficiency, Science 275 (1997) 533–536. [70] J.L. Gu, S. Muller, V. Mancino, S. Offermanns, M.I. Simon, Interaction of G alpha(12) with G alpha(13) and G alpha(q) signaling pathways, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 9352–9357. [71] K.M. Ruppel, D. Willison, H. Kataoka, A. Wang, Y.W. Zheng, I. Cornelissen, L. Yin, S.M. Xu, S.R. Coughlin, Essential role for Galpha13 in endothelial cells during embryonic development, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 8281–8286. [72] K. Harper, D. Arsenault, S. Boulay-Jean, A. Lauzier, F. Lucien, C.M. Dubois, Autotaxin promotes cancer invasion via the lysophosphatidic acid receptor 4: participation of the cyclic AMP/EPAC/Rac1 signaling pathway in invadopodia formation, Cancer Res. 70 (2010) 4634–4643. [73] Y.B. Liu, Y. Kharode, P.V. Bodine, P.J. Yaworsky, J.A. Robinson, J. Billiard, LPA induces osteoblast differentiation through interplay of two receptors: LPA1 and LPA4, J. Cell. Biochem. 109 (2010) 794–800. [74] Y. Daaka, L.M. Luttrell, R.J. Lefkowitz, Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A, Nature 390 (1997) 88–91. [75] S. Mittra, J.P. Bourreau, Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes, Am. J. Physiol. Heart Circ. Physiol. 290 (2006) H1842–H1847. [76] M. Jongsma, E. Matas-Rico, A. Rzadkowski, K. Jalink, W.H. Moolenaar, LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor, PLoS One 6 (2012) e29260. [77] S. Choi, M. Lee, A.L. Shiu, S.J. Yo, G.W. Aponte, Identification of a protein hydrolysate responsive G protein-coupled receptor in enterocytes, Am. J. Physiol. Gastrointest. Liver Physiol. 292 (2007) G98–G112. [78] A. Lundequist, J.A. Boyce, LPA5 is abundantly expressed by human mast cells and important for lysophosphatidic acid induced MIP-1beta release, PLoS One 6 (2011) e18192. [79] S. Koike, K. Keino-Masu, T. Ohto, F. Sugiyama, S. Takahashi, M. Masu, Autotaxin/lysophospholipase D-mediated lysophosphatidic acid signaling is required to form distinctive large lysosomes in the visceral endoderm cells of the mouse yolk sac, J. Biol. Chem. 284 (2009) 33561–33570. [80] S. Fotopoulou, N. Oikonomou, E. Grigorieva, I. Nikitopoulou, T. Paparountas, A. Thanassopoulou, Z. Zhao, Y. Xu, D.L. Kontoyiannis, E. Remboutsika, V. Aidinis, ATX expression and LPA signalling are vital for the development of the nervous system, Dev. Biol. 339 (2010) 451–464. [81] G. Ferry, A. Giganti, F. Coge, F. Bertaux, K. Thiam, J.A. Boutin, Functional invalidation of the autotaxin gene by a single amino acid mutation in mouse is lethal, FEBS Lett. 581 (2007) 3572–3578. [82] X. Ye, K. Hama, J.J. Contos, B. Anliker, A. Inoue, M.K. Skinner, H. Suzuki, T. Amano, G. Kennedy, H. Arai, J. Aoki, J. Chun, LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing, Nature 435 (2005) 104–108. [83] X. Ye, M.K. Skinner, G. Kennedy, J. Chun, Age-dependent loss of sperm production in mice via impaired lysophosphatidic acid signaling, Biol. Reprod. 79 (2008) 328–336. [84] H. Yukiura, K. Hama, K. Nakanaga, M. Tanaka, Y. Asaoka, S. Okudaira, N. Arima, A. Inoue, T. Hashimoto, H. Arai, A. Kawahara, H. Nishina, J. Aoki, Autotaxin

[85] [86]

[87] [88] [89]

[90]

[91] [92]

[93]

[94]

[95] [96] [97]

[98]

[99]

[100]

[101]

[102] [103]

[104]

[105]

[106]

[107]

[108]

[109]

[110]

41

regulates vascular development via multiple lysophosphatidic acid (LPA) receptors in zebrafish, J. Biol. Chem. 286 (2011) 43972–43983. S.J. Lee, T.H. Chan, T.C. Chen, B.K. Liao, P.P. Hwang, H. Lee, LPA1 is essential for lymphatic vessel development in zebrafish, FASEB J. 22 (2008) 3706–3715. I. Gennero, S. Laurencin-Dalicieux, F. Conte-Auriol, F. Briand-Mesange, D. Laurencin, J. Rue, N. Beton, N. Malet, M. Mus, A. Tokumura, P. Bourin, L. Vico, G. Brunel, R.O. Oreffo, J. Chun, J.P. Salles, Absence of the lysophosphatidic acid receptor LPA1 results in abnormal bone development and decreased bone mass, Bone 49 (2011) 395–403. J. Blackburn, J.P. Mansell, The emerging role of lysophosphatidic acid (LPA) in skeletal biology, Bone 50 (2012) 756–762. S. Bagga, K.S. Price, D.A. Lin, D.S. Friend, K.F. Austen, J.A. Boyce, Lysophosphatidic acid accelerates the development of human mast cells, Blood 104 (2004) 4080–4087. T. Hashimoto, H. Ohata, K. Honda, Lysophosphatidic acid (LPA) induces plasma exudation and histamine release in mice via LPA receptors, J. Pharmacol. Sci. 100 (2006) 82–87. T. Hashimoto, H. Ohata, K. Momose, K. Honda, Lysophosphatidic acid induces histamine release from mast cells and skin fragments, Pharmacology 75 (2005) 13–20. T. Hashimoto, H. Ohata, K. Momose, Itch-scratch responses induced by lysophosphatidic acid in mice, Pharmacology 72 (2004) 51–56. K. Mori, J. Kitayama, J. Aoki, Y. Kishi, D. Shida, H. Yamashita, H. Arai, H. Nagawa, Submucosal connective tissue-type mast cells contribute to the production of lysophosphatidic acid (LPA) in the gastrointestinal tract through the secretion of autotaxin (ATX)/lysophospholipase D (lysoPLD), Virchows Arch. 451 (2007) 47–56. M.L. Stracke, H.C. Krutzsch, E.J. Unsworth, A. Arestad, V. Cioce, E. Schiffmann, L.A. Liotta, Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein, J. Biol. Chem. 267 (1992) 2524–2529. M. Umezu-Goto, Y. Kishi, A. Taira, K. Hama, N. Dohmae, K. Takio, T. Yamori, G.B. Mills, K. Inoue, J. Aoki, H. Arai, Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production, J. Cell Biol. 158 (2002) 227–233. G.B. Mills, W.H. Moolenaar, The emerging role of lysophosphatidic acid in cancer, Nat. Rev. Cancer 3 (2003) 582–591. W.H. Moolenaar, L.A. van Meeteren, B.N. Giepmans, The ins and outs of lysophosphatidic acid signaling, Bioessays 26 (2004) 870–881. X. Xu, G. Yang, H. Zhang, G.D. Prestwich, Evaluating dual activity LPA receptor pan-antagonist/autotaxin inhibitors as anti-cancer agents in vivo using engineered human tumors, Prostaglandins Other Lipid Mediat. 89 (2009) 140–146. D.L. Baker, Y. Fujiwara, K.R. Pigg, R. Tsukahara, S. Kobayashi, H. Murofushi, A. Uchiyama, K. Murakami-Murofushi, E. Koh, R.W. Bandle, H.S. Byun, R. Bittman, D. Fan, M. Murph, G.B. Mills, G. Tigyi, Carba analogs of cyclic phosphatidic acid are selective inhibitors of autotaxin and cancer cell invasion and metastasis, J. Biol. Chem. 281 (2006) 22786–22793. M.K. Altman, V. Gopal, W. Jia, S. Yu, H. Hall, G.B. Mills, A.C. McGinnis, M.G. Bartlett, G. Jiang, D. Madan, G.D. Prestwich, Y. Xu, M.A. Davies, M.M. Murph, Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid, Mol. Cancer 9 (2010) 140. R. Gupte, A. Siddam, Y. Lu, W. Li, Y. Fujiwara, N. Panupinthu, T.C. Pham, D.L. Baker, A.L. Parrill, M. Gotoh, K. Murakami-Murofushi, S. Kobayashi, G.B. Mills, G. Tigyi, D.D. Miller, Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid, Bioorg. Med. Chem. Lett. 20 (2010) 7525–7528. R. Gupte, R. Patil, J. Liu, Y. Wang, S.C. Lee, Y. Fujiwara, J. Fells, A.L. Bolen, K. Emmons-Thompson, C.R. Yates, A. Siddam, N. Panupinthu, T.C. Pham, D.L. Baker, A.L. Parrill, G.B. Mills, G. Tigyi, D.D. Miller, Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity, ChemMedChem 6 (2011) 922–935. H. Ueda, Lysophosphatidic acid as the initiator of neuropathic pain, Biol. Pharm. Bull. 34 (2011) 1154–1158. M. Inoue, L. Ma, J. Aoki, J. Chun, H. Ueda, Autotaxin, a synthetic enzyme of lysophosphatidic acid (LPA), mediates the induction of nerve-injured neuropathic pain, Mol. Pain 4 (2008) 6. M. Inoue, M.H. Rashid, R. Fujita, J.J. Contos, J. Chun, H. Ueda, Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling, Nat. Med. 10 (2004) 712–718. L. Ma, H. Uchida, J. Nagai, M. Inoue, J. Chun, J. Aoki, H. Ueda, Lysophosphatidic acid-3 receptor-mediated feed-forward production of lysophosphatidic acid: an initiator of nerve injury-induced neuropathic pain, Mol. Pain 5 (2009) 64. M.E. Lin, R.R. Rivera, J. Chun, Targeted deletion of LPA5 identifies novel roles for LPA signaling in the development of neuropathic pain, J. Biol. Chem. 287 (2012) 17608–17617. T. Takahashi, A. Kamimura, T. Hamazono-Matsuoka, S. Honda, Phosphatidic acid has a potential to promote hair growth in vitro and in vivo, and activates mitogen-activated protein kinase/extracellular signal-regulated kinase kinase in hair epithelial cells, J. Invest. Dermatol. 121 (2003) 448–456. A. Kazantseva, A. Goltsov, R. Zinchenko, A.P. Grigorenko, A.V. Abrukova, Y.K. Moliaka, A.G. Kirillov, Z. Guo, S. Lyle, E.K. Ginter, E.I. Rogaev, Human hair growth deficiency is linked to a genetic defect in the phospholipase gene LIPH, Science 314 (2006) 982–985. Y. Shimomura, M. Wajid, Y. Ishii, L. Shapiro, L. Petukhova, D. Gordon, A.M. Christiano, Disruption of P2RY5, an orphan G protein-coupled receptor, underlies autosomal recessive woolly hair, Nat. Genet. 40 (2008) 335–339. S. Basit, G. Ali, N. Wasif, M. Ansar, W. Ahmad, Genetic mapping of a novel hypotrichosis locus to chromosome 7p21.3-p22.3 in a Pakistani family and screening of the candidate genes, Hum. Genet. 128 (2010) 213–220.