Secreted PLA2-III is a possible therapeutic target to treat neuropathic pain.

Lysophosphatidic acid (LPA) plays a critical role in developing and maintaining chronic pain in various animal models. Previous studies have reported that cytosolic and calcium-independent phospholipase A2 (PLA2) is involved in the LPA receptor-mediated amplification of LPA production in the spinal dorsal horn (SDH) after nerve injury, while the involvement of secreted PLA2 (sPLA2) remains unclear. The present study revealed that only sPLA2 -III among 11 species of PLA2 showed a significant upregulation of gene expression in the SDH. Intraspinal injection of adeno-associated virus-miRNA targeting sPLA2-III prevented hyperalgesia and unique hypoalgesia in mice treated with partial sciatic nerve ligation. In addition, intrathecal treatment with antisense oligodeoxynucleotide or siRNA targeting sPLA2-III significantly reversed the established thermal hyperalgesia. In the high-throughput screening of sPLA2-III inhibitors from the chemical library, we identified two hit compounds. Through in vitro characterization of PLA2 inhibitor profiles and in vivo assessment of the anti-hyperalgesic effects of known PLA2 inhibitors as well as hit compounds, sPLA2-III was found to be a novel therapeutic target molecule for the treatment of Neuropathic pain.

Evaluation of the pharmacokinetics of 2-carba-cyclic phosphatidic acid by liquid chromatography-triple quadrupole mass spectrometry.

Cyclic phosphatidic acid (cPA) is a lysophospholipid mediator that suppresses cancer metastasis and osteoarthritis. It also has neuroprotective roles in diseases such as multiple sclerosis and delayed neuronal death following transient ischemia. In order to take advantage of the properties of cPA for the development of new therapeutic strategies, we have synthesized several cPA derivatives and discovered 2-carba-cPA (2ccPA) as a promising candidate. To develop 2ccPA as a therapeutic agent, we investigated the pharmacokinetic profile of 2ccPA by liquid chromatography-triple quadrupole mass spectrometry in this study. When 2ccPA was administered intraperitoneally to mice at a dose of 1.6 mg/kg, the half-life of 2ccPA in plasma was 16 min. The 2ccPA, dosed intraperitoneally to mice at 16 mg/kg, distributed to each organ including brain at 20 min after dosing. It was found that 2ccPA was stable in neutral or alkaline conditions (e.g., intestine) but unstable in acidic conditions (e.g., stomach). When 2ccPA was orally administrated to rats as a gastro-resistant form using an enterosoluble capsule, plasma 2ccPA levels peaked at 2 h, slowly declined thereafter and persistently detected even at 10 h after administration. Here, we present the findings on the effect of the continuous release of 2ccPA from the capsule to reduce the lysophospholipase D activity and also decrease plasma levels of lysophosphatidic acid in rat. These findings will be useful in further studies for evaluating the application of 2ccPA in several disorders.

LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model.

Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.

Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARgamma by cyclic phosphatidic acid.

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

Myelin-related gene silencing mediated by LPA1 - Rho/ROCK signaling is correlated to acetylation of NFκB in S16 Schwann cells.

Lysophosphatidic acid (LPA) initiates demyelination following peripheral nerve injury, which causes neuropathic pain. Our previous in vivo and ex vivo studies using mice have demonstrated that LPA-induced demyelination of spinal dorsal roots is attributed by the LPA1-type receptor-mediated down-regulation of myelin-related molecules, such as MBP and MPZ. In this study using S16 mature-type Schwann cells, we found that LPA-induced down-regulation of myelin-related genes is attributed by the activation of LPA1 receptor, Rho kinase, and p300, leading to an acetylation of NFκB, which down-regulates the transcription of Sox10, MBP and MPZ genes.

Cyclic phosphatidic acid relieves osteoarthritis symptoms.

BACKGROUND: Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Natural cPA and its chemically stabilized cPA derivative, 2-carba-cPA (2ccPA), inhibit chronic and acute inflammation, and 2ccPA attenuates neuropathic pain. Osteoarthritis (OA) is a degenerative disease frequently associated with symptoms such as inflammation and joint pain. Because 2ccPA has obvious antinociceptive activity, we hypothesized that 2ccPA might relieve the pain caused by OA. We aimed to characterize the effects of 2ccPA on the pathogenesis of OA induced by total meniscectomy in the rabbit knee joint. RESULTS: Intra-articular injection of 2ccPA (twice a week for 42 days) significantly reduced pain and articular swelling. Histopathology showed that 2ccPA suppressed cartilage degeneration in OA. We also examined the effects of 2ccPA on the inflammatory and catabolic responses of human OA synoviocytes and chondrosarcoma SW1353 cells in vitro. 2ccPA stimulated synthesis of hyaluronic acid and suppressed production of the metalloproteinases MMP-1, -3, and -13. However, it had no effect on the production of interleukin (IL)-6, an inflammatory cytokine. The suppressive effect of 2ccPA on MMP-1 and -3 production in synoviocytes and on MMP-13 production in SW1353 cells was not mediated by the lysophosphatidic acid receptor, LPA1 receptor (LPA1R). CONCLUSIONS: Our results suggest that 2ccPA significantly reduces the pain response to OA by inducing hyaluronic acid production and suppressing MMP-1, -3, and -13 production in synoviocytes and chondrocytes.

Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface.

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.

Adenine nucleotide translocase 2, a putative target protein for 2-carba cyclic phosphatidic acid in microglial cells.

Lipid-protein interactions play essential roles in many biological phenomena. Lysophospholipid mediators, such as cyclic phosphatidic acid (cPA), have been recognized as secondary messengers, yet few cellular targets for cPA have been identified to date. Furthermore, the molecular mechanism that activates these downstream signaling events remains unknown. In this study, using metabolically stabilized cPA carba-derivative (2ccPA)-immobilized magnetic beads, we identified adenine nucleotide translocase 2 (ANT2) as a 2ccPA-interacting protein in microglial cells. 2ccPA was tested for its ability to inhibit apoptosis caused by phenylarsine oxide in microglial cells. This damage was significantly improved upon 2ccPA treatment, along with cell proliferation, apoptosis, reactive oxygen species production, and intracellular ATP levels. This is the first report to suggest the direct binding of 2ccPA to ANT2 in microglial cells and provides evidence for a new benefit of 2ccPA in protecting microglial cells from apoptotic death induced by the ANT2-mediated signaling pathway.

Lysophospholipid signaling: beyond the EDGs.

As our understanding of the myriads of biological effects caused by lysophospholipids expands, we become witnesses to another miracle of nature that has endowed the simplest lysophospholipids with functions seemingly ubiquitous to every mammalian cell. A decade after the discovery of the EDG family lysophospholipid receptors, the field has gained unimaginable impetus explaining the biological effects of sphingosine-1-phosphate and lysophosphatidic acid (LPA). The discovery of LPA receptors in the purinergic G-protein-coupled receptor (GPCR) gene cluster refined this picture and added complexity to our concepts of lysophospholipid cell signaling. The intracellular lysophospholipid targets - identified and not yet identified - make us realize the dual mediator and second messenger roles of lysophospholipids. In this paper we provide new data obtained concerning LPA-elicited responses using cell lines naturally lacking or intentionally knocked out of many of the known LPA GPCR, widely used by investigators in the field as cells with LPA receptor "null background." Our observations raise caution about the lack of LPA responsiveness in these cells and underline the unprecedented complexity and redundancy of lysophospholipid-evoked cellular responses.

Structure-based drug design identifies novel LPA3 antagonists.

Compound 5 ([5-(3-nitrophenoxy)-1,3-dioxo-1,3-dihydro-2-isoindol-2-yl]acetic acid) was identified as a weak selective LPA(3) antagonist (IC(50)=4504 nM) in a virtual screening effort to optimize a dual LPA(2 and 3) antagonist. Structure-based drug design techniques were used to prioritize similarity search matches of compound 5. This strategy rapidly identified 10 novel antagonists. The two most efficacious compounds identified inhibit activation of the LPA(3) receptor by 200 nM LPA with IC(50) values of 752 nM and 2992 nM. These compounds additionally define changes to our previously reported pharmacophore that will improve its ability to identify more potent and selective LPA(3) receptor antagonists. The results of the combined computational and experimental screening are reported.

Synthesis, pharmacology, and cell biology of sn-2-aminooxy analogues of lysophosphatidic acid.

An efficient enantioselective synthesis of sn-2-aminooxy (AO) analogues of lysophosphatidic acid (LPA) that possess palmitoyl and oleoyl acyl chains is presented. Both sn-2-AO LPA analogues are agonists for the LPA1, LPA2, and LPA4 G-protein-coupled receptors, but antagonists for the LPA3 receptor and inhibitors of autotaxin (ATX). Moreover, both analogues stimulate migration of intestinal epithelial cells in a scratch wound assay.

Identification of non-lipid LPA3 antagonists by virtual screening.

In the present study, we utilized virtual screening to identify LPA(3) antagonists. We have developed a three-point structure-based pharmacophore model based on known LPA(3) antagonists. This model was used to mine the NCI database. Docking, pharmacophore development, and database mining produced new, non-lipid leads. Experimental testing of seven computationally selected pharmacophore hits produced one potentiator and three antagonists, one of which displays both LPA(3) selectivity and nanomolar potency. Similarity searching in the ChemBridge database using the most promising lead as the search target produced four additional LPA(3) antagonists and a potent dual LPA(1&2) antagonist.

The AGP-PPARγ axis promotes oxidative stress and diabetic endothelial cell dysfunction.

Alkyl-glycerophosphate (AGP) accumulates in atherogenic oxidized-LDL and human atherosclerotic plaques and is a potent agonist of peroxisome-proliferator-activated receptor-gamma (PPARγ). Recent studies suggest a potential regulatory role for PPARγ in endothelial nitric oxide synthase (eNOS) expression/activation and nitrogen oxide (NO) generation in the vascular endothelium. Importantly, eNOS-induced NO and advanced glycation end-products (AGEs) are involved in blood-vessel damage, and diabetic patients exhibit high serum NO and AGE levels; however, the effect of AGP on NO- and AGE-mediated endothelium dysfunction remains unknown. Investigation of the AGP-specific effects on NO- and AGE-mediated dysfunction and the underlying molecular mechanisms revealed that AGP upregulated eNOS expression and NO production, and that eNOS silencing and PPARγ antagonism inhibited AGP-mediated eNOS upregulation and NO production. Moreover, AGP-PPARγ-axis-mediated NO production promoted the generation of reactive oxygen species and AGE formation. These results suggested that AGP plays a significant role in the initiation/progression of diabetes-related atherosclerosis through PPARγ activation.

Blockade of analgesic effects following systemic administration of N-methyl-kyotorphin, NMYR and arginine in mice deficient of preproenkephalin or proopiomelanocortin gene.

Kyotorphin is a unique biologically active neuropeptide (l-tyrosine-l-arginine), which is reported to have opioid-like analgesic actions through a release of Met-enkephalin from the brain slices. N-methyl-l-tyrosine-l-arginine (NMYR), an enzymatically stable mimetic of kyotorphin, successfully caused potent analgesic effects in thermal and mechanical nociception tests in mice when it was given through systemic routes. NMYR analgesia was abolished in µ-opioid receptor-deficient (MOP-KO) mice, and by intracerebroventricular (i.c.v.) injection of naloxone and of N-methyl l-leucine-l-arginine (NMLR), a kyotorphin receptor antagonist. In the Ca2+-mobilization assay using CHO cells expressing Gαqi5 and hMOPr or hDOPr, however, the addition of kyotorphin neither activated MOPr-mechanisms, nor affected the concentration-dependent activation of DAMGO- or Met-Enkephalin-induced MOPr activation, and Met-enkephalin-induced DOPr activation. NMYR-analgesia was significantly attenuated in preproenkephalin (PENK)- or proopioimelanocortin (POMC)-KO mice. The systemic administration of arginine, which is reported to elevate the level of endogenous kyotorphin selectively in midbrain and medulla oblongata, pain-related brain regions, caused significant analgesia, and the analgesia was reversed by i.c.v. injection of NMLR or naloxone. In addition, PENK- and POMC-KO mice also attenuated the arginine-induced analgesia. All these findings suggest that NMYR and arginine activate brain kyotorphin receptor in direct and indirect manner, respectively and both compounds indirectly cause the opioid-like analgesia through the action of endogenous opioid peptides.

Involvement of lysophosphatidic acid-induced astrocyte activation underlying the maintenance of partial sciatic nerve injury-induced neuropathic pain.

We have previously demonstrated that lysophosphatidic acid (LPA) plays key roles in the initial mechanisms for neuropathic pain (NeuP) development. Here, we examined whether LPA receptor mechanisms and LPA production are related to the glial activation at a late stage after partial sciatic nerve ligation (pSNL) by use of microglial inhibitor, Mac1-saporin or astrocyte inhibitor, and L-α-aminoadipate (L-AA). Although single intrathecal injection of LPA1/3 antagonist, Ki-16425 did not affect the pain threshold at day 7 after the spinal cord injury, repeated treatments of each compound gradually reversed the basal pain threshold to the control level. The intrathecal administration of a microglia inhibitor, Mac-1-saporin reversed the late hyperalgesia and LPA production at day 14 after the pSNL, whereas L-AA inhibited the hyperalgesia, but had no effect on LPA production. The involvement of LPA receptors in astrocyte activation in vivo was evidenced by the findings that Ki-16425 treatments abolished the upregulation of CXCL1 in activated astrocytes in the spinal dorsal horn of mice at day 14 after the pSNL, and that Ki-16425 reversed the LPA-induced upregulation of several chemokine gene expressions in primary cultured astrocytes. Finally, we found that significant hyperalgesia was observed with intrathecal administration of primary cultured astrocytes, which had been stimulated by LPA in a Ki-16425-reversible manner. All these findings suggest that LPA production and LPA1/3 receptor activation through differential glial mechanisms play key roles in the maintenance as well as initiation mechanisms in NeuP.

Farnesyl phosphates are endogenous ligands of lysophosphatidic acid receptors: inhibition of LPA GPCR and activation of PPARs.

Oligoprenyl phosphates are key metabolic intermediates for the biosynthesis of steroids, the side chain of ubiquinones, and dolichols and the posttranslational isoprenylation of proteins. Farnesyl phosphates are isoprenoid phosphates that resemble polyunsaturated fatty alcohol phosphates, which we have recently shown to be the minimal pharmacophores of lysophosphatidic acid (LPA) receptors. Here we examine whether farnesyl phosphates can interact with the cell surface and nuclear receptors for LPA. Both farnesyl phosphate and farnesyl diphosphate potently and specifically antagonized LPA-elicited intracellular Ca(2+)-mobilization mediated through the LPA(3) receptor, while causing only modest inhibition at the LPA(2) receptor and no measurable effect at the LPA(1) receptor. Farnesol also inhibited LPA(3) but was much less effective. The estimated dissociation constant of LPA(3) for farnesyl phosphate is 48+/-12 nM and 155+/-30 nM for farnesyl diphosphate. The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) binds to and is activated by LPA and its analogs including fatty alcohol phosphates. We found that both farnesyl phosphate and diphosphate, but not farnesol, compete with the binding of the synthetic PPARgamma agonist [(3)H]rosiglitazone and activate the PPARgamma-mediated gene transcription. Farnesyl monophosphate at 1 microM, but not diphosphate, activated PPARalpha and PPARbeta/delta reporter gene expression. These results indicate new potential roles for the oligoprenyl phosphates as potential endogenous modulators of LPA targets and show that the polyisoprenoid chain is recognized by some LPA receptors.

Phosphonothioate and fluoromethylene phosphonate analogues of cyclic phosphatidic acid: Novel antagonists of lysophosphatidic acid receptors.

Isoform-selective antagonists of the lysophosphatidic acid (LPA) G-protein coupled receptors (GPCRs) have important potential uses in cell biology and clinical applications. Novel phosphonothioate and fluoromethylene phosphonate analogues of carbacyclic phosphatidic acid (ccPA) were prepared by chemical synthesis. The pKa values of these amphilic phosphonolipids and the parent cyclic phosphonate were measured titrimetrically using the Yasuda-Shedlovsky extrapolation. The pharmacological properties of these and other ccPA analogues were characterized for LPA receptor (LPAR) subtype-specific agonist and antagonist activity using Ca2+-mobilization assays in RH7777 cells expressing the individual EDG-family GPCRs. In particular, the phosphonothioate ccPA analogue inhibited Ca2+ release through LPA1/LPA3 activation and was an LPA1/LPA3 antagonist. The monofluoromethylene phosphonate ccPA analogue was also a potent LPA1/LPA3 antagonist. In contrast, the difluoromethylene phosphonate ccPA analogue was a weak LPAR agonist, while ccPA itself had neither agonist nor antagonist activity.

Synthesis, structure-activity relationships, and biological evaluation of fatty alcohol phosphates as lysophosphatidic acid receptor ligands, activators of PPARgamma, and inhibitors of autotaxin.

We previously reported that fatty alcohol phosphates (FAP) represent a minimal pharmacophore required to interact with lysophosphatidic acid (LPA) receptors. To improve the activity of the first-generation saturated FAP series, a structure-activity relationship (SAR) study was carried out that includes modifications to the headgroup and alkyl side chain of the FAP pharmacophore. A series of unsaturated (C(10)-C(18)) FAP, headgroup-modified hydrolytically stable saturated (C(10)-C(18)) alkyl phosphonates, and saturated and unsaturated (C(10)-C(18)) thiophosphate analogues were synthesized and evaluated for activity in RH7777 cells transfected with individual LPA(1)(-3) receptors, in PC-3 cells and in human platelets that endogenously express all three isoforms. In this series we identified several LPA(1)- and LPA(3)-selective antagonists with IC(50) values in the nanomolar range. Oleoyl-thiophosphate (15g) was shown to be a pan-agonist, whereas tetradecyl-phosphonate (16c) was identified as a pan-antagonist. These compounds were also tested for the ability to activate the transcription factor PPARgamma, an intracellular receptor for LPA, in CV1 cells transfected with the PPRE-Acox-Rluc reporter gene. All the FAP tested, along with the previously reported LPA GPCR antagonists dioctanoyl glycerol pyrophosphate (2), Ki16425 (6), and the agonist OMPT (3), were activators of PPARgamma. The pan-agonist oleoyl-thiophosphate (15g) and pan-antagonist tetradecyl-phosphonate (16c) mimicked LPA in inhibiting autotaxin, a secreted lysophospholipase D that produces LPA in biological fluids.

Cyclic phosphatidic acid inhibits the secretion of vascular endothelial growth factor from diabetic human coronary artery endothelial cells through peroxisome proliferator-activated receptor gamma.

Atherosclerosis is a disease characterized by building up plaques formation and leads to a potentially serious condition in which arteries are clogged by fatty substances such as cholesterol. Increasing evidence suggests that atherosclerosis is accelerated in type 2 diabetes. Recent study reported that high level of alkyl glycerophosphate (AGP) was accumulated in atherosclerotic lesions. The presence of this phospholipid in mildly oxidized low-density lipoprotein (LDL) is likely to be involved in atherogenesis. It has been reported that the activation of peroxisome proliferator-activated receptor gamma plays a key role in developing atherosclerosis. Our previous result indicates that cyclic phosphatidic acid (cPA), one of bioactive lipids, potently suppresses neointima formation by inhibiting the activation of peroxisome proliferator-activated receptor gamma (PPARγ). However, the detailed mechanism is still unclear. In this study, to elucidate the mechanism of the cPA-PPARγ axis in the coronary artery endothelium, especially in patients with type 2 diabetes, we investigated the proliferation, migration, and secretion of VEGF in human coronary artery endothelial cells from diabetes patients (D-HCAECs). AGP induced cell growth and migration; however, cPA suppressed the AGP-elicited growth and migration in D-HCAECs. Moreover, AGP increased VEGF secretion from D-HCAECs, and this event was attenuated by cPA. Taken together, these results suggest that cPA suppresses VEGF-stimulated growth and migration in D-HCAECs. These findings could be important for regulatory roles of PPARγ and VEGF in the vascular processes associated with diabetes and atherosclerosis.

Autotaxin and LPA1 and LPA5 receptors exert disparate functions in tumor cells versus the host tissue microenvironment in melanoma invasion and metastasis.

UNLABELLED: Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein-coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors. IMPLICATIONS: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis.