Current progress in non-Edg family LPA receptor research

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.     

Role of LPA4/p2y9/GPR23 in negative regulation of cell motility

Lysophosphatidic acid (LPA) is a ligand of multiple G protein–coupled receptors. The LPA_1–3 receptors are members of the endothelial cell differentiation gene (Edg) family. LPA₄/p2y9/GPR23, a member of the purinergic receptor family, and recently identified LPA₅/GPR92 and p2y5 are structurally distant from the canonical Edg LPA receptors. Here we report targeted disruption of lpa₄ in mice. Although LPA₄-deficient mice displayed no apparent abnormalities, LPA₄-deficient mouse embryonic fibroblasts (MEFs) were hypersensitive to LPA-induced cell migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase pathway by LPA₄, LPA₄ deficiency potentiated Akt and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA₄ converted LPA₄-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA₄ strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA₁ in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA₄ attenuated LPA₁-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA₄ is a suppressor of LPA-dependent cell migration and invasion in contrast to the motility-stimulating Edg LPA receptors.     

The PDZ Protein GIPC Regulates Trafficking of the LPA1 Receptor from APPL Signaling Endosomes and Attenuates the Cell’s Response to LPA

Lysophosphatidic acid (LPA) mediates diverse cellular responses through the activation of at least six LPA receptors – LPA_1–6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA₁ contains a PDZ binding motif (SVV) identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA₁ but not that of other LPA receptors. LPA₁ colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA₁ to EEA1 early endosomes and promoted LPA₁ mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA₁ and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.     

Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3

Results

The lysophosphatidic acid receptors LPA₁, LPA₂, and LPA₃ were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA₁- and LPA₃-mediated effect, whereas that mediated by LPA₂ was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA₂ receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA_1–3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA_1–3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA₂ subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.

Conclusion

Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.     

EGF and angiotensin II modulate lysophosphatidic acid LPA1 receptor function and phosphorylation state

Background

Lysophosphatidic acid (LPA) is a local mediator that exerts its actions through G protein coupled receptors. Knowledge on the regulation of such receptors is scarce to date. Here we show that bidirectional cross-talk exits between LPA₁ and EGF receptors.

Methods

C9 cells expressing LPA₁ receptor fussed to the enhanced green fluorescent protein were used. We studied intracellular calcium concentration, Akt/PKB phosphorylation, LPA₁ and EGF receptor phosphorylation.

Results

EGF diminished LPA-mediated intracellular calcium response and induced LPA₁ receptor phosphorylation, which was sensitive to protein kinase C inhibitors. Angiotensin II and LPA induced EGF receptor transactivation as evidenced by Akt/PKB phosphorylation through metalloproteinase-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF receptors. This process was found to be of major importance in angiotensin II-induced LPA₁ receptor phosphorylation. Attempts to define a role for EGF receptor transactivation in homologous LPA₁ receptor desensitization and phosphorylation suggested that G protein-coupled receptor kinases are the major players in this process, overshadowing other events.

Conclusions

EGF receptors and LPA₁ receptors are engaged in an intense liaison, in that EGF receptors are capable of modulating LPA₁ receptor function through phosphorylation cascades. EGF transactivation plays a dual role: it mediates some LPA actions, and it modulates LPA₁ receptor function in inhibitory fashion.

General significance

EGF and LPA receptors coexist in many cell types and play key roles in maintaining the delicate equilibrium that we call health and in the pathogenesis of many diseases. The intense cross-talk described here has important physiological and pathophysiological implications.     

Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): Metabolically stabilized LPA receptor agonists

We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six LPA receptors (LPA_1–6) using a recently developed TGFα shedding assay. In general, the sn-2 radyl OMPT analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate (sn-1 OMPT) analogs for LPA_1–6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA₅ and LPA₆ agonists. The availability of sn-2 radyl OPMT analogs further refines the structure–activity relationships for ligand–receptor interactions for this class of GPCRs.     

Identifying lysophosphatidic acid receptor subtype 1 (LPA1) as a novel factor to modulate microglial activation and their TNF-α production by activating ERK1/2

Microglia regulate immune responses in the brain, and their activation is key to the pathogenesis of diverse neurological diseases. Receptor-mediated lysophosphatidic acid (LPA) signaling has been known to regulate microglial biology, but it is still unclear which receptor subtypes guide the biology, particularly, microglial activation. Here, we investigated the pathogenic aspects of LPA receptor subtype 1 (LPA₁) in microglial activation using a systemic lipopolysaccharide (LPS) administration-induced septic mouse model in vivo and LPS-stimulated rat primary microglia in vitro. LPA₁ knockdown in the brain with its specific shRNA lentivirus attenuated the sepsis-induced microglia activation, morphological transformation, and proliferation. LPA₁ knockdown also resulted in the downregulation of TNF-α, at both mRNA and protein levels in septic brains, but not IL-1β or IL-6. In rat primary microglia, genetic or pharmacological blockade of LPA₁ attenuated gene upregulation and secretion of TNF-α in LPS-stimulated cells. In particular, the latter was associated with the suppressed TNF-α converting enzyme (TACE) activity. We reaffirmed these biological aspects using a BV2 microglial cell line in which LPA₁ expression was negligible. LPA₁ overexpression in BV2 cells led to significant increments in TNF-α production upon stimulation with LPS, whereas inhibiting LPA₁ reversed the production. We further identified ERK1/2, but not p38 MAPK or Akt, as the underlying effector pathway after LPA₁ activation in both septic brains and stimulated microglia. The current findings of the novel role of LPA₁ in microglial activation along with its mechanistic aspects could be applied to understanding the pathogenesis of diverse neurological diseases that involve microglial activation.     

Different effects on cell proliferation and migration abilities of endothelial cells by LPA1 and LPA3 in mammary tumor FM3A cells

Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors and mediate a variety of cellular responses through the binding of LPA. So far, six types of LPA receptors (LPA receptor-1 (LPA₁) to LPA₆) have been identified. Recently, it has been demonstrated that each LPA receptor has opposite effects on malignant property of cancer cells. In this study, to evaluate an involvement of LPA receptors on angiogenic process in mammary tumor cells, we generated Lpar1- and Lpar3-expressing (FM3A-a1 and FM3A-a3A9, respectively) cells from FM3A cells, and investigated the effects on cell proliferation and migration abilities of endothelial F-2 cells by those cells. In Vegf-A and Vegf-C genes, FM3A-a1 cells indicated high expression and FM3A-a3A9 cells showed low expression, compared with control cells. When F-2 cells were cultured with a supernatant from FM3A-a1 cells, the cell growth rate and migration ability of F-2 cells was significantly higher than control cells. By contrast, a supernatant from FM3A-a3A9 cells significantly inhibited those abilities of F-2 cells. These results suggest that LPA₁ and LPA₃ may play opposite roles on the regulation of endothelial cells in mouse mammary tumor FM3A cells.     

Lysophosphatidic acid induces Ca2+ mobilization and c-Myc expression in mouse embryonic stem cells via the phospholipase C pathway

Embryonic stem cells (ESC) are pluripotent and could be maintained in vitro in a self-renewing state indefinitely, at the same time preserving their potential to differentiate towards more specific lineages. Despite the progress in the field, the complex network of signalling cascades involved in the maintenance of the self-renewing and pluripotent state remains not fully understood. In the present study, we have investigated the role of lysophosphatidic acid (LPA), a potent mitogen present in serum, in Ca²⁺ signalling and early gene activation in mouse ESC (mESC). In these cells, we detected the expression of the G-protein coupled LPA receptor subtypes LPA₁, LPA₂ and LPA₃. Using fluorescence Ca²⁺ imaging techniques, we showed that LPA induced an increase in intracellular Ca²⁺ concentration. This increase was also observed in the absence of extracellular Ca²⁺, suggesting the involvement of internal stores. Pre-treatment with BAPTA-AM, thapsigargin or U-73122 efficiently blocked this Ca²⁺ release, indicating that LPA was evoking Ca²⁺ mobilization from the endoplasmic reticulum via the phospholipase C (PLC) pathway. Interestingly, this signalling cascade initiated by LPA was involved in inducing the expression of the Ca²⁺-dependent early response gene c-myc, a key gene implicated in ESC self-renewal and pluripotency. Additionally, LPA increased the proliferation rate of mESC. Our findings therefore outline the physiological role of LPA in mESC.     

Role of lysophosphatidic acid receptor LPA2 in the development of allergic airway inflammation in a murine model of asthma

Background

Lysophosphatidic acid (LPA) plays a critical role in airway inflammation through G protein-coupled LPA receptors (LPA_1-3). We have demonstrated that LPA induced cytokine and lipid mediator release in human bronchial epithelial cells. Here we provide evidence for the role of LPA and LPA receptors in Th2-dominant airway inflammation.

Methods

Wild type, LPA₁ heterozygous knockout mice (LPA₁^+/-), and LPA₂ heterozygous knockout mice (LPA₂^+/-) were sensitized with inactivated Schistosoma mansoni eggs and local antigenic challenge with Schistosoma mansoni soluble egg Ag (SEA) in the lungs. Bronchoalveolar larvage (BAL) fluids and lung tissues were collected for analysis of inflammatory responses. Further, tracheal epithelial cells were isolated and challenged with LPA.

Results

BAL fluids from Schistosoma mansoni egg-sensitized and challenged wild type mice (4 days of challenge) showed increase of LPA level (~2.8 fold), compared to control mice. LPA₂^+/- mice, but not LPA₁^+/- mice, exposed to Schistosoma mansoni egg revealed significantly reduced cell numbers and eosinophils in BAL fluids, compared to challenged wild type mice. Both LPA₂^+/- and LPA₁^+/- mice showed decreases in bronchial goblet cells. LPA₂^+/- mice, but not LPA₁^+/- mice showed the decreases in prostaglandin E2 (PGE2) and LPA levels in BAL fluids after SEA challenge. The PGE2 production by LPA was reduced in isolated tracheal epithelial cells from LPA₂^+/- mice. These results suggest that LPA and LPA receptors are involved in Schistosoma mansoni egg-mediated inflammation and further studies are proposed to understand the role of LPA and LPA receptors in the inflammatory process.     

Ethionine regulates cell motile activity through LPA receptor-3 in liver epithelial WB-F344 cells

Lysophosphatidic acid (LPA) receptors (LPA₁ to LPA₆) indicate a variety of cellular responses, such as cell proliferation, migration, differentiation, and morphogenesis. However, the role of each LPA receptor is not functionally equivalent. Ethionine, an ethyl analog of methionine, is well known to be one of the potent liver carcinogens in rats. In this study, to assess whether ethionine may regulate cell motile activity through LPA receptors, rat liver epithelial (WB-F344) cells were treated with ethionine for 48 h. In cell motility assay with a cell culture insert, the treatment of ethionine at 1.0 and 10 μM enhanced significantly high cell motile activity, compared with untreated cells. The expression levels of LPA receptor genes in cells treated with ethionine were measured by quantitative real time RT-PCR analysis. The expression of the Lpar3 gene in ethionine-treated cells was significantly higher than that in untreated cells. Furthermore, to confirm an involvement of LPA₃ on cell motility increased by ethionine, the Lpar3 knockdown cells were also used. The cell motile activity by ethionine was completely suppressed in the Lpar3 knockdown cells. These results suggest that LPA signaling through LPA₃ may be involved in cell motile activity stimulated by ethionine in WB-F344 cells.     

Identification of Heparin-Binding EGF-Like Growth Factor (HB-EGF) as a Biomarker for Lysophosphatidic Acid Receptor Type 1 (LPA1) Activation in Human Breast and Prostate Cancers

Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA_1–6). LPA receptor type 1 (LPA₁) signaling influences the pathophysiology of many diseases including cancer, obesity, rheumatoid arthritis, as well as lung, liver and kidney fibrosis. Therefore, LPA₁ is an attractive therapeutic target. However, most mammalian cells co-express multiple LPA receptors whose co-activation impairs the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. LPA₁ is known to induce IL-6 and IL-8 secretion, as also do LPA₂ and LPA₃. In this work, we first determined the LPA induced early-gene expression profile in three unrelated human cancer cell lines expressing different patterns of LPA receptors (PC3: LPA_1,2,3,6; MDA-MB-231: LPA_1,2; MCF-7: LPA_2,6). Among the set of genes upregulated by LPA only in LPA₁-expressing cells, we validated by QPCR and ELISA that upregulation of heparin-binding EGF-like growth factor (HB-EGF) was inhibited by LPA_1–3 antagonists (Ki16425, Debio0719). Upregulation and downregulation of HB-EGF mRNA was confirmed in vitro in human MDA-B02 breast cancer cells stably overexpressing LPA₁ (MDA-B02/LPA₁) and downregulated for LPA₁ (MDA-B02/shLPA₁), respectively. At a clinical level, we quantified the expression of LPA₁ and HB-EGF by QPCR in primary tumors of a cohort of 234 breast cancer patients and found a significantly higher expression of HB-EGF in breast tumors expressing high levels of LPA₁. We also generated human xenograph prostate tumors in mice injected with PC3 cells and found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. All together our results demonstrate that HB-EGF is a new and relevant biomarker with potentially high value in quantifying LPA₁ activation state in patients receiving anti-LPA₁ therapies.     

Activation of fibroblast-like synoviocytes derived from rheumatoid arthritis via lysophosphatidic acid–lysophosphatidic acid receptor 1 cascade

Introduction

Lysophosphatidic acid (LPA) is a bioactive lipid that binds to G protein–coupled receptors (LPA_1–6). Recently, we reported that abrogation of LPA receptor 1 (LPA₁) ameliorated murine collagen-induced arthritis, probably via inhibition of inflammatory cell migration, Th17 differentiation and osteoclastogenesis. In this study, we examined the importance of the LPA–LPA₁ axis in cell proliferation, cytokine/chemokine production and lymphocyte transmigration in fibroblast-like synoviocytes (FLSs) obtained from the synovial tissues of rheumatoid arthritis (RA) patients.

Methods

FLSs were prepared from synovial tissues of RA patients. Expression of LPA_1–6 was examined by quantitative real-time RT-PCR. Cell surface LPA₁ expression was analyzed by flow cytometry. Cell proliferation was analyzed using a cell-counting kit. Production of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), chemokine (C-C motif) ligand 2 (CCL2), metalloproteinase 3 (MMP-3) and chemokine (C-X-C motif) ligand 12 (CXCL12) was measured by enzyme-linked immunosorbent assay. Pseudoemperipolesis was evaluated using a coculture of RA FLSs and T or B cells. Cell motility was examined by scrape motility assay. Expression of adhesion molecules was determined by flow cytometry.

Results

The expression of LPA₁ mRNA and cell surface LPA₁ was higher in RA FLSs than in FLSs from osteoarthritis tissue. Stimulation with LPA enhanced the proliferation of RA FLSs and the production of IL-6, VEGF, CCL2 and MMP-3 by FLSs, which were suppressed by an LPA₁ inhibitor (LA-01). Ki16425, another LPA₁ antagonist, also suppressed IL-6 production by LPA-stimulated RA FLSs. However, the production of CXCL12 was not altered by stimulation with LPA. LPA induced the pseudoemperipolesis of T and B cells cocultured with RA FLSs, which was suppressed by LPA₁ inhibition. In addition, LPA enhanced the migration of RA FLSs and expression of vascular cell adhesion molecule and intercellular adhesion molecule on RA FLSs, which were also inhibited by an LPA₁ antagonist.

Conclusions

Collectively, these results indicate that LPA–LPA₁ signaling contributes to the activation of RA FLSs.     

Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA₄. Here we report that p2y5 is a novel LPA receptor coupling to the G₁₃-Rho signaling pathway. “LPA receptor-null” RH7777 and B103 cells exogenously expressing p2y5 showed [³H]LPA binding, LPA-induced [³⁵S]guanosine 5′-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and G_s/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA₆.     

LPA1/3 signaling mediates tumor lymphangiogenesis through promoting CRT expression in prostate cancer

Lysophosphatidic acid (LPA) is a bioactive lipid growth factor which is present in high levels in serum and platelets. LPA binds to its specific G-protein-coupled receptors, including LPA₁ to LPA₆, thereby regulating various physiological functions, including cancer growth, angiogenesis, and lymphangiogenesis. Our previous study showed that LPA promotes the expression of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C in prostate cancer (PCa) cells. Interestingly, LPA has been shown to regulate the expression of calreticulin (CRT), a multifunctional chaperone protein, but the roles of CRT in PCa progression remain unclear. Here we investigated the involvement of CRT in LPA-mediated VEGF-C expression and lymphangiogenesis in PCa. Knockdown of CRT significantly reduced LPA-induced VEGF-C expression in PC-3 cells. Moreover, LPA promoted CRT expression through LPA receptors LPA₁ and LPA₃, reactive oxygen species (ROS) production, and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Tumor-xenografted mouse experiments further showed that CRT knockdown suppressed tumor growth and lymphangiogenesis. Notably, clinical evidence indicated that the LPA-producing enzyme autotaxin (ATX) is related to CRT and that CRT level is highly associated with lymphatic vessel density and VEGF-C expression. Interestingly, the pharmacological antagonist of LPA receptors significantly reduced the lymphatic vessel density in tumor and lymph node metastasis in tumor-bearing nude mice. Together, our results demonstrated that CRT is critical in PCa progression through the mediation of LPA-induced VEGF-C expression, implying that targeting the LPA signaling axis is a potential therapeutic strategy for PCa.     

Promotion of cell-invasive activity through the induction of LPA receptor-1 in pancreatic cancer cells

Abstract

Lysophosphatidic acid (LPA) is a simple biological lipid and mediates several biological functions with LPA receptors (LPA₁ to LPA₆). In the present study, to assess whether LPA receptors promote cell-invasive activity of pancreatic cancer cells, highly invasion PANC-R9 cells were established from PANC-1 cells, using Matrigel-coated Cell Culture Insert. The cell-invasive activity of PANC-R9 cells was shown to be approximately 15 times higher than that of PANC-1 cells. LPAR1 expression level was markedly elevated in PANC-R9 cells in comparison with PANC-1 cells, while LPAR3 expression level was reduced. The cell-invasive activity of PANC-R9 cells was enhanced by LPA, but LPA had no impact on PANC-1 cell invasion. Before initiation of the cell invasion assay, PANC-R9 cells were pretreated with dioctanoylglycerol pyrophosphate (DGPP), an antagonist of LPA₁/LPA₃. The invasive activity of PANC-R9 cells was markedly suppressed by DGPP. Autotaxin (ATX) is a key enzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to LPA. ATX expression level was elevated in PANC-R9 cells compared with PANC-1 cells. In the presence of LPC, the cell motile activity of PANC-R9 cells was markedly stimulated. In contrast, LPC did not affect the cell motile activity of PANC-1 cells. PANC-R9 cell motility was inhibited by an ATX inhibitor, PF-8380. These results suggest that LPA signaling via LPA₁ is a potent molecular target for the regulation of tumor progression in PANC-1 cells.     

A novel function of hepatocyte growth factor in the activation of checkpoint kinase 1 phosphorylation in colon cancer cells

Lysophosphatidic acid (LPA) is a simple biophysical lipid which interacts with at least six subtypes of G protein-coupled LPA receptors (LPA₁–LPA₆). In cancer cells, LPA signaling via LPA receptors is involved in the regulation of malignant properties, such as cell growth, motility, and invasion. The aim of this study was to assess whether LPA receptors regulate cellular functions of fibrosarcoma cells treated with anticancer drug. HT1080 cells were maintained by the stepwise treatment of cisplatin (CDDP) at a range of 0.01 to 1.0 µM for approximately 6 months. The cell motile and invasive activities of long-term CDDP-treated (HT-CDDP) cells were significantly stimulated by LPA treatment, while HT-CDDP cells in the static state showed the low cell motile and invasive activities in comparison with HT1080 cells. Since the expression level of LPAR2 gene was markedly elevated in HT-CDDP cells, LPA₂ knockdown cells were generated from HT-CDDP cells. The cell motile and invasive activities of HT-CDDP cells were reduced by LPA₂ knockdown. In colony assay, large-sized colonies formed by long-term CDDP treatment were suppressed by LPA₂ knockdown. In addition, LPA₂ knockdown cells reduced LPA production by autotaxin (ATX), correlating with ATX expression level. These results suggest that LPA signaling via LPA₂ may play an important role in the regulation of cellular functions in HT1080 cells treated with CDDP.