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.     

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.     

TNF-alpha promotes LPA1- and LPA3-mediated recruitment of leukocytes in vivo through CXCR2 ligand chemokines

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid present in low concentrations in serum and biological fluids but in high concentrations at sites of inflammation. LPA evokes a variety of cellular responses via binding to and activation of its specific G protein-coupled receptors (GPCR), namely LPA(1-6). Even though LPA is a chemoattractant for inflammatory cells in vitro, such a role for LPA in vivo remains largely unexplored. In the present study, we used the murine air pouch model to study LPA-mediated leukocyte recruitment in vivo using selective LPA receptor agonist/antagonist and LPA(3)-deficient mice. We report that 1) LPA injection into the air pouch induced leukocyte recruitment and that both LPA(1) and LPA(3) were involved in this process; 2) LPA stimulated the release of the pro-inflammatory chemokines keratinocyte-derived chemokine (KC) and interferon-inducible protein-10 (IP-10) in the air pouch; 3) tumor necrosis factor-α (TNF-α) injected into the air pouch prior to LPA strongly potentiated LPA-mediated secretion of cytokines/chemokines, including KC, IL-6, and IP-10, which preceded the enhanced leukocyte influx; and 4) blocking CXCR2 significantly reduced leukocyte infiltration. We suggest that LPA, via LPA(1) and LPA(3) receptors, may play a significant role in inducing and/or sustaining the massive infiltration of leukocytes during inflammation.     

Effects of bisphenol A and 4-nonylphenol on cellular responses through the different induction of LPA receptors in liver epithelial WB-F344 cells

Abstract

Lysophosphatidic acid (LPA) signaling via G protein-coupled transmembrane LPA receptors (LPA₁ to LPA₆) mediates a variety of cellular functions, including cell proliferation, migration, morphogenesis, and differentiation. Recently, we demonstrated that the different induction of LPA receptors by estrogens regulates cell motile activity of rat liver epithelial WB-F344 cells. In the present study, to assess whether endocrine disruptors (EDs) are involved in cellular functions through LPA signaling, we measured cell motile activity and LPA receptor expressions in WB-F344 cells treated with bisphenol A (BPA) and 4-nonylphenol (4-NP). Using quantitative real time RT-PCR analysis, the Lpar1 expression was elevated in BPA-treated cells, whereas the Lpar3 expression was decreased. In contrast, 4-NP increased the Lpar3 expression, but not the Lpar1 and Lpar2. For cell motility assay with a Cell Culture Insert, cell motile activity of BPA-treated cells was significantly lower than that of untreated cells. In contrast, 4-NP markedly enhanced cell motile activity. The effects of BPA and 4-NP on cell motility were inhibited by the Lpar1 or Lpar3 knockdown. These results suggest that BPA and 4-NP may regulate cell motile activity through the different induction of LPA receptors in WB-F344 cells.     

Genetics and cell biology of lysophosphatidic acid receptor-mediated signaling during cortical neurogenesis

Lysophosphatidic acid (LPA) is a small lysophospholipid that signals through G-protein coupled receptors (GPCRs) to mediate diverse cellular responses. Two LPA receptors, LPA(1) and LPA(2), show gene expression profiles in mouse embryonic cerebral cortex, suggesting roles for LPA signaling in cerebral cortical development. Here, we review loss-of-function and gain-of-function models that have been used to examine LPA signaling. Genetic deletion of lpa(1) or both lpa(1) and lpa(2) in mice results in 50-65% neonatal lethality, but not obvious cortical phenotypes in survivors, suggesting that compensatory signaling systems exist for regulating cortical development. A gain-of-function model, approached by increasing receptor activation through exogenous delivery of LPA, shows that LPA signaling regulates cerebral cortical growth and anatomy by affecting proliferation, differentiation and cell survival during embryonic development.     

EDG receptors and hepatic pathophysiology of LPA and S1P: EDG-ology of liver injury

The biological roles of phospholipid growth factors lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been broadly investigated. The cellular effects of LPA and S1P are mediated predominantly via endothelial differentiation gene (EDG) receptors. Yet, the biological significance of LPA, S1P and their EDG receptors in cells of the liver remains unclear. Recent data demonstrate the presence of EDG2 and EDG4 mRNA for LPA receptor in a murine hepatocyte cell line transformed with human TGF-alpha, and in primary mouse hepatocytes. EDG2 receptor protein is expressed in mouse liver, where it appears to be located in nonparenchymal cells. Moreover, we have obtained data suggesting that proliferation of small hepatocyte-progenitors and stem (oval) cells during liver injury is associated with the expression of EDG2 and EDG4 receptors. LPA, and possibly S1P, appear to be essential factors that control proliferation and motility of hepatic stellate cells (HSC) and hepatoma cells. It is proposed that LPA, S1P and their respective EDG receptors play important roles in pathophysiology of chronic liver injury and fibrogenesis. The underlying mechanisms recruited by LPA and S1P in pathogenesis of liver injury remain to be investigated.     

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.     

The role of Toll-like receptors in non-infectious lung injury

The role of Toll-like receptors （TLRs） in pathogen recognition has been expeditiously advanced in recent years. However, investigations into the function of TLRs in non-infectious tissue injury have just begun. Previously, we and others have demonstrated that fragmented hyaluronan （HA） accumulates during tissue injury. CD44 is required to clear HA during tissue injury, and impaired clearance of HA results in unremitting inflammation. Additionally, fragmented HA stimulates the expression of inflammatory genes by inflammatory cells at the injury site. Recently, we identified that HA fragments require both TLR2 and TLR4 to stimulate mouse macrophages to produce inflammatory chemokines and cytokines. In a non-infectious lung injury model, mice deficient in both TLR2 and TLR4 show an impaired transepithelial migration of inflammatory cells, increased tissue injury, elevated lung epithelial cell apoptosis, and decreased survival. Lung epithelial cell overexpression of high molecular mass HA protected mice against acute lung injury and apoptosis, in part through TLR-dependent basal activation of NF-κB. The exaggerated injury in TLR2 and TLR4 deficient mice appears to be due to impaired HA-TLR interactions on epithelial cells. These studies identify that host matrix component HA and TLR interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity, and promote recovery from acute lung injury.     

LPA induces osteoblast differentiation through interplay of two receptors: LPA1 and LPA4

The bioactive phospholipid, lysophosphatidic acid (LPA), acting through at least five distinct receptors LPA1–LPA5, plays important roles in numerous biological processes. Here we report that LPA induces osteoblastic differentiation of human mesenchymal stem cells hMSC‐TERT. We find that hMSC‐TERT mostly express two LPA receptors, LPA1 and LPA4, and undergo osteoblastic differentiation in serum‐containing medium. Inhibition of LPA1 with Ki16425 completely abrogates osteogenesis, indicating that this process is mediated by LPA in the serum through activation of LPA1. In contrast to LPA1, down‐regulation of LPA4 expression with shRNA significantly increases osteogenesis, suggesting that this receptor normally exerts negative effects on differentiation. Mechanistically, we find that in hMSC‐TERT, LPA induces a rise in both cAMP and Ca²⁺. The rise in Ca²⁺ is completely abolished by Ki16425, whereas LPA‐mediated cAMP increase is not sensitive to Ki16425. To test if LPA signaling pathways controlling osteogenesis in vitro translate into animal physiology, we evaluated the bones of LPA4‐deficient mice. Consistent with the ability of LPA4 to inhibit osteoblastic differentiation of stem cells, LPA4‐deficient mice have increased trabecular bone volume, number, and thickness. J. Cell. Biochem. 109: 794–800, 2010. © 2010 Wiley‐Liss, Inc.     

LPA in neural cell development

Lysophosphatidic acid (LPA) elicits diverse cellular responses through cell surface LPA receptors in nervous system-derived cells and cell lines. The developing nervous system is one of the major loci for LPA receptor expression. Recent studies have also revealed that metabolic pathways of LPA are present in the nervous system. A growing body of literature suggests a crucial role for LPA in neuronal development processes, including neurogenesis, neuronal migration, neuritogenesis, and myelination.     

Expression of Lysophosphatidic Acid Receptor in Rat Astrocytes: Mitogenic Effect and Expression of Neurotrophic Genes

Lysophosphatidic acid (LPA) is a phospholipid mediator with a variety of biological activities. It remains unknown, however, which cells in the brain express the LPA receptor. The present study was undertaken to identify cells in the rat brain expressing functional LPA receptors, and to explore biological roles of LPA in these cells. We found that the LPA receptor was most dominantly expressed in rat astrocytes, determined by LPA-induced Ca²⁺ imaging, and by Northern blot analyses. LPA induced a mitogenic response and expression of immediate early genes in astrocytes, through pertussis-toxin sensitive G-protein(s). LPA also stimulated the expression of various cytokine genes, including nerve growth factor, interleukin (IL)-1, IL-3 and IL-6. Thus, astrocytes are the major target of LPA in the brain. We propose that LPA may play important roles in neuronal development, gliosis and wound-healing process in the brain.     

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.     

Autotaxin induces lung epithelial cell migration through lysoPLD activity-dependent and -independent pathways

Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodelling and repair after lung injury. In the present paper we hypothesize that secreted ATX (autotaxin), which exhibits lysoPLD (lysophospholipase D) activity, stimulates lung epithelial cell migration through LPA (lysophosphatidic acid) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag overexpressed conditional medium had higher LPA levels compared with control medium and stimulated cell migration through G(αi)-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKC (protein kinase C) δ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to the LPA receptor and integrin β4 complex on A549 cell surface. Finally, intratracheal administration of LPS (lipopolysaccharide) into the mouse airway induced ATX release and LPA production in BAL (bronchoalveolar lavage) fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodelling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated association of ATX with the epithelial cell-surface LPA receptor and integrin β4.     

Lysophosphatidic acid‐triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary‐like structures, migration, and proliferation. The HTR‐8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR‐8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein‐coupled receptors, LPA₁ and LPA₃, were expressed in HTR‐8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA₃. In addition, cyclooxygenase‐2 and inducible nitric oxide synthase pathways participated in LPA‐stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase‐2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration‐dependent manner. Finally, we observed that cyclooxygenase‐2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPA‐triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal‐fetal interface.     

Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA₁–LPA₆) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA₁ inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA₅ in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA₁ and LPA₅ on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA₅ may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA₁.     

The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak

Aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses have yet to be fully identified. We show that lysophosphatidic acid levels increase in bronchoalveolar lavage fluid following lung injury in the bleomycin model of pulmonary fibrosis, and that mice lacking one of its receptors, LPA1, are markedly protected from fibrosis and mortality in this model. The absence of LPA1 led to reduced fibroblast recruitment and vascular leak, two responses that may be excessive when injury leads to fibrosis rather than to repair, whereas leukocyte recruitment was preserved during the first week after injury. In persons with idiopathic pulmonary fibrosis, lysophosphatidic acid levels in bronchoalveolar lavage fluid were also increased, and inhibition of LPA1 markedly reduced fibroblast responses to the chemotactic activity of this fluid. LPA1 therefore represents a new therapeutic target for diseases in which aberrant responses to injury contribute to fibrosis, such as idiopathic pulmonary fibrosis.     

Biological roles of lysophosphatidic acid signaling through its production by autotaxin

Lysophosphatidic acid (LPA) exhibits a wide variety of biological functions as a bio-active lysophospholipid through G-protein-coupled receptors specific to LPA. Currently at least six LPA receptors are identified, named LPA₁ to LPA₆, while the existence of other LPA receptors has been suggested. From studies on knockout mice and hereditary diseases of these LPA receptors, it is now clear that LPA is involved in various biological processes including brain development and embryo implantation, as well as patho-physiological conditions including neuropathic pain and pulmonary and renal fibrosis. Unlike sphingosine 1-phosphate, a structurally similar bio-active lysophospholipid to LPA and produced intracellularly, LPA is produced by multiple extracellular degradative routes. A plasma enzyme called autotaxin (ATX) is responsible for the most of LPA production in our bodies. ATX converts lysophospholipids such as lysophosphatidylcholine to LPA by its lysophospholipase D activity. Recent studies on ATX have revealed new aspects of LPA. In this review, we highlight recent advances in our understanding of LPA functions and several aspects of ATX, including its activity, expression, structure, biochemical properties, the mechanism by which it stimulates cell motility and its pahto-physiological function through LPA production.