Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish.

Functional lysophosphatidic acid receptors expressed in Oryzias latipes.

Lysophosphatidic acid (LPA) signaling is known to play biological and pathophysiological roles in many types of animals. Medaka (Oryzias latipes) is an experimental fish that can be easily maintained, propagated, and analyzed, and whose genome has been completely sequenced. However, there is limited information available regarding medaka LPA receptors. Here, using information from the medaka genome database, we examine the genomic structures, expression, and functions of six LPA receptor genes, Lpar1-Lpar6. Our analyses reveal that the genomic structures of Lpar1 and Lpar4 are different from those deduced from the database. Functional analyses using a heterologous expression system demonstrate that all medaka LPA receptors except for LPA5b respond to LPA treatment with cytoskeletal changes. These findings provide useful information on the structure and function of medaka LPA receptor genes, and identify medaka as a useful experimental model for exploration of the biological significance of LPA signaling.

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 - LPA(1), LPA(2), and LPA(3). 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 LPA(4), LPA(5), and LPA(6) 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.

Non-Edg family LPA receptors: the cutting edge of LPA research.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator with diverse physiological and pathological actions on many types of cells. Originally, LPA was thought to elicit its biological functions through three subtypes of endothelial differentiation gene (Edg) family G protein-coupled receptors (LPA1, LPA2 and LPA3) until our group identified a fourth subtype, LPA4. The discovery of this receptor, which is structurally distinct from the Edg family LPA receptors, led to the identification of two additional LPA receptors, LPA5 and LPA6, homologous to LPA4. These 'non-Edg family' LPA receptors now provide a new framework for understanding the diverse functions of LPA, including vascular development, platelet activation and hair growth. In this review, we summarize the identification, intracellular signalling and biological functions of this novel cluster of LPA receptors.

The lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptor gene families: cloning and comparative expression analysis in Xenopus laevis.

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are endogenous bioactive lipids which mediate a variety of biological cell responses such as cell proliferation, migration, differentiation and apoptosis. Their actions are mediated by binding to the G-protein-coupled endothelial differentiation gene (Edg) receptor subfamily, referred to as S1P1-5 and LPA1-5, and regulate a variety of signalling pathways involved in numerous physiological processes and pathological conditions. Their importance during embryogenesis has been demonstrated by the generation of knock-out mice and specific roles have been assigned to these receptors. However, potential functional redundancy and the lethality of some mutants have complicated functional analysis in these models. Here we report the cloning of the S1P and LPA receptors in Xenopus laevis and tropicalis. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. We have conducted a comparative expression analysis of these receptors during development and in the adult frog, by both RT-PCR and whole mount in situ hybridisation. In particular, we show that S1P1, 2 and 5 display distinct embryonic specific expression patterns, suggesting potentially different developmental roles for these receptors, and therefore for their ligands, during amphibian embryogenesis.

LPA(3), a unique G protein-coupled receptor for lysophosphatidic acid.

Lysophosphatidic acid (LPA; 1- or 2-acyl-sn-glycerol-3-phosphate) is a phospholipid that is involved in numerous normal physiological and pathological processes such as brain development, blood vessel formation, embryo implantation, hair growth, neuropathic pain, lung fibrosis and colon cancer. Most of these functions are mediated by G protein-coupled receptors (GPCRs) specific to LPA. So far, six GPCRs for LPA have been identified: LPA(1)/Edg2, LPA(2)/Edg4, LPA(3)/Edg7, LPA(4)/GPR23/P2Y9, LPA(5)/GPR92 and LPA(6)/P2Y5. An intracellular target of LPA has also been proposed. Among the LPA receptors, LPA(3) is unique in that it is activated significantly by a specific form of LPA (2-acyl LPA with unsaturated fatty acids) and is expressed in a limited number of tissues such as the reproductive organs. Recent studies have shown that LPA(3)-mediated LPA signaling is essential for proper embryo implantation and have revealed an unexpected genetic linkage between LPA and prostaglandin signaling. Here we review recent advances in the study of LPA(3), especially studies using LPA(3)-deficient mice. In addition, we focus on the agonists and antagonists that are specific to each LPA receptor as important tools for the functional study of LPA signaling.

LPA receptors: subtypes and biological actions.

Lysophosphatidic acid (LPA) is a small, ubiquitous phospholipid that acts as an extracellular signaling molecule by binding to and activating at least five known G protein-coupled receptors (GPCRs): LPA(1)-LPA(5). They are encoded by distinct genes named LPAR1-LPAR5 in humans and Lpar1-Lpar5 in mice. The biological roles of LPA are diverse and include developmental, physiological, and pathophysiological effects. This diversity is mediated by broad and overlapping expression patterns and multiple downstream signaling pathways activated by cognate LPA receptors. Studies using cloned receptors and genetic knockout mice have been instrumental in uncovering the significance of this signaling system, notably involving basic cellular processes as well as multiple organ systems such as the nervous system. This has further provided valuable proof-of-concept data to support LPA receptors and LPA metabolic enzymes as targets for the treatment of medically important diseases that include neuropsychiatric disorders, neuropathic pain, infertility, cardiovascular disease, inflammation, fibrosis, and cancer.

Different expressions and DNA methylation patterns of lysophosphatidic acid receptor genes in mouse tumor cells.

OBJECTIVE: Lysophosphatidic acid (LPA) receptors act as several biological effectors through LPA, which is a bioactive phospholipid. Recently, aberrant expressions of LPA receptor genes due to DNA methylation have been detected in several tumor cells. In this study, we measured expression levels and DNA methylation status of LPA receptor genes in mouse tumor cells, LL/2 lung carcinoma, B16F0 melanoma, FM3A mammary carcinoma and L1210 leukemia cells, compared with normal tissues. METHODS: Total RNAs were extracted and RT-PCR analysis was performed. For DNA methylation status, bisulfite sequencing analysis was carried out, comparing outcomes with other tumor cells and normal tissues. RESULTS: The expressions of LPA1 gene were shown in LL/2, but not in B16F0, FM3A and L1210 cells. While the LPA2 gene was expressed in all 4 tumor cells, the LPA3 gene was unexpressed in them. The LPA1 and LPA3 unexpressed cells were highly methylated, although normal tissues were all unmethylated. The DNA methylation status was correlated with gene expression levels in cancer cells. CONCLUSION: The present results demonstrate that DNA methylation patterns of LPA receptor genes are dependent on cancer cell types, suggesting that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention.

Lysophosphatidic Acid signals through specific lysophosphatidic Acid receptor subtypes to control key regenerative responses of human gingival and periodontal ligament fibroblasts.

BACKGROUND: We showed that the pluripotent platelet growth factor and mediator lysophosphatidic acid (LPA) controls key regenerative responses of human gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) and positively modulates their responses to platelet-derived growth factor (PDGF). This study determined which LPA receptor (LPAR) subtype(s) LPA signals through to stimulate mitogenic extracellular signal-regulated kinase (ERK) 1/2 signaling and chemotaxis and to elicit intracellular Ca(2+) increases in GFs and PDLFs because many healing responses are calcium-dependent. METHODS: Activation of mitogen-activated protein kinase was determined using Western blotting with an antibody to phosphorylated ERK1/2. Migration responses were measured using a microchemotaxis chamber. GF and PDLF intracellular Ca(2+) mobilization responses to multiple LPA species and LPAR subtype-specific agonists were measured by using a cell-permeable fluorescent Ca(2+) indicator dye. RESULTS: LPA stimulated ERK1/2 phosphorylation via LPA(1)(-3). For GFs, LPA(1) preferentially elicited chemotaxis, and LPA(1-3) for PDLFs, as confirmed using subtype-specific agonists. Elevation of intracellular calcium seems to be mediated through LPA(1) and LPA(3), with little, if any, contribution from LPA(2). CONCLUSIONS: To the best of our knowledge, this study provides the first evidence that LPA signals through specific LPAR subtypes to stimulate human oral fibroblast regenerative responses. These data, in conjunction with our previous findings showing that LPA modulates GF and PDLF responses to PDGF, suggest that LPA is a factor of emerging importance to oral wound healing.

Toward the three-dimensional structure and lysophosphatidic acid binding characteristics of the LPA(4)/p2y(9)/GPR23 receptor: a homology modeling study.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that initiates a broad array of biological processes, including those involved in cell proliferation, survival and migration via activation of specific G protein-coupled receptors located on the cell surface. To date, at least five receptor subtypes (LPA(1-5)) have been identified. The LPA(1-3) receptors are members of the endothelial cell differentiation gene (Edg) family. LPA(4), a member of the purinergic receptor family, and the recently identified LPA(5) are structurally distant from the canonical Edg LPA(1-3) receptors. LPA(4) and LPA(5) are linked to G(q), G(12/13) and G(s) but not G(i), while LPA(1-3) all couple to G(i) in addition to G(q) and G(12/13). There is also evidence that LPA(4) and LPA(5) are functionally different from the Edg LPA receptors. Computational modeling has provided useful information on the structure-activity relationship (SAR) of the Edg LPA receptors. In this work, we focus on the initial analysis of the structural and ligand-binding properties of LPA(4), a prototype non-Edg LPA receptor. Three homology models of the LPA(4) receptor were developed based on the X-ray crystal structures of the ground state and photoactivated bovine rhodopsin and the recently determined human beta(2)-adrenergic receptor. Docking studies of LPA in the homology models were then conducted, and plausible LPA binding loci were explored. Based on these analyses, LPA is predicted to bind to LPA(4) in an orientation similar to that reported for LPA(1-3), but through a different network of hydrogen bonds. In LPA(1-3), the ligand polar head group is reported to interact with residues at positions 3.28, 3.29 and 7.36, whereas three non-conserved amino acid residues, S114(3.28), T187(EL2) and Y265(6.51), are predicted to interact with the polar head group in the LPA(4) receptor models.

LPA1 is essential for lymphatic vessel development in zebrafish.

Lysophosphatidic acid (LPA) has long been implicated in regulating vascular development via endothelial cell-expressed G protein-coupled receptors. However, because of a lack of notable vascular defects reported in LPA receptor knockout mouse studies, the regulation of vasculature by LPA receptors in vivo is still uncertain. Using zebrafish as a model, we studied the gene expression patterns and functions of an LPA receptor, LPA(1), during embryonic development, in particular, vascular formation. Whole-mount in situ hybridization experiments revealed that zebrafish lpa(1) (zlpa(1)) was ubiquitously expressed early in development, and its expression domains were later localized to the head region and the vicinity of the dorsal aorta. The expression of zlpa(1) surrounding the dorsal aorta suggests its role in vasculature development. Knocking down of zLPA(1) by injecting morpholino (MO) oligonucleotides at 0.625-1.25 ng per embryo resulted in the absence of thoracic duct and edema in pericardial sac and trunk in a dose-dependent manner. These zlpa(1)-MO-resulted defects could be specifically rescued by ectopic expression of zlpa(1). In addition, overexpression of vegf-c, a well-known lymphangiogenic factor, also partially ameliorated the inhibition of thoracic duct development. Taken together, these results demonstrate that LPA(1) is necessary for lymphatic vessel formation during embryonic development in zebrafish.

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor.

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y(10) receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y(10) receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca(2+) increases in the CHO cells stably expressing the P2Y(10) fused with a G(16alpha) protein. These Ca(2+) responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y(10) receptor into the P2Y(10)-CHO cells effectively blocked both S1P- and LPA-induced Ca(2+) increases. RT-PCR analysis showed that the mouse P2Y(10) was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y(10) receptor is the first receptor identified as a dual lysophospholipid receptor.

The type 1 lysophosphatidic acid receptor is a target for therapy in bone metastases.

Platelet-derived lysophosphatidic acid (LPA) supports the progression of breast and ovarian cancer metastasis to bone. The mechanisms through which LPA promotes bone metastasis formation are, however, unknown. Here we report that silencing of the type 1 LPA receptor (LPA(1)) in cancer cells blocks the production of tumor-derived cytokines that are potent activators of osteoclast-mediated bone destruction and significantly reduces the progression of osteolytic bone metastases. Moreover, functional blockade of LPA action on its cognate receptor LPA(1) using a pharmacological antagonist mimics the effects of silencing LPA(1) in tumor cells in vitro and substantially reduces bone metastasis progression in animals. Overall, these results suggest that inhibition of platelet-derived LPA action on LPA(1) expressed by tumor cells may be a promising therapeutic target for patients with bone metastases.