Role of lysophosphatidic acid in the regulation of uterine leiomyoma cell proliferation by phospholipase D and autotaxin

Phospholipase D (PLD) hydrolyzes phosphatidylcholine into phosphatidic acid (PA), a lipidic mediator that may act directly on cellular proteins or may be metabolized into lysophosphatidic acid (LPA). We previously showed that PLD contributed to the mitogenic effect of endothelin-1 (ET-1) in a leiomyoma cell line (ELT3 cells). In this work, we tested the ability of exogenous PA and PLD from Streptomyces chromofuscus (scPLD) to reproduce the effect of endogenous PLD in ELT3 cells and the possibility that these agents acted through LPA formation. We found that PA, scPLD, and LPA stimulated thymidine incorporation. LPA and scPLD induced extracellular signal-regulated kinase (ERK(1/2)) mitogen-activated protein kinase activation. Using Ki16425, an LPA(1)/LPA(3) receptor antagonist and small interfering RNA targeting LPA(1) receptor, we demonstrated that scPLD acted through LPA production and LPA(1) receptor activation. We found that scPLD induced LPA production by hydrolyzing lysophosphatidylcholine through its lysophospholipase D (lysoPLD) activity. Autotaxin (ATX), a naturally occurring lysoPLD, reproduced the effects of scPLD. By contrast, endogenous PLD stimulated by ET-1 failed to produce LPA. These results demonstrate that scPLD stimulated ELT3 cell proliferation by an LPA-dependent mechanism, different from that triggered by endogenous PLD. These data suggest that in vivo, an extracellular lysoPLD such as ATX may participate in leiomyoma growth through local LPA formation.     

Polyamines are essential for the synthesis of 2-ricinoleoyl phosphatidic acid in developing seeds of castor

The major fatty acid component of castor (Ricinus communis L.) oil is ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid), and unsaturated hydroxy acid accounts for >85% of the total fatty acids in triacylglycerol (TAG). TAG had a higher ricinoleate content at position 2 than at positions 1 and 3. Although lysophosphatidic acid (LPA) acyltransferase (EC 2.3.1.51), which catalyzes acylation of LPA at position 2, was expected to utilize ricinoleoyl-CoA preferentially over other fatty acyl-CoAs, no activity was found for ricinoleoyl-CoA in vitro at concentrations at which other unsaturated acyl-CoAs were incorporated rapidly. However, activity for ricinoleoyl-CoA appeared with addition of polyamines (putrescine, spermidine, and spermine), while polyamines decreased the rates of incorporation of other acyl-CoAs into position 2. The order of effect of polyamines on LPA acyltransferase activity was spermine > spermidine >> putrescine. At concentrations of spermine and spermidine of >0.1 mM, ricinoleoyl-CoA served as an effective substrate for LPA acyltransferase reaction. The concentrations of spermine and spermidine in the developing seeds were estimated at ∼0.09 and ∼0.63 mM, respectively. These stimulatory effects for incorporation of ricinoleate were specific to polyamines, but basic amino acids were ineffective as cations. In contrast, in microsomes from safflower seeds that do not contain ricinoleic acid, spermine and spermidine stimulated the LPA acyltransferase reaction for all acyl-CoAs tested, including ricinoleoyl-CoA. Although the fatty acid composition of TAG depends on both acyl-CoA composition in the cell and substrate specificity of acyltransferases, castor bean polyamines are crucial for incorporation of ricinoleate into position 2 of LPA. Polyamines are essential for synthesis of 2-ricinoleoyl phosphatidic acid in developing castor seeds.     

Identification of a putative membrane receptor for the bioactive phospholipid, lysophosphatidic acid.

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid with hormone- and growth factor-like activities. Exogenous LPA stimulates GTP-dependent phosphoinositide hydrolysis and inhibits adenylate cyclase in its target cells, but the site of action of LPA is unknown. We now report the identification by photoaffinity labeling of a putative LPA membrane receptor in various LPA-responsive cell types. A 32P-labeled LPA analogue containing a photoreactive fatty acid, [32P]diazirine-LPA, labels a membrane protein of apparent molecular mass of 38-40 kDa in various cell types, including neuronal cells, brain homogenates, carcinoma cells, leukemic cells and normal fibroblasts. Labeling of the 38-40 kDa protein is competitively inhibited by unlabeled 1-oleoyl-LPA (IC50 approximately 10 nM), but not by other phospholipids. Specific labeling is not detected in rat liver membranes or in human neutrophils, which are physiologically unresponsive to LPA. Suramin, an inhibitor of both early and late events in the action of LPA, completely inhibits the binding of photoreactive LPA. We suggest that the 38-40 kDa protein represents a specific LPA cell surface receptor mediating at least part of the multiple cellular responses to LPA.     

Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid

Immobilised 1,3-specific lipase from Rhizopus arrhizus was used as catalyst for the esterification of -glycero-3-phosphate and fatty acid or fatty acid vinyl ester in a solvent-free system. With lauric acid vinyl ester as acyl donor, a_w<0.53 favored the synthesis of lysophosphatidic acid (1-acyl-rac-glycero-3-phosphate, LPA1) and the spontaneous acyl migration of the fatty acid on the molecule. Subsequent acylation by the enzyme resulted in high phosphatidic acid (1,2-diacyl-rac-glycero-3-phosphate, PA) formation and high total conversions (>95%). With oleic acid, maximum conversions of 55% were obtained at low water activities. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/(PA+LPA)]. Thus, LPA was the only product with lauric acid vinyl ester as acyl donor at 25°C. Increased substrate ratio ( -glycero-3-phosphate/fatty acid) from 0.05 to 1 resulted in a higher ratio of LPA to PA formed, but a lower total conversion of -glycero-3-phosphate. Increased amounts of enzyme preparation did not result in higher esterification rates, probably due to high mass-transfer limitations.     

Lysophosphatidic acid stimulates astrocyte proliferation through LPA1

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions through multiple types of LPA receptors. Here we explore the role of LPA receptor subtypes in cortical astrocyte functions. Astrocytes cultured under serum-free conditions were found to express the genes of five LPA receptor subtypes, lpa1 to lpa5. When astrocytes were treated with dibutyryl cyclic adenosine monophosphate, a reagent inducing astrocyte differentiation or activation, lpa1 expression levels remained unchanged, but those of other LPA receptor subtypes were relatively reduced. LPA stimulated DNA synthesis in both undifferentiated and differentiated astrocytes, but failed to do so in astrocytes prepared from mice lacking lpa1 gene. LPA also inhibited [3H]-glutamate uptake in both undifferentiated and differentiated astrocytes; and LPA-induced inhibition of glutamate uptake was still observed in lpa1-deficient astrocytes. Taken together, these observations demonstrate that LPA1 mediates LPA-induced stimulation of cell proliferation but not inhibition of glutamate uptake in astrocytes.     

Role for 18:1 lysophosphatidic acid as an autocrine mediator in prostate cancer cells

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in growth and survival of carcinomas. In this study, LPA production and response were characterized in two human prostate cancer (CaP) cell lines: PC-3 and Du145. Bombesin, a neuroendocrine peptide that is mitogenic for CaP cells, stimulated focal adhesion kinase phosphorylation and activated the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Similar responses were elicited by 18:1 LPA (oleoyl-LPA). Studies using radioisotopic labeling revealed that both PC-3 and Du145 generate LPA and that LPA production is increased by bombesin. The kinetics of bombesin-induced phospholipase D activation and LPA production were similar. Using electrospray ionization mass spectrometry, 18:1 LPA was found to be an abundant LPA species in CaP cell medium. Structure activity studies of acyl-LPAs revealed that 18:1 LPA is most efficacious for activation of extracellular signal-regulated kinase and phospholipase D in CaP cells. Incubation with 18:1 LPA caused homologous desensitization of LPA response, whereas bombesin caused heterologous desensitization. LPA was present at nanomolar levels in medium from bombesin-treated cells. LPA extracted from the medium induced calcium mobilization in CaP cells. These results demonstrate that bioactive LPA is generated by CaP cells in response to a mitogen and suggest that 18:1 LPA can act as an autocrine mediator.     

Lysophosphatidic acid, but not phosphatidic acid, is a potent Ca2(+)-mobilizing stimulus for fibroblasts. Evidence for an extracellular site of action

Lysophosphatidic acid (LPA) is a potent mitogen for quiescent fibroblasts. Among the earliest detectable responses to LPA is GTP-dependent phosphoinositide hydrolysis (van Corven, E. J., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W. H. (1989) Cell 59, 45-54). Here we describe the Ca2(+)-mobilizing properties of LPA in human fibroblasts and present evidence suggesting that previously reported Ca2(+)-mobilizing effects of phosphatidic acid are attributable to contamination with LPA. Addition of LPA (1-oleoyl or 1-palmitoyl) to fibroblasts evokes the formation of inositol 1,4,5-trisphosphate accompanied by an immediate but transient rise in [Ca2+]i which originates primarily from intracellular stores. The Ca2+ response is dose-dependent with a half-maximal effect at LPA concentrations as low as 10 ng/ml, far below the reported half-maximal effect for DNA synthesis (5-10 micrograms/ml). LPA-induced Ca2+ release is also observed in various other cell types, both normal and transformed, but not in Jurkat T cells and neutrophils. The Ca2(+)-mobilizing action of LPA is specific, in that 1,2-dioleoyl-phosphatidic acid (when prepared free of LPA contaminants), other lysophospholipids, monoacylglycerol, and free fatty acids have no effect. Furthermore, LPA, unlike lysophosphatidylcholine, does not cause detectable membrane leakiness, even when added at high concentrations (500 micrograms/ml). The LPA-induced Ca2+ signal is blocked completely by tetradecanoylphorbol acetate, but is not affected by prior stimulation of the cells with Ca2(+)-mobilizing agonists such as bradykinin or histamine. In contrast, pretreating the cells with a low dose of LPA desensitizes the Ca2+ response to subsequent addition of higher doses. This homologous desensitization is not inhibited by staurosporine, nor by down-regulating protein kinase C with tetradecanoylphorbol acetate, suggesting independence of functional protein kinase C activity. Addition of La3+ instantaneously blocks inositol phosphate production and Ca2+ mobilization in response to LPA, but not to bradykinin, most likely due to formation of inactive La3(+)-LPA complexes, suggesting that LPA acts at an extracellular site on the plasma membrane to trigger GTP-dependent phosphoinositide breakdown.     

Novel clusters of receptors for sphingosine-1-phosphate, sphingosylphosphorylcholine, and (lyso)-phosphatidic acid: new receptors for "old" ligands

The (lyso)phospholipid mediators sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC), and phosphatidic acid (PA) regulate diverse cellular responses such as proliferation, survival and death, cytoskeletal rearrangements, cell motility, and differentiation among many others. Signaling is complex and many signaling events are mediated through the activation of cell surface seven transmembrane (7TM) G protein coupled receptors. Five high affinity receptors for S1P have been identified so far and named S1P(1, 2,3,4,5) (formerly referred to as endothelial differentiation gene (edg)1, 5, 3, 6, 8). Recently, the orphan receptor GPR63 was identified a low affinity S1P receptor structurally distant from the S1P(1-5) family. The orphan GPR3, 6, 12 cluster, phylogenetically related to the edg and melanocortin receptors appears to be subject to modulation by S1P and SPC although all three receptors are strong constitutive stimulators of the Galphas-adenylyl cyclase (AC) pathway and would not require additional ligand stimulation but rather inverse agonism to control activity. Ovarian cancer G protein coupled receptor 1 (OGR1) and GPR4, two structurally closely related receptors were assigned in functional and binding studies as high affinity molecular targets for SPC. Very recently, however, both OGR1 and GPR4 were described as receptors endowed with the ability to signal cells in response to protons. LPA exerts its biological effects through the activation of G protein coupled LPA(1-3) receptors (formerly referred to as edg2, 4, 7). A fourth high affinity LPA receptor has been identified: P2Y9 (GPR23) structurally related to nucleotide receptors and phylogenetically quite distant from the high affinity LPA(1-3) cluster. This review attempts to give an overview about the existing families of lysophosholipid receptors and the spectrum of lipid agonists they use as high or low affinity ligands to relay extracellular signals into intracellular responses. Recently deorphaned lipid receptors, within and outside the known lipid receptor clusters will receive particular attention.     

The lysophosphatidic acid 2 receptor mediates down-regulation of Siva-1 to promote cell survival

Lysophosphatidic acid (LPA) promotes cell survival through the activation of G protein-coupled LPA receptors. However, whether different LPA receptors activate distinct anti-apoptotic signaling pathways is not yet clear. Here we report a novel mechanism by which the LPA(2) receptor targets the proapoptotic Siva-1 protein for LPA-dependent degradation, thereby attenuating Siva-1 function in DNA damage response. The carboxyl-terminal tail of the LPA(2) receptor, but not LPA(1) or LPA(3) receptor, specifically associates with the carboxyl cysteine-rich domain of Siva-1. Prolonged LPA stimulation promotes the association of Siva-1 with the LPA(2) receptor and targets both proteins for ubiquitination and degradation. As a result, adriamycin-induced Siva-1 protein stabilization is attenuated by LPA in an LPA(2)-dependent manner, and the function of Siva-1 in promoting DNA damage-induced apoptosis is inhibited by LPA pretreatment. Consistent with this result, inhibition of the LPA(2) receptor expression increases Siva-1 protein levels and augments adriamycin-induced caspase-3 cleavage and apoptosis. Together, these findings reveal a critical and specific role for the LPA(2) receptor through which LPA directly inactivates a critical component of the death machinery to promote cell survival.     

A lysophosphatidic acid receptor lacking the PDZ-binding domain is constitutively active and stimulates cell proliferation

Lysophosphatidic acid (LPA) is an extracellular signaling lipid that regulates cell proliferation, survival, and motility of normal and cancer cells. These effects are produced through G protein-coupled LPA receptors, LPA(1) to LPA(5). We generated an LPA(1) mutant lacking the SerValVal sequence of the C-terminal PDZ-binding domain to examine the role of this domain in intracellular signaling and other cellular functions. B103 neuroblastoma cells expressing the mutant LPA(1) showed rapid cell proliferation and tended to form colonies under serum-free conditions. The enhanced cell proliferation of the mutant cells was inhibited by exogenous expression of the plasmids inhibiting G proteins including G(betagamma), G(alphai) and G(alphaq) or G(alpha12/13), or treatment with pertussis toxin, phosphoinositide 3-kinase (PI3K) inhibitors or a Rho inhibitor. We confirmed that the PI3K-Akt and Rho pathways were intrinsically activated in mutant cells by detecting increases in phosphorylated Akt in western blot analyses or by directly measuring Rho activity. Interestingly, expression of the mutant LPA(1) in non-tumor mouse fibroblasts induced colony formation in a clonogenic soft agar assay, indicating that oncogenic pathways were activated. Taken together, these observations suggest that the mutant LPA(1) constitutively activates the G protein signaling leading to PI3K-Akt and Rho pathways, resulting in enhanced cell proliferation.     

Molecular cloning and characterization of a novel human G-protein-coupled receptor, EDG7, for lysophosphatidic acid.

Lysophosphatidic acid (LPA), together with sphingosine 1-phosphate, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC12 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation.     

ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase

CoA-dependent transacylation activity in microsomes is known to catalyze the transfer of fatty acids between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acids. We previously found a novel acyl-CoA synthetic pathway, ATP-independent acyl-CoA synthesis from phospholipids. We proposed that: 1) the ATP-independent acyl-CoA synthesis is due to the reverse reaction of acyl-CoA:lysophospholipid acyltransferases and 2) the reverse and forward reactions of acyltransferases can combine to form a CoA-dependent transacylation system. To test these proposals, we examined whether or not recombinant mouse acyl-CoA:1-acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT) could catalyze ATP-independent acyl-CoA synthetic activity and CoA-dependent transacylation activity. ATP-independent acyl-CoA synthesis was indeed found in the membrane fraction from Escherichia coli cells expressing mouse LPAAT, whereas negligible activity was observed in mock-transfected cells. Phosphatidic acid (PA), but not free fatty acids, served as an acyl donor for the reaction, and LPA was formed from PA in a CoA-dependent manner during acyl-CoA synthesis. These results indicate that the ATP-independent acyl-CoA synthesis was due to the reverse reaction of LPAAT. In addition, bacterial membranes containing LPAAT catalyzed CoA-dependent acylation of LPA; PA but not free fatty acid served as an acyl donor. These results indicate that the CoA-dependent transacylation of LPA consists of 1) acyl-CoA synthesis from PA through the reverse action of LPAAT and 2) the transfer of the fatty acyl moiety of the newly formed acyl-CoA to LPA through the forward reaction of LPAAT.     

Differential effects of sphingosine 1-phosphate and lysophosphatidic acid on endothelial cells

This review discusses multiple effects of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) on endothelial cells and proposes that S1P and LPA are important regulators of the vascular system. Two physiologic sources of S1P and LPA are platelets and lipoproteins. S1P is an inducer of angiogenesis in vivo whereas LPA is not. S1P and LPA act through endothelial cell surface Edg receptors. S1P stimulates endothelial cell migration, but inhibits migration of most nonendothelial cells. Edg1 and Edg3 receptors, working through G(i), play an important role in regulation of S1P-stimulated endothelial cell migration. LPA effects on endothelial cells are more restricted than the effects of S1P on endothelial cells. LPA stimulates migration of certain endothelial cells on certain extracellular matrix proteins. However, LPA acts like S1P in its effects on the endothelial cell cytoskeleton, proliferation, cell-cell adhesion molecule expression, and vascular permeability. LPA receptors on endothelial cells are likely Edg2 and Edg4. Future studies should better delineate the roles of Edg receptors and downstream pathways on effects of extracellular S1P and LPA and the contributions of intracellularly generated S1P and nitric oxide (NO).     

Lysophospholipids and chemokines activate distinct signal transduction pathways in T helper 1 and T helper 2 cells

We demonstrate the expression of S1P(1,3,4,5) the receptors for sphingosine 1-phosphate (S1P), and LPA(1,2,3) the receptors for lysophosphatidic acid (LPA) in T helper 1 (Th1) and T helper 2 (Th2) cells. S1P and LPA induce the chemotaxis of Th1 and Th2 cells, an activity that is resistant to pertussis toxin (PTX) pretreatment in Th1, but is sensitive in Th2 cells. Also, I-TAC-induced Th1 and eotaxin-induced Th2 cell chemotaxis are blocked by PTX pretreatment. LPA but not S1P induces calcium flux response in Th1 and Th2 cells, which is due to the influx of extracellular calcium and is mediated by receptor activation, since EGTA and suramin (SUR) completely abrogate LPA-induced the release of calcium. No cross-desensitization is observed between thapsigargin (TG) and LPA in both cell types. PTX and SUR but not EGTA inhibit I-TAC- or eotaxin-induced [Ca(2+)](i) release in Th1 and Th2 cells. Our results indicate that lysophospholipids and chemokines stimulate different signal transduction pathways.     

Rho-dependent, Rho kinase-independent inhibitory regulation of Rac and cell migration by LPA1 receptor in Gi-inactivated CHO cells

Lysophosphatidic acid (LPA) is a major serum lysophospholipid that stimulates cell migration in diverse cell types including ovarian cancer cells. We report here that in the absence of Gi function, LPA induces inhibition, rather than stimulation, of cellular Rac activity, lamellipodium formation, and cell migration in response to insulin like growth factor I (IGF-I) in Chinese hamster ovary (CHO) cells, which solely express LPA1 as a LPA receptor. The inhibitory effects of LPA are abrogated by the expression of either Galpha13 C-terminal peptide or C3 toxin pretreatment, but not a Rho kinase inhibitor. Without PTX pretreatment, LPA stimulates Rac and cell migration yet similarly activates Rho, indicating that Rho activation by itself is not sufficient for inhibition of cell migration. Conversely, the expression of a dominant negative Rac mutant sufficiently mimics the LPA inhibition of cell migration. LPA inhibits IGF I-induced Akt activation by only 40% in a manner dependent on Rho kinase. These results demonstrate that inhibition of Gi function converts LPA regulation on Rac and cell migration to an inhibitory mode, which is mediated by G13 and Rho but not Rho kinase, and raise a possibility of Gi as a new therapeutic target for LPA-dependent tumor progression.     

Extracellular and intracellular productions of lysophosphatidic acids and cyclic phosphatidic acids by lysophospholipase D from exogenously added lysophosphatidylcholines to cultured NRK52E cells

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that is a notable biomarker of kidney injury. However, it is not clear how LPA is produced in renal cells. In this study, we explored LPA generation and its enzymatic pathway in a rat kidney-derived cell, NRK52E cells. Culturing of NRK52E cells with acyl lysophosphatidylcholine (acyl LPC), or lyso-platelet activating factor (lysoPAF, alkyl LPC) was resulted in increased extracellular level of choline, co-product with LPA by lysophospholipase D (lysoPLD). Their activities were enhanced by addition of calcium ions to the cell culture medium, but failed to be inhibited by S32826, an autotaxin (ATX)-specific inhibitor. Liquid chromatography-tandem mass spectrometric analysis revealed the small, but significant extracellular production of acyl LPA/cyclic phosphatidic acid (cPA) and alkyl LPA/cPA. The mRNA expression of glycerophosphodiesterase (GDE) 7 with lysoPLD activity was elevated in confluent NRK52E cells cultured over 3 days. GDE7 plasmid-transfection of NRK52E cells augmented both extracellular and intracellular productions of LPAs (acyl and alkyl) as well as extracellular productions of cPAs (acyl and alkyl) from exogenous LPCs (acyl and alkyl). These results suggest that intact NRK52E cells are able to produce choline and LPA/cPA from exogenous LPCs through the enzymatic action of GDE7 that is located on the plasma membranes and intracellular membranes.     

Convergent Regulation of Neuronal Differentiation and Erk and Akt Kinases in Human Neural Progenitor Cells by Lysophosphatidic Acid,Sphingosine 1-Phosphate,and LIF: Specific Roles for the LPA1 Receptor

The bioactive lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have diverse effects on the developing nervous system and neural progenitors, but the molecular basis for their pleiotropic effects is poorly understood. We previously defined LPA and S1P signaling in proliferating human neural progenitor (hNP) cells, and the current study investigates their role in neuronal differentiation of these cells. Differentiation in the presence of LPA or S1P significantly enhanced cell survival and decreased expression of neuronal markers. Further, the LPA receptor antagonist Ki16425 fully blocked the effects of LPA, and differentiation in the presence of Ki16425 dramatically enhanced neurite length. LPA and S1P robustly activated Erk, but surprisingly both strongly suppressed Akt activation. Ki16425 and pertussis toxin blocked LPA activation of Erk but not LPA inhibition of Akt, suggesting distinct receptor and G-protein subtypes mediate these effects. Finally, we explored cross talk between lysophospholipid signaling and the cytokine leukemia inhibitory factor (LIF). LPA/S1P effects on neuronal differentiation were amplified in the presence of LIF. Similarly, the ability of LPA/S1P to regulate Erk and Akt was impacted by the presence of LIF; LIF enhanced the inhibitory effect of LPA/S1P on Akt phosphorylation, while LIF blunted the activation of Erk by LPA/S1P. Taken together, our results suggest that LPA and S1P enhance survival and inhibit neuronal differentiation of hNP cells, and LPA1 is critical for the effect of LPA. The pleiotropic effects of LPA may reflect differences in receptor subtype expression or cross talk with LIF receptor signaling.     

Lysophosphatidic acid (LPA) protects primary chronic lymphocytic leukemia cells from apoptosis through LPA receptor activation of the anti-apoptotic protein AKT/PKB

Lysophosphatidic acid (LPA) protects epithelial and fibroblast cell lines from apoptosis. In B-cells, LPA acts as a growth factor promoting cell proliferation. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+ B-lymphocytes primarily through a block in apoptosis. The mechanisms underlying this defect are not fully understood. We investigated whether LPA could be a survival factor in CLL cells. Herein, we demonstrate that LPA protects B-cell lines BJAB and I-83 and primary CLL cells but not normal B-cells from fludarabine- and etoposide-induced apoptosis. Furthermore, LPA prevented spontaneous apoptosis in primary CLL cells. The LPA1 expression was found to be increased in primary CLL cells compared with normal B-cells correlating with LPA prevention of apoptosis. Treatment of primary CLL cells with the LPA receptor antagonist, diacylglycerol pyrophosphate, reverses the protective effect of LPA against apoptosis, and down-regulation of the LPA1 by siRNA blocked LPA-mediated protection against spontaneous apoptosis in primary CLL cells. The protective effect of LPA was inhibited by blocking activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. These results indicate that LPA is a survival factor in B-cell lines and primary CLL cells but not normal B-cells. Thus, drugs targeting the LPA receptors might be an effective therapy against B-cell-derived malignancies such as CLL.     

Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity

Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.