Lysophosphatidic acid increases phosphatidic acid formation, phospholipase D activity and degranulation by human neutrophils

I-oleoyl-sn-glycero-3-phosphate, a lysophosphatidic acid (LPA), in serum is a biologically active lipid and has multiple functions depending on the cell types. Several studies have shown that LPA stimulates phospholipase D (PLD) activity in fibroblasts and prostate cancer cells in culture. PLD plays a central role in regulating neutrophil functions. One of the functions of the lipid product, phosphatidic acid (PA), of PLD action in neutrophils is to promote degranulation. In the present study, we examined the effect of LPA on PLD activity and degranulation by human neutrophils. The results show that exogenous LPA increased PA formation, PLD activity and degranulation by human neutrophils in a time and concentration dependent manner. These findings suggest that LPA released from activated platelets during blood clotting may participate in bacterial killing and wound healing process. On the other hand, augmented LPA production might be involved in inflammation, causing damage of the host tissues.     

Mechanisms of lysophosphatidic acid production

Lysophosphatidic acid is one of the most attractive phospholipid mediator with multiple biological functions and is implicated in various human diseases. In the past ten years much has been learned about the physiological roles of LPA through series of studies on LPA actions and its receptors. However, the molecular mechanisms of LPA have been poorly understood. LPA is produced in various conditions both in cells and in biological fluids, where multiple synthetic reactions occur. At least two pathways are postulated. In serum and plasma, LPA is mainly converted from lysophospholipids. By contrast, in platelets and some cancer cells, LPA is converted from phosphatidic acid. In each pathway, at least two phospholipase activities are required: phospholipase A1 (PLA1)/PLA2 plus lysophospholipase D (lysoPLD) activities are involved in the first pathway and phospholipase D (PLD) plus PLA1/PLA2 activities are involved in the second pathway. Now multiple phospholipases are identified that account for PLA1, PLA2, PLD, and lysoPLD activities. In the absence of specific inhibitors and genetically modified animals and individuals, the contribution of each phospholipase to LPA production can not be easily determined. However, apparently certain extracellular phospholipases such as secretory PLA2 (sPLA2-IIA), membrane-associated PA-selective PLA1 (mPA-PLA1), lecithin-cholesterol acyltransferase (LCAT), and lysoPLD are involved in LPA production.     

Altered activation of phospholipase D by lysophosphatidic acid and endothelin-1 in mouse embryo fibroblasts lacking phospholipase C-gamma1

Lysophosphatidic acid (LPA) and endothelin-1 (ET-1) activate phospholipase D (PLD) in many cell types. To see if phospholipase C-gamma1 plays a role, we used embryonic fibroblasts from mice in which the PLCgamma1 gene was disrupted. Surprisingly, the effect of LPA on inositol phosphate accumulation was increased in these PLCgamma1-/- cells, whereas that of ET-1 was completely abrogated. When PLD activity was measured, the response to LPA was also enhanced and the response to ET-1 lost in the PLCgamma1-/- cells. Treatment of these cells with ionomycin and oleoyl acetyl glycerol to mimic PLC stimulation restored PLD activity. Treatment of either PLCgamma1+/+ and PLCgamma1-/- cells with tyrosine kinase inhibitors did not inhibit LPA- or ET-1-induced PLD activity. Moreover, LPA and ET-1 treatment of PLCgamma1+/+ and PLCgamma1-/- cells did not cause tyrosine phosphorylation of PLC-gamma1 or PLC-gamma2. In summary, these results show that the altered PLD responses to LPA and ET-1 in PLCgamma1-/- are due to changes in PLC activity and do not involve tyrosine kinase activity.     

Phospholipase D and extracellular signal-regulated kinase in hepatic stellate cells: effects of platelet-derived growth factor and extracellular nucleotides

We have previously provided evidence suggesting that phosphatidic acid, possibly derived from the hydrolysis of phosphatidylcholine by phospholipase D (PLD), is involved in platelet-derived growth factor (PDGF)-mediated increases in extracellular signal-regulated kinase (ERK) activity and DNA synthesis in rat hepatic stellate cells (HSC), the primary fibrogenic cells of the liver. A recent study has shown the presence of P2Y nucleotide receptors on HSC that are coupled to contraction and synthesis of the matrix component, alpha1-procollagen, leading to the suggestion that they may represent a new therapeutic target in the treatment of liver fibrosis. However, although extracellular nucleotides have been shown to stimulate both PLD and ERK, and to elicit proliferation of fibrogenic cells outside the liver, their effect on these parameters in HSC have not yet been investigated. PLD activity was determined by [3H]choline release and [3H]phosphatidylbutanol production, ERK activity by Western blotting, and DNA synthesis by [3H]thymidine incorporation. We report here, for the first time in HSC, that extracellular nucleotides stimulate PLD activity and a sustained activation of ERK. However, in contrast to PDGF, nucleotides had negligible effects on DNA synthesis. Moreover, the effects of PDGF and nucleotides on PLD and ERK were not additive, suggesting activation of the same PLD isoform and pool of ERK. The data demonstrate that nucleotide-stimulated PLD and ERK activities are not coupled to DNA synthesis in HSC. Instead, these responses may be linked to other phenotypic changes associated with activated HSC such as increases in contraction, motility, or extracellular matrix deposition.     

Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s,mPA-PLA1alpha and mPA-PLA1beta

We have identified a novel phospholipase A1, named mPA-PLA1beta, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA1alpha. The sequence of mPAPLA1beta encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA1, mPA-PLA1alpha. mPA-PLA1beta contains a short lid and deleted beta9 loop, which are characteristics of PLA1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA1alpha and phosphatidylserine-specific PLA1. Both mPA-PLA1beta and mPA-PLA1alpha recombinant proteins exhibited PA-specific PLA1 activity and were vanadate-sensitive. When mPAPLA1beta-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA1alpha and beta-expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA1alpha protein was recovered from the cell supernatant. By contrast, mPA-PLA1beta was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA1beta has higher affinity to heparin than mPA-PLA1alpha. We also found that the membrane-associated mPA-PLA1s were insoluble in solubilization by 1% Triton X-100 and were detected in Triton X-100-insoluble buoyant fractions of sucrose gradients. The present study raises the possibility that production of LPA by mPA-PLA1alpha and -beta occurs on detergent-resistant membrane domains of the cells where they compete with lipid phosphate phosphatase for PA.     

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.     

Positive feedback between vascular endothelial growth factor-A and autotaxin in ovarian cancer cells

Tumor cell migration, invasion, and angiogenesis are important determinants of tumor aggressiveness, and these traits have been associated with the motility stimulating protein autotaxin (ATX). This protein is a member of the ectonucleotide pyrophosphatase and phosphodiesterase family of enzymes, but unlike other members of this group, ATX possesses lysophospholipase D activity. This enzymatic activity hydrolyzes lysophosphatidylcholine to generate the potent tumor growth factor and motogen lysophosphatidic acid (LPA). In the current study, we show a link between ATX expression, LPA, and vascular endothelial growth factor (VEGF) signaling in ovarian cancer cell lines. Exogenous addition of VEGF-A to cultured cells induces ATX expression and secretion, resulting in increased extracellular LPA production. This elevated LPA, acting through LPA(4), modulates VEGF responsiveness by inducing VEGF receptor (VEGFR)-2 expression. Down-regulation of ATX secretion in SKOV3 cells using antisense morpholino oligomers significantly attenuates cell motility responses to VEGF, ATX, LPA, and lysophosphatidylcholine. These effects are accompanied by decreased LPA(4) and VEGFR2 expression as well as by increased release of soluble VEGFR1. Because LPA was previously shown to increase VEGF expression in ovarian cancer, our data suggest a positive feedback loop involving VEGF, ATX, and its product LPA that could affect tumor progression in ovarian cancer cells.     

Hippocalcin increases phospholipase D2 expression through extracellular signal-regulated kinase activation and lysophosphatidic acid potentiates the hippocalcin-induced phospholipase D2 expression

We have previously isolated a 22 kDa protein from a rat brain which was found to be involved in activating phospholipsae D (PLD), and identified the protein as hippocalcin through sequence analysis. Nevertheless, the function of hippocalcin for PLD activation still remains to be resolved. Here, we proposed that hippocalcin was involved in extracellular signal-regulated kinase (ERK)-mediated PLD2 expression. To elucidate a role of hippocalcin, we made hippocalcin transfected NIH3T3 cells and showed that the expression of PLD2 and basal PLD activity were increased in hippocalcin transfected cells. We performed PLD assay with dominant negative PLD2 (DN-PLD2) and hippocalcin co-transfected cells. DN-PLD2 suppressed increase of basal PLD activity in hippocalcin transfected cells, suggesting that increased basal PLD activity is due to PLD2 over-expression. Hippocalcin is a Ca2+-binding protein, which is expressed mainly in the hippocampus. Since it is known that lysophosphatidic acid (LPA) increases intracellular Ca2+, we investigated the possible role of hippocalcin in the LPA-induced elevation of intracellular Ca2+. When the intracellular Ca2+ level was increased by LPA, hippocalcin was translocated to the membrane after LPA treatment in hippocalcin transfected cells. In addition, treatment with LPA in hippocalcin transfected cells markedly potentiated PLD2 expression and showed morphological changes of cell shape suggesting that increased PLD2 expression acts as one of the major factors to cause change of cell shape by making altered membrane lipid composition. Hippocalcin-induced PLD2 expression potentiated by LPA in hippocalcin transfected cells was inhibited by a PI-PLC inhibitor, U73122 and a chelator of intracellular Ca2+, BAPTA-AM suggesting that activation of hippocalcin caused by increased intracellular Ca2+ is important to induce over-expression of PLD2. However, downregulation of PKC and treatment of a chelator of extracellular Ca2+, EGTA had little or no effect on the inhibition of hippocalcin-induced PLD2 expression potentiated by LPA in the hippocalcin transfected cells. Interestingly, when we over-express hippocalcin, ERK was activated, and treatment with LPA in hippocalcin transfected cells significantly potentiated ERK activation. Specific inhibition of ERK dramatically abolished hippocalcin-induced PLD2 expression. Taken together, these results suggest for the first time that hippocalcin can induce PLD2 expression and LPA potentiates hippocalcin-induced PLD2 expression, which is mediated by ERK activation.     

Lysophosphatidic acid increases intracellular H2O2 by phospholipase D and RhoA in rat-2 fibroblasts.

We have investigated the possible roles of phospholipase D (PLD) and RhoA in the production of intracellular H2O2 and actin polymerization in response to lysophosphatidic acid (LPA) in Rat-2 fibroblasts. LPA increased intracellular H2O2, with a maximal increase at 30 min, which was blocked by the catalase from Aspergillus niger. The LPA-stimulated production of H2O2 was inhibited by 1-butanol or PKC-downregulation, but not by 2-butanol. Purified phosphatidic acid (PA) also increased intracellular H2O2 and the increase was inhibited by the catalase. The role of RhoA was studied by the scrape-loading of C3 transferase into the cells. The C3 toxin, which inhibited stress fiber formation stimulated by LPA, blocked the H2O2 production in response to LPA or PA, but had no inhibitory effect on the activation of PLD by LPA. Exogenous H2O2 increased F-actin content by stress fiber formation. In addition, catalase inhibited actin polymerization activated by LPA, PA, or H2O2, indicated the role of H2O2 in actin polymerization. These results suggest that LPA increased intracellular H2O2 by the activation of PLD and RhoA, and that intracellular H2O2 was required for the LPA-stimulated stress fiber formation.     

Endogenous RGS proteins attenuate Galpha(i)-mediated lysophosphatidic acid signaling pathways in ovarian cancer cells

Lysophosphatidic acid is a bioactive phospholipid that is produced by and stimulates ovarian cancer cells, promoting proliferation, migration, invasion, and survival. Effects of LPA are mediated by cell surface G-protein coupled receptors (GPCRs) that activate multiple heterotrimeric G-proteins. G-proteins are deactivated by Regulator of G-protein Signaling (RGS) proteins. This led us to hypothesize that RGS proteins may regulate G-protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared LPA activity in SKOV-3 ovarian cancer cells expressing G(i) subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS wild-type (RGSwt) counterparts. Both forms of the G-protein contained a point mutation rendering them insensitive to inhibition with pertussis toxin, and cells were treated with pertussis toxin prior to experiments to eliminate endogenous G(i/o) signaling. The potency and efficacy of LPA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi G(i) proteins as compared to RGSwt G(i). We further showed that LPA signaling that is subject to RGS regulation terminates much faster than signaling thru RGS insensitive G-proteins. Finally, LPA-stimulated SKOV-3 cell migration, as measured in a wound-induced migration assay, was enhanced in cells expressing Galpha(i2) RGSi as compared to cells expressing Galpha(i2) RGSwt, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate this LPA effect. These data establish RGS proteins as novel regulators of LPA signaling in ovarian cancer cells.     

Phospholipase D stimulation is required for sphingosine-1-phosphate activation of actin stress fibre assembly in human airway epithelial cells.

In human airway epithelial cells, sphingosine-1-phosphate (SPP) and lysophosphatidic acid (LPA) stimulated the production of phosphatidic acid (PA), which was inhibited by the primary alcohol butan-1-ol, but not by the inactive butan-2-ol, clearly indicating phospholipase D (PLD) involvement. Both SPP and LPA stimulated actin stress fibre formation, which was also butan-2-ol-insensitive and inhibited by butan-1-ol. SPP-induced PLD activation and cytoskeletal remodelling were insensitive to brefeldin A and toxin B from Clostridium difficile, which conversely blocked the effect of LPA, suggesting that the monomeric GTPases ADP ribosylation factor (ARF) and Rho are involved in LPA, but not in SPP responses. Pertussis toxin inhibited SPP- but not LPA-induced effects. PLD activation and stress fibre formation by both lysolipids were abolished by the tyrosine kinase inhibitor genistein. Addition of PA to cells caused a massive stress fibre assembly. In conclusion, PLD is one of the signalling components linking SPP-receptor activation to assembly of actin stress fibres.     

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.     

Activation of AMP-activated protein kinase is essential for lysophosphatidic acid-induced cell migration in ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-β3 (PLC-β3) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-β3, or (CaMKKβ) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.     

Lysophosphatidic acid activates lipogenic pathways and de novo lipid synthesis in ovarian cancer cells

One of the most common molecular changes in cancer is the increased endogenous lipid synthesis, mediated primarily by overexpression and/or hyperactivity of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC). The changes in these key lipogenic enzymes are critical for the development and maintenance of the malignant phenotype. Previous efforts to control oncogenic lipogenesis have been focused on pharmacological inhibitors of FAS and ACC. Although they show anti-tumor effects in culture and in mouse models, these inhibitors are nonselective blockers of lipid synthesis in both normal and cancer cells. To target lipid anabolism in tumor cells specifically, it is important to identify the mechanism governing hyperactive lipogenesis in malignant cells. In this study, we demonstrate that lysophosphatidic acid (LPA), a growth factor-like mediator present at high levels in ascites of ovarian cancer patients, regulates the sterol regulatory element binding protein-FAS and AMP-activated protein kinase-ACC pathways in ovarian cancer cells but not in normal or immortalized ovarian epithelial cells. Activation of these lipogenic pathways is linked to increased de novo lipid synthesis. The pro-lipogenic action of LPA is mediated through LPA(2), an LPA receptor subtype overexpressed in ovarian cancer and other malignancies. Downstream of LPA(2), the G(12/13) and G(q) signaling cascades mediate LPA-dependent sterol regulatory element-binding protein activation and AMP-activated protein kinase inhibition, respectively. Moreover, inhibition of de novo lipid synthesis dramatically attenuated LPA-induced cell proliferation. These results demonstrate that LPA signaling is causally linked to the hyperactive lipogenesis in ovarian cancer cells, which can be exploited for development of new anti-cancer therapies.     

Lysophosphatidic acid stimulates phospholipase D activity and cell proliferation in PC-3 human prostate cancer cells

Phospholipase D (PLD) is activated in mammalian cells in response to a variety of growth factors and may play a role in cell proliferation. Lysophosphatidic acid (LPA) is a bioactive metabolite potentially generated as a result of PLD activation. Two human prostate cancer cell lines, PC-3 and LNCaP, express membrane PLD activity. The effects of LPA on PLD activity and proliferation were examined in PC-3 cells, which express hPLD1a/1b. Phorbol 12-myristate 13-acetate (PMA) induced a prolonged activation of PLD, as detected in both intact cells and membranes. LPA induced a transient activation of PLD that was maximal by 10 minutes. The EC₅₀ for LPA-induced PLD activation was approximately 1 μM. Pertussis toxin did not inhibit activation of PLD by LPA or PMA. Ro-31-8220 and bisindolylmaleimide I, inhibitors of protein kinase C, blocked activation by PLD by both PMA and LPA. PMA-induced activation of PLD did not appear to require translocation of PLDs from cytosol to membrane. LPA stimulated proliferation of PC-3 cells with an EC₅₀ of approximately 0.2 μM; this response was not inhibited by pertussis toxin. Perillyl alcohol, an anti-cancer drug, reversibly inhibited proliferation in response to either serum or LPA but did not inhibit activation of PLD by PMA or LPA. These data establish that LPA activates PLD and stimulates proliferation via G_i-independent pathways in a human prostate cancer cell line. J. Cell. Physiol. 174:261–272, 1998. © 1998 Wiley-Liss, Inc.     

A novel phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid

Lysophosphatidic acid (LPA) is a lipid mediator with diverse biological properties, although its synthetic pathways have not been completely solved. We report the cloning and characterization of a novel phosphatidic acid (PA)-selective phospholipase A(1) (PLA(1)) that produces 2-acyl-LPA. The PLA(1) was identified in the GenBank(TM) data base as a close homologue of phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)). When expressed in insect Sf9 cells, this enzyme was recovered from the Triton X-100-insoluble fraction and did not show any catalytic activity toward exogenously added phospholipid substrates. However, culture medium obtained from Sf9 cells expressing the enzyme was found to activate EDG7/LPA(3), a cellular receptor for 2-acyl-LPA. The activation of EDG7 was further enhanced when the cells were treated with phorbol ester or a bacterial phospholipase D, suggesting involvement of phospholipase D in the process. In the latter condition, an increased level of LPA, but not other lysophospholipids, was confirmed by mass spectrometry analyses. Expression of the enzyme is observed in several human tissues such as prostate, testis, ovary, pancreas, and especially platelets. These data show that the enzyme is a membrane-associated PA-selective PLA(1) and suggest that it has a role in LPA production.     

Biosynthesis of alkyl lysophosphatidic acid by diacylglycerol kinases

Lysophosphatidic acid (LPA) designates a family of bioactive phosphoglycerides that differ in the length and degree of saturation of their radyl chain. Additional diversity is provided by the linkage of the radyl chain to glycerol: acyl, alkyl, or alk-1-enyl. Acyl-LPAs are the predominate species in tissues and biological fluids. Alkyl-LPAs exhibit distinct pharmacodynamics at LPA receptors, potently drive platelet aggregation, and contribute to ovarian cancer aggressiveness. Multiple biosynthetic pathways exist for alkyl-LPA production. Herein we report that diacylglycerol kinases (DGKs) contribute to cell-associated alkyl-LPA production involving phosphorylation of 1-alkyl-2-acetyl glycerol and document the biosynthesis of alkyl-LPA by DGKs in SKOV-3 ovarian cancer cells, specifically identifying the contribution of DGKα. Concurrently, we discovered that treating SKOV-3 ovarian cancer cell with a sphingosine analog stimulates conversion of exogenous 1-alkyl-2-acetyl glycerol to alkyl-LPA, indicating that DGKα contributes significantly to the production of alkyl-LPA in SKOV-3 cells and identifying cross-talk between the sphingolipid and glycerol lipid pathways.     

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca(2+)](i)) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.