Expression and regulation of phospholipase D isoforms in mammalian cell lines

Phospholipase D (PLD) is activated in mammalian cells in response to diverse stimuli that include growth factors, activators of protein kinase C, and agonists binding to G-protein-coupled receptors. Two forms of mammalian PLD, PLD1 and PLD2, have been identified. Expression of mRNA and protein for PLD1 and PLD2 was analyzed in the following cell lines: A7r5 (rat vascular smooth muscle); EL4 (mouse thymoma); HL-60 (human myeloid leukemia); Jurkat (human leukemia); PC-3 (human prostate adenocarcinoma); PC-12K (rat phaeochromocytoma); and Rat-1 HIR (rat fibroblast). All, with the exception of EL4, express agonist-activated PLD activity. PLD1 is expressed in A7r5, HL-60, PC-3, and Rat-1, while PLD2 is expressed in A7r5, Jurkat, PC12K, PC-3, and Rat-1. Neither isoform is expressed in EL4. Guanine nucleotide-independent PLD activity is present in membranes from all cells expressing PLD2. In PC12K cells, which express only PLD2, treatment with nerve growth factor causes neurite outgrowth and increases expression of PLD2 mRNA and protein within 6-12 h. A corresponding increase is observed in membrane PLD activity and in phorbol-12-myristate-13-acetate (PMA)-stimulated PLD activity in intact cells. These results show that PLD2 can be regulated both pretranslationally and posttranslationally by agonists.     

The cross talk between protein kinase A- and RhoA-mediated signaling in cancer cells

The cross talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and RhoA-mediated signal transductions and the effect of this cross talk on biologic features of human prostate and gastric cancer cells were investigated. In the human gastric cancer cell line, SGC-7901, lysophosphatidic acid (LPA) increased RhoA activity in a dose-dependent manner. The cellular permeable cAMP analog, 8-chlorophenylthio-cAMP (CPT-cAMP), inhibited the LPA-induced RhoA activation and caused phosphorylation of RhoA at serine(188). Immunofluorescence microscopy, Western blotting, and green fluorescent protein (GFP)-tagged RhoA location assay in live cells revealed that RhoA was distributed in both the cytoplasm and nucleus of SGC-7901 cells. Treatment with LPA and/or CPT-cAMP did not induce obvious translocation of RhoA in the cells. The LPA treatment caused formation of F-actin in SGC-7901 cells, and CPT-cAMP inhibited the formation. In a modified Boyden chamber assay, LPA stimulated the migration of SGC-7901 cells, and CPT-cAMP dose-dependently inhibited the stimulating effect of LPA. In soft agar assay, LPA stimulated early proliferation of SGC-7901 cells, and CPT-cAMP significantly inhibited the growth of LPA-stimulated cells. In the prostate cancer cell line, PC-3, LPA caused morphologic changes from polygonal to round, and transfection with plasmid DNA encoding constitutively active RhoA(63L) caused a similar change. Treatment with CPT-cAMP inhibited the changes in both cases. However, in PC-3 cells transfected with a plasmid encoding mutant RhoA188A, LPA induced rounding, but CPT-cAMP could not prevent the change. Results of this experiment indicated that cAMP/PKA inhibited RhoA activation, and serine188 phosphorylation on RhoA was necessary for PKA to exert its inhibitory effect on RhoA activation. The cross talk between cAMP/PKA and RhoA-mediated signal transductions had significant affect on biologic features of gastric and prostate cancer cells, such as morphologic and cytoskeletal change, migration, and anchorage-independent growth. The results may be helpful in implementing novel therapeutic strategies for invasive and metastatic prostate and gastric cancers.     

Inflammatory Cytokines and Survival Factors from Serum Modulate Tweak-Induced Apoptosis in PC-3 Prostate Cancer Cells

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the tumor necrosis factor superfamily. TWEAK activates the Fn14 receptor, and may regulate cell death, survival and proliferation in tumor cells. However, there is little information on the function and regulation of this system in prostate cancer. Fn14 expression and TWEAK actions were studied in two human prostate cancer cell lines, the androgen-independent PC-3 cell line and androgen-sensitive LNCaP cells. Additionally, the expression of Fn14 was analyzed in human biopsies of prostate cancer. Fn14 expression is increased in histological sections of human prostate adenocarcinoma. Both prostate cancer cell lines express constitutively Fn14, but, the androgen-independent cell line PC-3 showed higher levels of Fn14 that the LNCaP cells. Fn14 expression was up-regulated in PC-3 human prostate cancer cells in presence of inflammatory cytokines (TNFα/IFNγ) as well as in presence of bovine fetal serum. TWEAK induced apoptotic cell death in PC-3 cells, but not in LNCaP cells. Moreover, in PC-3 cells, co-stimulation with TNFα/IFNγ/TWEAK induced a higher rate of apoptosis. However, TWEAK or TWEAK/TNFα/IFNγ did not induce apoptosis in presence of bovine fetal serum. TWEAK induced cell death through activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-3, release of mitochondrial cytochrome C and an increased Bax/BclxL ratio. TWEAK/Fn14 pathway activation promotes apoptosis in androgen-independent PC-3 cells under certain culture conditions. Further characterization of the therapeutic target potential of TWEAK/Fn14 for human prostate cancer is warranted.     

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.     

Phospholipase D activity is required for actin stress fiber formation in fibroblasts

Phospholipase D (PLD) is a ubiquitously expressed enzyme of ill-defined function. In order to explore its cellular actions, we inactivated the rat PLD1 (rPLD1) isozyme by tagging its C terminus with a V5 epitope (rPLD1-V5). This was stably expressed in Rat-2 fibroblasts to see if it acted as a dominant-negative mutant for PLD activity. Three clones that expressed rPLD1-V5 were selected (Rat2V16, Rat2V25, and Rat2V29). Another clone (Rat2V20) that lost expression of rPLD1-V5 was also obtained. In the three clones expressing rPLD1-V5, PLD activity stimulated by phorbol myristate acetate (PMA) or lysophosphatidic acid (LPA) was reduced by ~50%, while the PLD activity of Rat2V20 cells was normal. Changes in the actin cytoskeleton in response to LPA or PMA were examined in these clones. All three clones expressing rPLD1-V5 failed to form actin stress fibers after treatment with LPA. However, Rat2V20 cells formed stress fibers in response to LPA to the same extent as wild-type Rat-2 cells. In contrast, there was no significant change in membrane ruffling induced by PMA in the cells expressing rPLD1-V5. Since Rho is an activator both of rPLD1 and stress fiber formation, the activation of Rho was monitored in wild-type Rat-2 cells and Rat2V25 cells, but no significant difference was detected. The phosphorylation of vimentin mediated by Rho-kinase was also intact in Rat2V25 cells. Rat2V25 cells also showed normal vinculin-containing focal adhesions. However, the translocation of alpha-actinin to the cytoplasm and to the detergent-insoluble fraction in Rat2V25 cells was reduced. These results indicate that PLD activity is required for LPA-induced rearrangement of the actin cytoskeleton to form stress fibers and that PLD might be involved in the cross-linking of actin filaments mediated by alpha-actinin.     

Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid

We made stable cell lines overexpressing PLD1 (GP-PLD1) from GP+envAm12 cell, a derivative of NIH 3T3 cell. PLD1 activity and extracellular signal-regulated kinase (ERK) phosphorylation were enhanced in GP-PLD1 cells by the treatment of lysophosphatidic acid (LPA). In contrast, these LPA-induced effects were attenuated with the pretreatment of pertussis toxin (PTX) or protein kinase C (PKC) inhibitor. Moreover, accumulation of phosphatidic acid (PA), a product of PLD action, potentiated the LPA-induced ERK activation in GP-PLD1 cells while blocking of PA production with the treatment of 1-butanol attenuated LPA-induced ERK phosphorylation. From these results, we suggest that LPA activate PLD1 through pertussis toxin-sensitive G protein and PKC-dependent pathways, then PA produced from PLD1 activation facilitate ERK phosphorylation.     

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC(50) value of 39.0 microM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0-50 microM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.     

A novel acylglycerol kinase that produces lysophosphatidic acid modulates cross talk with EGFR in prostate cancer cells

The bioactive phospholipids, lysophosphatidic acid (LPA) and phosphatidic acid (PA), regulate pivotal processes related to the pathogenesis of cancer. Here, we report characterization of a novel lipid kinase, designated acylglycerol kinase (AGK), that phosphorylates monoacylglycerol and diacylglycerol to form LPA and PA, respectively. Confocal microscopy and subcellular fractionation suggest that AGK is localized to the mitochondria. AGK expression was up-regulated in prostate cancers compared with normal prostate tissues from the same patient. Expression of AGK in PC-3 prostate cancer cells markedly increased formation and secretion of LPA. This increase resulted in concomitant transactivation of the EGF receptor and sustained activation of extracellular signal related kinase (ERK) 1/2, culminating in enhanced cell proliferation. AGK expression also increased migratory responses. Conversely, down-regulating expression of endogenous AGK inhibited EGF- but not LPA-induced ERK1/2 activation and progression through the S phase of the cell cycle. Hence, AGK can amplify EGF signaling pathways and may play an important role in the pathophysiology of prostate cancer.     

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.     

Antioxidation and Antiapoptosis Characteristics of Heme Oxygenase-1 Enhance Tumorigenesis of Human Prostate Carcinoma Cells

Heme oxygenase-1 (HO-1) has antiinflammatory and antioxidant properties and is deemed as a tissue protector. However, effects of HO-1 in prostate cancer remain in controversy. We evaluated the role of HO-1 in prostate carcinoma in vitro and in vivo.Overexpression of HO-1 did not affect prostate cell proliferation in the normal condition but enhanced cell proliferation under serum starvation. HO-1 overexpression enhanced cell invasion of PC-3 cells through epithelial–mesenchymal transition (EMT) induction, which was supported by increased Slug, N-cadherin, and vimentin expressions. In the xenograft animal study, HO-1 overexpression enhanced PC-3 cell tumor growth in vivo. HO-1 attenuated reactive oxygen species induced by H₂O₂ or pyocyanin treatment in PC-3 and DU145 cells. HO-1 further reduced PC-3 and DU145 cell apoptosis induced by H₂O₂ or serum starvation. Our results suggested that HO-1 was able to increase prostate carcinoma cell invasion in vitro and tumor growth in vivo. The EMT induction and antioxidant and antiapoptotic effects of HO-1 in the prostate carcinoma cells may be responsible for these findings.     

Involvement of phospholipase D2 in lysophosphatidate-induced transactivation of platelet-derived growth factor receptor-beta in human bronchial epithelial cells

Lysophosphatidate (LPA) mediates multiple cellular responses via heterotrimeric G protein coupled LPA-1, LPA-2, and LPA-3 receptors. Many G protein-coupled receptors stimulate ERK following tyrosine phosphorylation of growth factor receptors; however, the mechanism(s) of transactivation of receptor tyrosine kinases are not well defined. Here, we provide evidence for the involvement of phospholipase D (PLD) in LPA-mediated transactivation of platelet-derived growth factor receptor-beta (PDGF-R beta). In primary cultures of human bronchial epithelial cells (HBEpCs), LPA stimulated tyrosine phosphorylation of PDGF-R beta and threonine/tyrosine phosphorylation of ERK1/2. The LPA-mediated activation of ERK and tyrosine phosphorylation of PDGF-R beta was attenuated by tyrphostin AG 1296, an inhibitor of PDGF-R kinase, suggesting transactivation of PDGF-R by LPA. Furthermore, LPA-, but not PDGF beta-chain homodimer-induced tyrosine phosphorylation of PDGF-R beta was partially blocked by pertussis toxin, indicating coupling of LPA-R(s) to Gi. Exposure of HBEpCs to LPA activated PLD. Butan-1-ol, which acts as an acceptor of phosphatidate generated by the PLD pathway, blocked LPA-mediated transactivation of PDGF-R beta. This effect was not seen with butan-3-ol, suggesting PLD involvement. The role of PLD1 and PLD2 in the PDGF-R beta transactivation by LPA was investigated by infection of cells with adenoviral constructs of wild type and catalytically inactive mutants of PLD. LPA activated both PLD1 and PLD2 in HBEpCs; however, infection of cells with cDNA for wild type PLD2, but not PLD1, increased the tyrosine phosphorylation of PDGF-R beta in response to LPA. Also, the LPA-mediated tyrosine phosphorylation of PDGF-R beta was attenuated by the catalytically inactive mutant mPLD2-K758R. Infection of HBEpCs with adenoviral constructs of wild type hPLD1, mPLD2, and the inactive mutants of hPLD1 and mPLD2 resulted in association of PLD2 wild type and inactive mutant proteins with the PDGF-R beta compared with PLD1. These results show for the first time that transactivation of PDGF-R beta by LPA in HBEpCs is regulated by PLD2.     

Granulocyte-macrophage colony-stimulating factor primes phospholipase D activity in human neutrophils in vitro: role of calcium, G-proteins and tyrosine kinases.

The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human peripheral blood neutrophils primes phospholipase D (PLD) to subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). The present investigation was directed at the elucidation of the pathway(s) involved in the regulation of the activity of PLD in untreated as well as in GM-CSF-primed neutrophils. Pretreatment with pertussis toxin (PT) totally inhibited fMLP-induced activation of PLD in control or GM-CSF-treated cells. PT did not affect the activation of PLD by PMA but inhibited the priming effect of GM-CSF. Activation of PLD by fMLP was dose-dependently inhibited by erbstatin, an inhibitor of tyrosine kinases. Furthermore, pre-incubation with GM-CSF accelerated the tyrosine phosphorylation response to fMLP (as analysed by protein immunoblot with antiphosphotyrosine antibodies). In PMA-stimulated neutrophils, erbstatin antagonized the priming effect of GM-CSF on PLD without affecting the direct effects of the phorbol ester. Buffering cytoplasmic calcium with the chelator BAPTA inhibited fMLP-induced activation of PLD as monitored by the formation of phosphatidylethanol. The stimulation of PLD by PMA was partially attenuated in BAPTA-loaded cells while the priming effect of GM-CSF was abolished. Thus, priming of human neutrophil PLD by GM-CSF may be mediated by G-proteins, by increases in the levels of cytosolic free calcium, and by stimulation of protein kinase C and/or tyrosine kinase(s).     

Effect of lysophosphatidic acid on motility, polarisation and metabolic burst of human neutrophils

Abstract The effect of lysophosphatidic acid (LPA) on human neutrophil activation was examined by a combination of automated tracking assays, cell shape measurements and assays of the metabolic burst by means of 7-dimethylamino-naphthalene-1,2-dicarbonic acid hydrazide (DNDH)-dependent chemiluminescence. LPA powerfully stimulated polarisation and motility. Polarisation became detectable at 2 μM LPA and virtually 100% of cells were polarised at 20 μM LPA. Cell motility increased with the degree of polarisation, and was diminished at high LPA concentration, but this decrease was reversed by albumin. LPA also inhibited the metabolic burst response to both n -formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Inhibition of the PMA-induced metabolic burst by LPA was not affected by pertussis toxin, showing that the effect was not mediated by the pertussis toxin-sensitive heterotrimeric G protein, and that inhibition of the PMA-stimulated metabolic burst by LPA could result from a direct action of LPA on the small cytosolic GTP-binding proteins. These results indicate that lysophosphatidic acid production by thrombin-activated platelets could play a significant role in the regulation of the inflammatory response.     

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.     

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.     

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.     

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.     

Autophagy researchers

Lysophosphatidic acid (LPA) is a platelet-enriched bioactive lysophospholipid. By binding to its cognitive G protein-coupled receptors, which are encoded by endothelial differentiation genes (Edg), LPA regulates various cellular activities including proliferation, survival, and migration. Currently, little is known about the influences of LPA on autophagy, a pivotal mechanism for cell survival during conditions of starvation. Herein we present data indicating that LPA attenuates starvation-induced autophagy, by monitoring the percentage of LC3-II, an autophagy indicator, in human prostate PC-3 cells. In addition, by using cells stably expressing EGFP-LC3, LPA is shown to inhibit the formation of autophagosomes in serum-starved conditions. Our results suggest that in these conditions, LPA inhibits autophagy, which might facilitate early cancer development.

Addendum to:

Lysophosphatidic Acid (LPA) Inhibits Serum Starvation Induced-Autophagy of PC-3 Human Prostate Cancer Cell-Line,br>W.-P. Huang, J.-J. Liao and H. Lee

FASEB J 2006; 20:12     

Vascular smooth muscle migration and proliferation in response to lysophosphatidic acid (LPA) is mediated by LPA receptors coupling to Gq

Many G protein-coupled receptors can couple to multiple G proteins to convey their intracellular signaling cascades. The receptors for lysophosphatidic acid (LPA) possess this ability. LPA receptors are important mediators of a wide variety of biological actions including cell migration, proliferation and survival which are processes that can all have a considerable impact on vascular smooth muscle (VSM) and blood vessels. To date, confirmation of G proteins involved has mostly relied on the inhibition of Gi-mediated signaling via pertussis toxin (PTx). We were interested in the specific involvement of LPA-Gq-mediated signaling therefore we isolated aorta VSM cells (VSMCs) from transgenic mice that express a peptide inhibitor of Gq, GqI, exclusively in VSM. We detected both LPA1 and LPA2 receptor expression in mouse VSM whereas LPA1 and LPA3 were expressed in rat VSM. SM22-GqI did not alter LPA-induced migration but it was sufficient to attenuate LPA-induced proliferation. GqI expression also attenuated LPA-induced ERK1/2 and Akt activation by 40-50%. To test the feasibility of this peptide as a potential therapeutic agent, we also generated adenovirus encoding the GqI. Transient expression of GqI was capable of inhibiting both LPA-induced migration and proliferation of VSMCs isolated from rat and mouse. Furthermore, ERK activation in response to LPA was also attenuated in VSMCs with Adv-GqI. Therefore, LPA receptors couple to Gq in VSMC and mediate migration and proliferation which may be mediated through activation of ERK1/2 and Akt. Our data also suggest that both chronic and transient expression of the GqI peptide is an effective strategy to lower Gq-mediated LPA signaling and may be a successful therapeutic strategy to combat diseases with enhanced VSM growth such as occurs following angioplasty or stent implantation.