Mutagenesis of a plastidial lysophosphatidic acid acyltransferase

A combination of site-directed and random mutagenesis generated sequence variants of a plastidial lysophosphatidic acid acyltransferase. Alanine substitutions of residues present within two conserved motifs including the putative catalytic histidine resulted in a loss of acyltransferase activity assessed as complementation competence. Substitutions at five sites within the central core resulted in reduced or loss of activity. Truncation mutants reveal that sequences in the C-terminal moiety are essential for function.     

Adiponutrin functions as a nutritionally regulated lysophosphatidic acid acyltransferase

Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.     

Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids-protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity

Recently, we isolated a subset of glycolipoproteins from Panax ginseng, that we designated gintonin, and demonstrated that it induced [Ca2+]i transients in cells via G protein-coupled receptor (GPCR) signaling pathway(s). However, active components responsible for Ca2+ mobilization and the corresponding receptor(s) were unknown. Active component(s) for [Ca2+]i transients of gintonin were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry and ion-mobility mass spectrometry, respectively. The corresponding receptor(s)were investigated through gene expression assays. We found that gintonin contains LPA C18:2 and other LPAs. Proteomic analysis showed that ginseng major latex-like protein and ribonuclease-like storage proteins are protein components of gintonin. Gintonin induced [Ca2+]i transients in B103 rat neuroblastoma cells transfected with human LPA receptors with high affinity in order of LPA2 >LPA5 > LPA1 > LPA3 > LPA4. The LPA1/LPA3 receptor antagonist Ki16425 blocked gintonin action in cells expressing LPA1 or LPA3. Mutations of binding sites in the LPA3 receptor attenuated gintonin action. Gintonin acted via pertussis toxin (PTX)-sensitive and -insensitive G protein-phospholipase C (PLC)-inositol 1,4,5-trisphosphate (IP3)-Ca2+ pathways. However, gintonin had no effects on other receptors examined. In human umbilical vein endothelial cells (HUVECs) gintonin stimulated cell proliferation and migration. Gintonin stimulated ERK1/2 phosphorylation. PTX blocked gintonin-mediated migration and ERK1/2 phosphorylation. In PC12 cells gintonin induced morphological changes, which were blocked by Rho kinase inhibitorY-27632. Gintonin contains GPCR ligand LPAs in complexes with ginseng proteins and could be useful in the development of drugs targeting LPA receptors.     

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.     

A novel lysophosphatidic acid acyltransferase enzyme (LPAAT4) with a possible role for incorporating docosahexaenoic acid into brain glycerophospholipids

Glycerophospholipids are important components of cellular membranes, required for constructing structural barriers, and for providing precursors of bioactive lipid mediators. Lysophosphatidic acid acyltransferases (LPAATs) are enzymes known to function in the de novo glycerophospholipid biosynthetic pathway (Kennedy pathway), using lysophosphatidic acid (LPA) and acyl-CoA to form phosphatidic acid (PA). Until now, three LPAATs (LPAAT1, 2, and 3) have been reported from the 1-acyl-glycerol-3-phosphate O-acyltransferase (AGPAT) family. In this study, we identified a fourth LPAAT enzyme, LPAAT4, previously known as an uncharacterized enzyme AGPAT4 (LPAATδ), from the AGPAT family. Although LPAAT4 was known to contain AGPAT motifs essential for acyltransferase activities, detailed biochemical properties were unknown. Here, we found that mouse LPAAT4 (mLPAAT4) possesses LPAAT activity with high acyl-CoA specificity for polyunsaturated fatty acyl-CoA, especially docosahexaenoyl-CoA (22:6-CoA, DHA-CoA). mLPAAT4 was distributed in many tissues, with relatively high expression in the brain, rich in docosahexaenoic acid (DHA, 22:6). mLPAAT4 siRNA in a neuronal cell line, Neuro 2A, caused a decrease in LPAAT activity with 22:6-CoA, suggesting that mLPAAT4 functions endogenously. siRNA in Neuro 2A cells caused a decrease in 18:0–22:6 PC, whereas mLPAAT4 overexpression in Chinese hamster ovary (CHO)-K1 cells caused an increase in this species. Although DHA is considered to have many important functions for the brain, the mechanism of its incorporation into glycerophospholipids is unknown. LPAAT4 might have a significant role for maintaining DHA in neural membranes. Identification of LPAAT4 will possibly contribute to understanding the regulation and the biological roles of DHA-containing glycerophospholipids in the brain.     

GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.     

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.     

Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae

The incorporation of unsaturated acyl chains into phospholipids during de novo synthesis is primarily mediated by the 1-acyl-sn-glycerol-3-phosphate acyltransferase reaction. In Saccharomyces cerevisiae, Slc1 has been shown to mediate this reaction, but distinct activity remains after its removal from the genome. To identify the enzyme that mediates the remaining activity, we performed synthetic genetic array analysis using a slc1Delta strain. One of the genes identified by the screen, LPT1, was found to encode for an acyltransferase that uses a variety of lysophospholipid species, including 1-acyl-sn-glycerol-3-phosphate. Deletion of LPT1 had a minimal effect on 1-acyl-sn-glycerol-3-phosphate acyltransferase activity, but overexpression increased activity 7-fold. Deletion of LPT1 abrogated the esterification of other lysophospholipids, and overexpression increased lysophosphatidylcholine acyltransferase activity 7-fold. The majority of this activity co-purified with microsomes. To test the putative role for this enzyme in selectively incorporating unsaturated acyl chains into phospholipids in vitro, substrate concentration series experiments were performed with the four acyl-CoA species commonly found in yeast. Although the saturated palmitoyl-CoA and stearoyl-CoA showed a lower apparent Km, the monounsaturated palmitoleoyl-CoA and oleoyl-CoA showed a higher apparent Vmax. Arachidonyl-CoA, although not abundant in yeast, also had a high apparent Vmax. Pulse-labeling of lpt1Delta strains showed a 30% reduction in [3H]oleate incorporation into phosphatidylcholine only. Therefore, Lpt1p, a member of the membrane-bound o-acyltransferase gene family, seems to work in conjunction with Slc1 to mediate the incorporation of unsaturated acyl chains into the sn-2 position of phospholipids.     

YPR139c/LOA1 encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets and involved in TAG homeostasis

For many years, lipid droplets (LDs) were considered to be an inert store of lipids. However, recent data showed that LDs are dynamic organelles playing an important role in storage and mobilization of neutral lipids. In this paper, we report the characterization of LOA1 (alias VPS66, alias YPR139c), a yeast member of the glycerolipid acyltransferase family. LOA1 mutants show abnormalities in LD morphology. As previously reported, cells lacking LOA1 contain more LDs. Conversely, we showed that overexpression results in fewer LDs. We then compared the lipidome of loa1Δ mutant and wild-type strains. Steady-state metabolic labeling of loa1Δ revealed a significant reduction in triacylglycerol content, while phospholipid (PL) composition remained unchanged. Interestingly, lipidomic analysis indicates that both PLs and glycerolipids are qualitatively affected by the mutation, suggesting that Loa1p is a lysophosphatidic acid acyltransferase (LPA AT) with a preference for oleoyl-CoA. This hypothesis was tested by in vitro assays using both membranes of Escherichia coli cells expressing LOA1 and purified proteins as enzyme sources. Our results from purification of subcellular compartments and proteomic studies show that Loa1p is associated with LD and active in this compartment. Loa1p is therefore a novel LPA AT and plays a role in LD formation.     

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.     

Cloning and expression of a novel lysophospholipase which structurally resembles lecithin cholesterol acyltransferase

Lecithin cholesterol acyltransferase (LCAT) is the key enzyme in the esterification of plasma cholesterol and in the reverse cholesterol transport on high-density lipoprotein (HDL). We have found a novel LCAT-related gene among differentially expressed cDNA fragments between two types of foam cells derived from THP-1 cells, which are different in cholesterol efflux ability, using a subtractive PCR technique. The deduced 412-amino-acid sequence has 49% amino acid sequence similarity with human LCAT. In contrast to the liver-specific expression of LCAT, mRNA expression of the gene was observed mainly in peripheral tissues including kidney, placenta, pancreas, testis, spleen, heart, and skeletal muscle. The protein exists in human plasma and is probably associated with HDL. Moreover, we discovered that the recombinant protein hydrolyzed lysophosphatidylcholine (lysoPC), a proatherogenic lipid, to glycerophosphorylcholine and a free fatty acid. We have therefore named this novel enzyme LCAT-like lysophospholipase (LLPL), through which a new catabolic pathway for lysoPC on lipoproteins could be elucidated.     

Identification of a novel Ras-regulated proapoptotic pathway

BACKGROUND: The Ras-GTPase controls cell fate decisions through the binding of an array of effector molecules, such as Raf and PI 3-kinase, in a GTP-dependent manner. NORE1, a noncatalytic polypeptide, binds specifically to Ras-GTP and to several other Ras-like GTPases. NORE is homologous to the putative tumor suppressor RASSF1 and to the Caenorhabditis elegans polypeptide T24F1.3. RESULTS: We find that all three NORE-related polypeptides bind selectively to the proapoptotic protein kinase MST1, a member of the Group II GC kinases. Endogenous NORE and MST1 occur in a constitutive complex in vivo that associates with endogenous Ras after serum stimulation. Targeting recombinant MST1 to the membrane, either through NORE or myristoylation, augments the apoptotic efficacy of MST1. Overexpression of constitutively active Ki-RasG12V promotes apoptosis in a variety of cell lines; Ha-RasG12V is a much less potent proapoptotic agent; however, a Ha-RasG12V effector loop mutant (E37G) that binds NORE, but not Raf or PI 3-kinase, exhibits proapoptotic efficacy approaching that of Ki-RasG12V. The apoptotic action of both Ki-RasG12V and Ha-RasG12V, E37G is suppressed by overexpression of the MST1 carboxy-terminal noncatalytic segment or by the NORE segment that binds MST1. CONCLUSIONS: MST1 is a phylogenetically conserved partner of the NORE/RASSF polypeptide family, and the NORE-MST1 complex is a novel Ras effector unit that mediates the apoptotic effect of Ki-RasG12V.     

Membrane glycerophospholipid biosynthesis in Neisseria meningitidis and Neisseria gonorrhoeae: identification, characterization, and mutagenesis of a lysophosphatidic acid acyltransferase

Lysophosphatidic acid (LPA) acyltransferases of Neisseria meningitidis and Neisseria gonorrhoeae were identified which share homology with other prokaryotic and eukaryotic LPA acyltransferases. In Escherichia coli, the conversion of LPA to phosphatidic acid, performed by the 1-acyl-sn-glycerol-3-phosphate acyltransferase PlsC, is a critical intermediate step in the biosynthesis of membrane glycerophospholipids. A Tn916-generated mutant of a serogroup B meningococcal strain was identified that exhibited increased amounts of capsular polysaccharide, as shown by colony immunoblots, and a threefold increase in the number of assembled pili. The single, truncated 3.8 kb Tn916 insertion in the meningococcal mutant was localized within a 771 bp open reading frame. The gonococcal equivalent of this gene was identified by transformation with the cloned meningococcal mutant gene. In N. gonorrhoeae, the mutation increased piliation fivefold. The insertions were found to be within a gene that was subsequently designated nIaA (n eisserial L PA acyltransferase). The predicted neisserial LPA acyltransferases were homologous (>20% identity,>40% amino acid similarity) to the family of PlsC protein homologues. A cloned copy of the meningococcal nIaA gene complemented in trans a temperature-sensitive E. coli PlsC^ts? mutant. Tn916 and Ω-cassette insertional inactivations of the neisserial nIaA genes altered the membrane glycerophospholipid compositions of both N. meningitidis and N. gonorrhoeae but were not lethal. Therefore, the pathogenic Neisseria spp. appear to be able to utilize alternative enzyme(s) to produce phosphatidic acid. This hypothesis is supported by the observation that, although the amounts of mature glycerophospholipids were altered in the meningococcal and the gonococcal nIaA mutants, glycerophospholipid synthesis was detectable at significant levels. In addition, acyltransferase enzymatic activity, while reduced in the gonococcal nIaA mutant, was increased in the meningococcal nIaA mutant. We postulate that the pathogenic Neisseria spp. are able to utilize alternate acyltransferases to produce glycerophospholipids in the absence of nIaA enzymatic activity.Implementation of these secondary enzymes results in alterations of glycerophospholipid composition that lead to pleiotropic effects on the cell surface components, including effects on capsule and piliation.     

Oxytocin and lysophosphatidic acid induce stress fiber formation in human myometrial cells via a pathway involving Rho-kinase

The actin cytoskeleton is important for stress fiber formation and contributes to the initiation and maintenance of smooth muscle contraction. To determine if oxytocin and lysophosphatidic acid (LPA) induce stress fiber formation, cultured human myometrial cells were exposed to oxytocin (10(-5) M) or LPA (10(-6) M), and filamentous (F) and globular (G) actin pools were stained with fluorescein isothiocyanate-phalloidin and Texas red DNase I, respectively. The F- to G-actin fluorescent-staining ratio was measured by fluorescence microscopy. Oxytocin and LPA increased stress fiber formation, as indicated by an increase in the F- to G-actin fluorescent-staining ratio. The Rho-kinase inhibitor Y-27632 markedly attenuated this increase. Oxytocin-induced stress fiber formation was completely inhibited in the presence of the oxytocin antagonist compound VI. Tyrosine kinase inhibition with tyrphostin A23 partially blocked the increase induced by oxytocin but had no effect on LPA-induced stress fiber formation. Stress fiber formation was not blocked by pertussis toxin, mitogen-activated protein kinase, or protein kinase C inhibitors. Our results show that human myometrial cells respond to oxytocin and LPA with the formation of stress fibers that may be involved in the maintenance of uterine contractions. Rho-kinase appears to be a key signaling factor in this pathway.     

Identification of an EDG7 variant, HOFNH30, a G-protein-coupled receptor for lysophosphatidic acid

We have identified a cDNA, designated HOFNH30, which encodes a 354 amino acid G-protein-coupled receptor (GPCR). This receptor has 96% amino acid identity to the Jurkat-T cell-derived EDG7 and could be a splice variant. RT-PCR analysis demonstrated that HOFNH30 mRNA is expressed in placenta whereas EDG7 mRNA shows highest expression in prostate. The HOFNH30 gene is localized to human chromosome 1p22. 3-1p31.1. When HOFNH30 was expressed in RBL-2H3 cells, LPA and phosphatidic acid (PA) induced a calcium mobilization response with EC(50) values of 13 nM and 3 microM, respectively. LPA also induced phosphorylation of mitogen-activated protein kinase (p42(MAPK) and p44(MAPK)) in HOFNH30-transfected but not vector-transfected RBL-2H3 cells. In the present study, we have identified a novel variant from the EDG receptor family, a GPCR for which LPA is a high-affinity endogenous ligand.     

Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli

Complete separation of glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase from Escherichia coli was obtained by sequential extraction with Triton X-100. Solubilized glycerophosphate acyltransferase was reconstituted by the cholate dispersion and gel filtration method in small unilamellar vesicles. 1-Acylglycerophosphate acyltransferase could not be solubilized from the membranes and was used in endogenous membrane fragments after detergent removal. Mixing of the two preparations and subsequent incubation in the presence of glycerol 3-phosphate, palmitoyl-CoA and oleoyl-CoA resulted in the efficient synthesis of phosphatidic acid. Inclusion of exogenous lysophosphatitic acid in the assay medium resulted in a dilution of the newly synthesized lysophosphatidate. By contrast, the synthesis of phosphatidic acid from glycerol 3-phosphate by the acyltransferases present in native membrane vesicles was barely influenced by the presence of exogenous lysophosphatidic acid. When comparing the utilization of membrane-associated 14C-labeled and newly generated 3H-labeled lysophosphatidic acid, the latter appeared to be the preferred substrate. These results indicate that lysophosphatidic acid, synthesized by glycerophosphate acyltransferase, is utilized by 1-acylglycerophosphate acyltransferase without prior mixing with the total membrane-associated pool of lysophosphatidic acid, and suggest a close proximity of the two enzymes in native E. coli membranes. This property of the acyltransferases is lost upon separation and reconstitution of enzyme activities.     

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.     

Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induces the generation of reactive oxygen species (ROS) by upregulating the expression of NADPH oxidase 2 and its regulatory proteins (p22(phox), p67(phox), p40(phox), p47(phox), and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to the activation of mTOR signaling, as pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of the type I insulin-like growth factor receptor (IGFR) β subunit, which was abrogated by NAC. Wortmannin, a phosphoinositide 3'-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1, as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of the mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/PI3K and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for the prevention of Cd-induced neurodegenerative diseases.     

Identification of human plasma lysophospholipase D,a lysophosphatidic acid-producing enzyme,as autotaxin,a multifunctional phosphodiesterase

We purified human plasma lysophospholipase D that produces physiologically active lysophosphatidic acid and showed that it is a soluble form of autotaxin, an ecto-nucleotide pyrophosphatase/phosphodiesterase, originally found as a tumor cell motility-stimulating factor. Its lower K(m) value for a lysophosphatidylcholine than that for a synthetic substrate of nucleotide suggests that lysophosphatidylcholine is a more likely physiological substrate for autotaxin and that its predicted physiological and pathophysiological functions could be mediated by its activity to produce lysophosphate acid, an intercellular mediator. Recombinant autotaxin was found to have lysophospholipase D activity; its substrate specificity and metal ion requirement were the same as those of the purified plasma enzyme. The activity of lysophospholipase D for exogenous lysophosphatidylcholine in human serum was found to increase in normal pregnant women at the third trimester of pregnancy and to a higher extent in patients in threatened preterm delivery, suggesting its roles in induction of parturition.