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1.  Lysophosphatidylcholine Acyltransferase 3 Is the Key Enzyme for Incorporating Arachidonic Acid into Glycerophospholipids during Adipocyte Differentiation 
Cellular membranes contain glycerophospholipids, which have important structural and functional roles in cells. Glycerophospholipids are first formed in the de novo pathway (Kennedy pathway) and are matured in the remodeling pathway (Lands’ cycle). Recently, lysophospholipid acyltransferases functioning in Lands’ cycle were identified and characterized. Several enzymes involved in glycerophospholipid biosynthesis have been reported to have important roles in adipocytes. However, the role of Lands’ cycle in adipogenesis has not yet been reported. Using C3H10T1/2, a cell line capable of differentiating to adipocyte-like cells in vitro, changes of lysophospholipid acyltransferase activities were investigated. Lysophosphatidylcholine acyltransferase (LPCAT), lysophosphatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylserine acyltransferase (LPSAT) activities were enhanced, especially with 18:2-CoA and 20:4-CoA as donors. Correspondingly, mRNA expression of LPCAT3, which possesses LPCAT, LPEAT and LPSAT activities with high specificity for 18:2- and 20:4-CoA, was upregulated during adipogenesis. Analysis of acyl-chain compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) showed a change in their profiles between preadipocytes and adipocytes, including an increase in the percentage of arachidonic acid-containing phospholipids. These changes are consistent with the activities of LPCAT3. Therefore, it is possible that enhanced phospholipid remodeling by LPCAT3 may be associated with adipocyte differentiation.
doi:10.3390/ijms131216267
PMCID: PMC3546689  PMID: 23208369
glycerophospholipid; lysophospholipid acyltransferase; adipocyte; C3H10T1/2; LPCAT3; Lands’ cycle; arachidonic acid
2.  Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions* 
The Journal of Biological Chemistry  2013;288(52):36902-36914.
Background: Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine.
Results: Plant LPCATs were expressed in yeast and biochemically characterized.
Conclusion: LPCATs can edit acyl composition of phosphatidylcholine through their combined forward and reverse reactions.
Significance: Plant LPCATs play a role in editing both sn-positions of PC and remove ricinoleic acid with high selectivity from this lipid.
Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.
doi:10.1074/jbc.M113.521815
PMCID: PMC3873549  PMID: 24189065
Arabidopsis; Enzymes; Lipid Metabolism; Phosphatidylcholine; Plant Biochemistry; Lysophosphatidylcholine Acyltransferases; Phosphatidylcholine Metabolism; Plant Lipid Biochemistry; Enzyme
3.  The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics 
Biology of the Cell  2011;103(Pt 11):499-517.
Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization.
Results. Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet.
Conclusions. We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.
doi:10.1042/BC20110024
PMCID: PMC3181828  PMID: 21787361
3-β-hydroxysteroid dehydrogenase; apolipoprotein A-IV; Caco-2/TC7 cell; cytosolic lipid droplet; enterocyte; lysophosphatidylcholine acyltransferase 2 (LPCAT2); proteome; 3BHS1, 3-β-hydroxysteroid dehydrogenase 1; ABHD5, α/β-hydrolase-domain-containing protein 5; ACSL3, acyl-CoA synthetase long-chain 3; ApoA-IV, apolipoprotein A-IV; CCT-α, choline-phosphate cytidylyltransferase A; CE, cholesterol ester; CLD, cytosolic lipid droplet; DAPI, 4′,6-diamidino-2-phenylindole; DGAT, diacylglycerol acyltransferase; DGE, diacylglyceryl ether; emPAI, exponentially modified protein abundance index; ER, endoplasmic reticulum; GM130, Golgi matrix 130; HSD17B11, 17-β-hydroxysteroid dehydrogenase type 11; HSP60, heat-shock protein 60 kDa; LC-MS/MS, liquid chromatography coupled with tandem MS; LDH, lactate dehydrogenase; LPCAT2, lysophosphatidylcholine acyltransferase 2; MALDI, matrix-assisted laser-desorption ionization; MGAT, monoacylglycerol acyltransferase; MGLL, monoacylglycerol lipase; TAG, triacylglycerol; MTTP, microsomal TAG transfer protein; NSDHL, NAD(P)-dependent steroid dehydrogenase-like; OA, oleic acid; PC, phosphatidylcholine; PDI, protein disulfide-isomerase; PFA, paraformaldehyde; PL, phospholipid; PLIN, perilipin; TOF, time-of-flight; TRL, TAG-rich lipoprotein; VLDL, very-low-density lipoprotein
4.  Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2[S] 
Journal of Lipid Research  2014;55(7):1386-1396.
Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid mediator. In response to extracellular stimuli, PAF is rapidly biosynthesized by lyso-PAF acetyltransferase (lyso-PAFAT). Previously, we identified two types of lyso-PAFATs: lysophosphatidylcholine acyltransferase (LPCAT)1, mostly expressed in the lungs where it produces PAF and dipalmitoyl-phosphatidylcholine essential for respiration, and LPCAT2, which biosynthesizes PAF and phosphatidylcholine (PC) in the inflammatory cells. Under inflammatory conditions, LPCAT2, but not LPCAT1, is activated and upregulated to produce PAF. Thus, it is important to develop inhibitors specific for LPCAT2 in order to ameliorate PAF-related inflammatory diseases. Here, we report the first identification of LPCAT2-specific inhibitors, N-phenylmaleimide derivatives, selected from a 174,000-compound library using fluorescence-based high-throughput screening followed by the evaluation of the effects on LPCAT1 and LPCAT2 activities, cell viability, and cellular PAF production. Selected compounds competed with acetyl-CoA for the inhibition of LPCAT2 lyso-PAFAT activity and suppressed PAF biosynthesis in mouse peritoneal macrophages stimulated with a calcium ionophore. These compounds had low inhibitory effects on LPCAT1 activity, indicating that adverse effects on respiratory functions may be avoided. The identified compounds and their derivatives will contribute to the development of novel drugs for PAF-related diseases and facilitate the analysis of LPCAT2 functions in phospholipid metabolism in vivo.
doi:10.1194/jlr.M049205
PMCID: PMC4076079  PMID: 24850807
TSI-01; LPCAT2 inhibitor; N-phenylmaleimide derivatives; platelet-activating factor; high-throughput screening; lyso-PAF acetyltransferase; lipid mediator; inflammation; fluorescent probe; lysophospholipid acyltransferase
5.  Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein 
Background
Oxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced.
Methods
Human and rabbit LDLs were pretreated with or without a selective platelet-activating factor acetylhydrolase (PAF-AH) inhibitor. LDL was oxidized by incubation with copper sulfate, and PC profiles were analyzed by liquid chromatography-tandem mass spectrometry.
Results
When human LDL was oxidized, the peak areas for polyunsaturated fatty acid (PUFA)-containing PC species dramatically decreased after a short lag period, concomitantly lysoPC species increased sharply. Although a variety of oxPC species containing oxidized fatty acyl groups or cleaved acyl chains are formed during LDL oxidation, only a few oxPC products accumulated in oxLDL: 1-palmitoyl-2-(9-oxo-nonanoyl) PC and long-chain oxPC with two double bonds. Pretreatment of LDL with the PAF-AH inhibitor greatly reduced lysoPC production while it had no effect on lipid peroxidation reactions and oxPC profiles. Rabbit LDL, which has a different composition of PC molecular species and needs a longer time to reach achieve full oxidation than human LDL, also accumulated lysoPC during oxidation. The increase in lysoPC in rabbit oxLDL was suppressed by pretreatment with the PAF-AH inhibitor. The major oxPC species formed in rabbit oxLDL were almost the same as human oxLDL.
Conclusions
These results suggest that lysoPC species are the major products and PAF-AH activity is crucial for lysoPC generation during oxidation of LDL. The oxPC species accumulated are limited when LDL is oxidized with copper ion in vitro.
doi:10.1186/1476-511X-13-48
PMCID: PMC4007754  PMID: 24625108
Oxidized LDL; PC molecular species; LC-MS/MS; Oxidized PC; PAF-AH; Pefabloc; lysoPC; PONPC; Rabbit LDL
6.  LPCAT1 regulates surfactant phospholipid synthesis and is required for transitioning to air breathing in mice 
The Journal of Clinical Investigation  2010;120(5):1736-1748.
Respiratory distress syndrome (RDS), which is the leading cause of death in premature infants, is caused by surfactant deficiency. The most critical and abundant phospholipid in pulmonary surfactant is saturated phosphatidylcholine (SatPC), which is synthesized in alveolar type II cells de novo or by the deacylation-reacylation of existing phosphatidylcholine species. We recently cloned and partially characterized a mouse enzyme with characteristics of a lung lysophosphatidylcholine acyltransferase (LPCAT1) that we predicted would be involved in surfactant synthesis. Here, we describe our studies investigating whether LPCAT1 is required for pulmonary surfactant homeostasis. To address this issue, we generated mice bearing a hypomorphic allele of Lpcat1 (referred to herein as Lpcat1GT/GT mice) ufsing a genetrap strategy. Newborn Lpcat1GT/GT mice showed varying perinatal mortality from respiratory failure, with affected animals demonstrating hallmarks of respiratory distress such as atelectasis and hyaline membranes. Lpcat1 mRNA levels were reduced in newborn Lpcat1GT/GT mice and directly correlated with SatPC content, LPCAT1 activity, and survival. Surfactant isolated from dead Lpcat1GT/GT mice failed to reduce minimum surface tension to wild-type levels. Collectively, these data demonstrate that full LPCAT1 activity is required to achieve the levels of SatPC essential for the transition to air breathing.
doi:10.1172/JCI38061
PMCID: PMC2860922  PMID: 20407208
7.  LysoPC acyltransferase/PC transacylase activities in plant plasma membrane and plasma membrane-associated endoplasmic reticulum 
BMC Plant Biology  2007;7:64.
Background
The phospholipids of the plant plasma membrane are synthesized in the endoplasmic reticulum (ER). The majority of these lipids reach the plasma membrane independently of the secretory vesicular pathway. Phospholipid delivery to the mitochondria and chloroplasts of plant cells also bypasses the secretory pathway and here it has been proposed that lysophospholipids are transported at contact sites between specific regions of the ER and the respective organelle, followed by lysophospholipid acylation in the target organelle. To test the hypothesis that a corresponding mechanism operates to transport phospholipids to the plasma membrane outside the secretory pathway, we investigated whether lysolipid acylation occurs also in the plant plasma membrane and whether this membrane, like the chloroplasts and mitochondria, is in close contact with the ER.
Results
The plant plasma membrane readily incorporated the acyl chain of acyl-CoA into phospholipids. Oleic acid was preferred over palmitic acid as substrate and acyl incorporation occurred predominantly into phosphatidylcholine (PC). Phospholipase A2 stimulated the reaction, as did exogenous lysoPC when administered in above critical micellar concentrations. AgNO3 was inhibitory. The lysophospholipid acylation reaction was higher in a membrane fraction that could be washed off the isolated plasma membranes after repeated freezing and thawing cycles in a medium with lowered pH. This fraction exhibited several ER-like characteristics. When plasma membranes isolated from transgenic Arabidopsis expressing green fluorescent protein in the ER lumen were observed by confocal microscopy, membranes of ER origin were associated with the isolated plasma membranes.
Conclusion
We conclude that a lysoPC acylation activity is associated with plant plasma membranes and cannot exclude a PC transacylase activity. It is highly plausible that the enzyme(s) resides in a fraction of the ER, closely associated with the plasma membrane, or in both. We suggest that this fraction might be the equivalent of the mitochondria associated membrane of ER origin that delivers phospholipids to the mitochondria, and to the recently isolated ER-derived membrane fraction that is in close contact with chloroplasts. The in situ function of the lysoPC acylation/PC transacylase activity is unknown, but involvement in lipid delivery from the ER to the plasma membrane is suggested.
doi:10.1186/1471-2229-7-64
PMCID: PMC2241621  PMID: 18045483
8.  Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer 
The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p<10−8) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [14C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p<10−5) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.
doi:10.1007/s00109-008-0409-0
PMCID: PMC2614561  PMID: 18974965
Colorectal cancer; Lysophosphatidic acyltransferase; Microarrays; Lipid metabolism; Phosphatidylcholine
9.  Triacylglycerol synthesis by PDAT1 in the absence of DGAT1 activity is dependent on re-acylation of LPC by LPCAT2 
BMC Plant Biology  2012;12:4.
Background
The Arabidopsis thaliana dgat1 mutant, AS11, has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1, PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis.
Methods
Biochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity.
Results
Through microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1/lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11.
Conclusions
Collectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn-2 moieties to the sn-3 position of the enlarged AS11 DAG pool.
doi:10.1186/1471-2229-12-4
PMCID: PMC3310826  PMID: 22233193
dgat1 mutant AS11, LPCAT1, LPCAT2, PDAT1, Oil biosynthesis Seed lines from Nottingham Arabidopsis Stock Centre WT (ecotype Columbia-0); dgat1, AS11 (CS3861); A7 (SALK_039456); lpcat1 (SALK_123480); lpcat2 (SAIL_357_H01) (all in a Columbia background)
10.  Role of lysophosphatidylcholine in the inhibition of endothelial cell motility by oxidized low density lipoprotein. 
Journal of Clinical Investigation  1996;97(12):2736-2744.
Endothelial cell (EC) movement is required for the development and repair of blood vessels. We have previously shown that LDL oxidized by transition metals almost completely suppressed the wound-healing migratory response of vascular EC in vitro. We now report that lysophosphatidylcholine (lysoPC), a lipid component of oxidized LDL, has an important role in the antimigratory activity of the lipoprotein. Purified 1-palmitoyl lysoPC inhibited movement with a half-maximal activity at 12-15 micrometers, and near complete inhibition at 20 micrometers; the inhibitory concentration of lysoPC was consistent with its abundance in oxidized LDL. The inhibition was not due to cytotoxicity since protein synthesis was unaffected and since EC movement was restored after removal of lysoPC. Lysophospholipid activity was dependent on lipid structure. LysoPC's containing 1-position C16 or C18 saturated fatty acids were antimigratory, but those containing C < or = 14 saturated fatty acids or polyunsaturated fatty acids were not. The activity of 1-palmitoyl lysolipids with various head groups was examined. Lysophosphatidylinositol was more antimigratory than lysophosphatidylglycerol and lysophosphatidylcholine, which were more potent than lysophosphatidylserine and lysophosphatidylethanolamine. Monoglyceride was inactive while lysophosphatidate had promigratory activity. These results are consistent with head group size rather than charge as a critical determinant of activity. To show that lysophospholipids within an intact lipoprotein were active, LDL was treated with bee venom phospholipase A2 (PLA2). The modified lipoprotein inhibited EC movement to the same extent as iron-oxidized LDL and antimigratory activity correlated with the amount of lysoPC formed. To determine antimigratory activity of lysoPC present in oxidized LDL, lipid extracts from oxidized LDL were fractionated by normal phase HPLC. The fraction comigrating with lysoPC had nearly the same activity as the total extract confirming that lysoPC (or a co-eluting lipid) was a major antimigratory molecule in oxidized LDL. These studies demonstrate that lysoPC in oxidized LDL limit EC wound healing responses in vitro, and suggest a possible role for lysolipids in limiting endothelial regeneration after a denuding injury in vivo.
PMCID: PMC507366  PMID: 8675684
11.  Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation 
Molecular Vision  2010;16:252-271.
Purpose
During retinal development, post-mitotic neural progenitor cells must activate thousands of genes to complete synaptogenesis and terminal maturation. While many of these genes are known, others remain beyond the sensitivity of expression microarray analysis. Some of these elusive gene activation events can be detected by mapping changes in RNA polymerase-II (Pol-II) association around transcription start sites.
Methods
High-resolution (35 bp) chromatin immunoprecipitation (ChIP)-on-chip was used to map changes in Pol-II binding surrounding 26,000 gene transcription start sites during photoreceptor maturation of the mouse neural retina, comparing postnatal age 25 (P25) to P2. Coverage was 10–12 kb per transcription start site, including 2.5 kb downstream. Pol-II-active regions were mapped to the mouse genomic DNA sequence by using computational methods (Tiling Analysis Software-TAS program), and the ratio of maximum Pol-II binding (P25/P2) was calculated for each gene. A validation set of 36 genes (3%), representing a full range of Pol-II signal ratios (P25/P2), were examined with quantitative ChIP assays for transcriptionally active Pol-II. Gene expression assays were also performed for 19 genes of the validation set, again on independent samples. FLT-3 Interacting Zinc-finger-1 (FIZ1), a zinc-finger protein that associates with active promoter complexes of photoreceptor-specific genes, provided an additional ChIP marker to highlight genes activated in the mature neural retina. To demonstrate the use of ChIP-on-chip predictions to find novel gene activation events, four additional genes were selected for quantitative PCR analysis (qRT–PCR analysis); these four genes have human homologs located in unidentified retinal disease regions: Solute carrier family 25 member 33 (Slc25a33), Lysophosphatidylcholine acyltransferase 1 (Lpcat1), Coiled-coil domain-containing 126 (Ccdc126), and ADP-ribosylation factor-like 4D (Arl4d).
Results
ChIP-on-chip Pol-II peak signal ratios >1.8 predicted increased amounts of transcribing Pol-II and increased expression with an estimated 97% accuracy, based on analysis of the validation gene set. Using this threshold ratio, 1,101 genes were predicted to experience increased binding of Pol-II in their promoter regions during terminal maturation of the neural retina. Over 800 of these gene activations were additional to those previously reported by microarray analysis. Slc25a33, Lpcat1, Ccdc126, and Arl4d increased expression significantly (p<0.001) during photoreceptor maturation. Expression of all four genes was diminished in adult retinas lacking rod photoreceptors (Rd1 mice) compared to normal retinas (90% loss for Ccdc126 and Arl4d). For rhodopsin (Rho), a marker of photoreceptor maturation, two regions of maximum Pol-II signal corresponded to the upstream rhodopsin enhancer region and the rhodopsin proximal promoter region.
Conclusions
High-resolution maps of Pol-II binding around transcription start sites were generated for the postnatal mouse retina; which can predict activation increases for a specific gene of interest. Novel gene activation predictions are enriched for biologic functions relevant to vision, neural function, and chromatin regulation. Use of the data set to detect novel activation increases was demonstrated by expression analysis for several genes that have human homologs located within unidentified retinal disease regions: Slc25a33, Lpcat1, Ccdc126, and Arl4d. Analysis of photoreceptor-deficient retinas indicated that all four genes are expressed in photoreceptors. Genome-wide maps of Pol-II binding were developed for visual access in the University of California, Santa Cruz (UCSC) Genome Browser and its eye-centric version EyeBrowse (National Eye Institute-NEI). Single promoter resolution of Pol-II distribution patterns suggest the Rho enhancer region and the Rho proximal promoter region become closely associated with the activated gene’s promoter complex.
PMCID: PMC2822553  PMID: 20161818
12.  Allochromatium vinosum DsrC: Solution-State NMR Structure, Redox Properties and Interaction with DsrEFH, a Protein Essential for Purple Sulfur Bacterial Sulfur Oxidation 
Journal of molecular biology  2008;382(3):692-707.
Summary
Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under non-denaturing conditions evidence was obtained for interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and the Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur-substrate binding/transferring protein as well as with an electron-transfer function via thiol-disulfide interchanges.
doi:10.1016/j.jmb.2008.07.022
PMCID: PMC2637153  PMID: 18656485
dissimilatory sulfite reductase; DsrC; sulfur oxidation; NMR solution structure; anoxygenic phototrophic sulfur bacteria
13.  Impact of Lysophosphatidylcholine on the Plasminogen Activator System in Cultured Vascular Smooth Muscle Cells 
Journal of Korean Medical Science  2012;27(7):803-810.
The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expressions of PAI-1 but did not affect the protein expression of t-PA. Fibrin overlay zymography revealed that lysoPC increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA. Vitamin E inhibited the lysoPC-induced PAI-1 expression. Further, lysoPC increased the intracellular reactive oxygen species (ROS) formation. Caffeic acid phenethyl ester, an inhibitor of NF-κB, blocked this lysoPC effect. Indeed, lysoPC induced the NF-κB-mediated transcriptional activity as measured by luciferase reporter assay. In addition, genistein, an inhibitor of protein-tyrosine kinase (PTK), diminished the lysoPC effect, while 7,12-dimethylbenz[a]anthracene, a stimulator of PTK, stimulated PAI-1 production. In conclusion, lysoPC does not affect t-PA expression but induces PAI-1 expression in the VSMC by mediating NF-κB and the genistein-sensitive PTK signaling pathways via oxidative stress. Importantly, lysoPC stimulates the enzyme activity of PAI-1 and suppresses that of t-PA.
doi:10.3346/jkms.2012.27.7.803
PMCID: PMC3390732  PMID: 22787379
Lysophosphatidylcholines; NF-kappa B; Oxidative Stress; Plasminogen Activator Inhibitor 1; Protein-Tyrosine Kinase; Muscle, Smooth, Vascular
14.  Cell signalling by oxidized lipids and the role of reactive oxygen species in the endothelium 
Biochemical Society transactions  2005;33(Pt 6):1385-1389.
The controlled formation of ROS (reactive oxygen species) and RNS (reactive nitrogen species) is now known to be critical in cellular redox signalling. As with the more familiar phosphorylation-dependent signal transduction pathways, control of protein function is mediated by the post-translational modification at specific amino acid residues, notably thiols. Two important classes of oxidant-derived signalling molecules are the lipid oxidation products, including those with electrophilic reactive centres, and decomposition products such as lysoPC (lysophosphatidylcholine). The mechanisms can be direct in the case of electrophiles, as they can modify signalling proteins by post-translational modification of thiols. In the case of lysoPC, it appears that secondary generation of ROS/RNS, dependent on intracellular calcium fluxes, can cause the secondary induction of H2O2 in the cell. In either case, the intracellular source of ROS/RNS has not been defined. In this respect, the mitochondrion is particularly interesting since it is now becoming apparent that the formation of superoxide from the respiratory chain can play an important role in cell signalling, and oxidized lipids can stimulate ROS formation from an undefined source. In this short overview, we describe recent experiments that suggest that the cell signalling mediated by lipid oxidation products involves their interaction with mitochondria. The implications of these results for our understanding of adaptation and the response to stress in cardiovascular disease are discussed.
doi:10.1042/BST20051385
PMCID: PMC1413972  PMID: 16246125
4-hydroxynonenal (4-HNE); lysophosphatidylcholine; mitochondrion; oxidized lipid; reactive oxygen species (ROS); thiol
15.  Up-regulation of platelet-activating factor synthases and its receptor in spinal cord contribute to development of neuropathic pain following peripheral nerve injury 
Molecular Pain  2012;8:8.
Background
Platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator derived from cell membrane. It has been reported that PAF is involved in various pathological conditions, such as spinal cord injury, multiple sclerosis, neuropathic pain and intrathecal administration of PAF leads to tactile allodynia. However, the expression of PAF synthases and its receptor in the spinal cord following peripheral nerve injury is unknown.
Methods
Using the rat spared nerve injury (SNI) model, we investigated the expression of PAF synthases (LPCAT1 and 2) and PAF receptor (PAFr) mRNAs in the spinal cord. Reverse transcription polymerase chain reaction (RT-PCR) and double-labeling analysis of in situ hybridization histochemistry (ISHH) with immunohistochemistry (IHC) were employed for the analyses. Pain behaviors were also examined with PAFr antagonist (WEB2086).
Results
RT-PCR showed that LPCAT2 mRNA was increased in the ipsilateral spinal cord after injury, but not LPCAT1 mRNA. Double-labeling of ISHH with IHC revealed that LPCAT1 and 2 mRNAs were constitutively expressed by a subset of neurons, and LPCAT2 mRNA was increased in spinal microglia after nerve injury. RT-PCR showed that PAFr mRNA was dramatically increased in the ipsilateral spinal cord after nerve injury. Double-labeling analysis of ISHH with IHC revealed that after injury PAFr mRNA was predominantly colocalized with microglia in the spinal cord. Continuous intrathecal administration of the PAFr antagonist suppressed mechanical allodynia following peripheral nerve injury. Delayed administration of a PAFr antagonist did not reverse the mechanical allodynia.
Conclusions
Our data show the histological localization of PAF synthases and its receptor in the spinal cord following peripheral nerve injury, and suggest that PAF/PAFr signaling in the spinal cord acts in an autocrine or paracrine manner among the activated microglia and neurons, thus contributing to development of neuropathic pain.
doi:10.1186/1744-8069-8-8
PMCID: PMC3293010  PMID: 22296727
PAF; Synthase; Receptor; Microglia; Neuron; Neuropathic pain
16.  Lysophosphatidylcholine Hydrolases of Human Erythrocytes, Lymphocytes and Brain: Sensitive Targets of Conserved Specificity for Organophosphorus Delayed Neurotoxicants 
Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC50 values of 0.13–85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1–3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes and brain remain to be defined.
doi:10.1016/j.taap.2007.06.008
PMCID: PMC2682731  PMID: 17663017
erythrocytes; lysophosphatidylcholine; lysophosphatidylcholine hydrolase; neuropathy target esterase; organophosphorus
17.  Plasma Metabolomics Reveal Alterations of Sphingo- and Glycerophospholipid Levels in Non-Diabetic Carriers of the Transcription Factor 7-Like 2 Polymorphism rs7903146 
PLoS ONE  2013;8(10):e78430.
Aims/Hypothesis
Polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene have been shown to display a powerful association with type 2 diabetes. The aim of the present study was to evaluate metabolic alterations in carriers of a common TCF7L2 risk variant.
Methods
Seventeen non-diabetic subjects carrying the T risk allele at the rs7903146 TCF7L2 locus and 24 subjects carrying no risk allele were submitted to intravenous glucose tolerance test and euglycemic-hyperinsulinemic clamp. Plasma samples were analysed for concentrations of 163 metabolites through targeted mass spectrometry.
Results
TCF7L2 risk allele carriers had a reduced first-phase insulin response and normal insulin sensitivity. Under fasting conditions, carriers of TCF7L2 rs7903146 exhibited a non-significant increase of plasma sphingomyelins (SMs), phosphatidylcholines (PCs) and lysophosphatidylcholines (lysoPCs) species. A significant genotype effect was detected in response to challenge tests in 6 SMs (C16:0, C16:1, C18:0, C18:1, C24:0, C24:1), 5 hydroxy-SMs (C14:1, C16:1, C22:1, C22:2, C24:1), 4 lysoPCs (C14:0, C16:0, C16:1, C17:0), 3 diacyl-PCs (C28:1, C36:6, C40:4) and 4 long-chain acyl-alkyl-PCs (C40:2, C40:5, C44:5, C44:6).
Discussion
Plasma metabolomic profiling identified alterations of phospholipid metabolism in response to challenge tests in subjects with TCF7L2 rs7903146 genotype. This may reflect a genotype-mediated link to early metabolic abnormalities prior to the development of disturbed glucose tolerance.
doi:10.1371/journal.pone.0078430
PMCID: PMC3813438  PMID: 24205231
18.  Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro. 
Journal of Clinical Investigation  1996;97(7):1696-1704.
Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.
PMCID: PMC507234  PMID: 8601635
19.  Redox Factor-1 Activates Endothelial SIRTUIN1 through Reduction of Conserved Cysteine Sulfhydryls in Its Deacetylase Domain 
PLoS ONE  2013;8(6):e65415.
Apurinic/Apyrmidinic Endonuclease 1/Redox Factor-1 (APE1/Ref-1) is a reductant which is important for vascular homeostasis. SIRTUIN1 (SIRT1) is a lysine deacetylase that also promotes endothelium-dependent vasorelaxation. We asked if APE1/Ref-1 governs the redox state and activity of SIRT1, and whether SIRT1 mediates the effect of APE1/Ref-1 on endothelium-dependent vascular function. APE1/Ref-1 maintains sulfhydryl (thiol) groups of cysteine residues in SIRT1 in the reduced form and promotes endothelial SIRT1 activity. APE1/Ref-1 stimulates SIRT1 activity by targeting highly conserved vicinal thiols 371 and 374 which form a zinc tetra-thiolate motif in the deacetylase domain of SIRT1. Cysteine residues in the N-terminal redox domain of APE1/Ref-1 are essential for reducing SIRT1 and stimulating its activity. APE1/Ref-1 protects endothelial SIRT1 from hydrogen peroxide-induced oxidation of sulfhydryls and from inactivation. APE1/Ref-1 also promotes lysine deacetylation of the SIRT1 target endothelial nitric oxide synthase (eNOS). SIRT1 mutated at cysteines 371 and 374, which renders it non-reducible by APE1/Ref-1, prevents lysine deacetylation of eNOS by APE1/Ref-1. SIRT1 free thiol (reduced sulfhydryl) content and deacetylase activity are diminished in all examined tissues of APE1/Ref-1+/− mice, including the vasculature. Overexpression of SIRT1 in aortas of APE1/Ref-1+/− mice restores endothelium-dependent vasorelaxation and bioavailable nitric oxide (NO) to levels similar to those observed in wild-type mice. Thus, APE1/Ref-1, by maintaining functionally important cysteine sulfhydryls in SIRT1 in the reduced form, promotes endothelial SIRT1 activity. This reductive activation of endothelial SIRT1 by APE1/Ref-1 mediates the effect of APE1/Ref-1 on eNOS acetylation, promoting endothelium-derived NO and endothelium-dependent vasorelaxation.
doi:10.1371/journal.pone.0065415
PMCID: PMC3670896  PMID: 23755229
20.  Lysophosphatidylcholine Activates a Novel PKD2-Mediated Signaling Pathway That Controls Monocyte Migration 
Objective
Monocyte activation and migration are crucial events in the development of atherosclerosis and other inflammatory diseases. This study examined the role of protein kinase D (PKD) in monocyte migration.
Method and Results
PKD2 is the predominant isoform of PKD expressed in monocytic THP-1 cells and primary human monocytes. Lysophosphatidylcholine (lysoPC), a prominent component of oxidized low density lipoprotein, induces rapid and marked PKD activation in these cells. Using multiple approaches, including dominant-negative mutants and small interfering RNA knock-down, we found that lysoPC-induced PKD2 activation was required for the activation of both ERK and p38 MAPK. p38 MAPK mediation of lysoPC-induced monocytic cell migration was reported previously; our results reveal that the lysoPC-induced PKD2-p38 pathway controls monocyte migration.
Conclusions
This study provides the first evidence that 1) lysoPC activates PKD, 2) PKD2 has a novel role in p38 activation, and 3) the PKD2-activated p38 pathway is responsible for lysoPC-induced migration of THP-1 cells and human monocytes. Thus, PKD is a novel and functional intracellular regulator in both lysoPC signaling and monocyte migration. These results suggest a new role for PKD2 in the development of atherosclerosis and other inflammatory diseases.
doi:10.1161/ATVBAHA.109.191585
PMCID: PMC3073140  PMID: 19520973
protein kinase; signaling pathway; lysophosphatidylcholine; monocyte migration
21.  Host Derived Inflammatory Phospholipids Regulate rahU (PA0122) Gene, Protein, and Biofilm Formation in Pseudomonas aeruginosa 
Cellular immunology  2011;270(2):95-102.
This study describes the role of “inflammatory” oxidative (Ox) phospholipids in regulation of rahU (PA0122) expression and biofilm formation in Pseudomonas aeruginosa (383) wild type (rahU+) and rahU mutant (rahU−). Functional analysis of RahU protein from P. aeruginosa in presence of Ox-phospholipids show: (a) LysoPC modulates RahU gene-protein expression in rahU+ cells; (b) rahU promoter activity is increased by lysoPC and inhibited by PAPC, Ox-PAPC and arachidonic acid; the latter inhibitory effect can be reversed by lysoPC, which was enzymatically derived from PAPC; (c) biofilm formation increased in rahU− cells as compared to rahU+; and (d) inhibition of rahU promoter activity by PAPC and AA (but not lysoPC) showed significantly augmented biofilm formation in rahU+ but not in rahU− cells. This study shows that host derived Ox-phospholipids affect P. aeruginosa-rahU gene and protein expression, which in turn modulates biofilm formation. The accompanying paper describes the role of RahU in eukaryotic-host cells.
doi:10.1016/j.cellimm.2011.04.011
PMCID: PMC3415270  PMID: 21679933
Pseudomonas; Inflammation; Phospholipids; Biofilm; Gene Expression
22.  Lipopolysaccharide triggers nuclear import of Lpcat1 to regulate inducible gene expression in lung epithelia 
AIM: To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene transcription.
METHODS: Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation.
RESULTS: Lpcat1 translocates into the nucleus from the cytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli, two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overexpressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment.
CONCLUSION: These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.
doi:10.4331/wjbc.v3.i7.159
PMCID: PMC3421133  PMID: 22905292
Lipopolysaccharide; Nuclear import; Lysophosphatidylcholine acyltransferase 1; Gene expression; Lung epithelia; Epigenetic code; Quantitative reverse transcription polymerase chain reaction; Haemophilus influenza; Escherichia coli
23.  Decrease in Membrane Phospholipid Unsaturation Induces Unfolded Protein Response* 
The Journal of Biological Chemistry  2010;285(29):22027-22035.
Various kinds of fatty acids are distributed in membrane phospholipids in mammalian cells and tissues. The degree of fatty acid unsaturation in membrane phospholipids affects many membrane-associated functions and can be influenced by diet and by altered activities of lipid-metabolizing enzymes such as fatty acid desaturases. However, little is known about how mammalian cells respond to changes in phospholipid fatty acid composition. In this study we showed that stearoyl-CoA desaturase 1 (SCD1) knockdown increased the amount of saturated fatty acids and decreased that of monounsaturated fatty acids in phospholipids without affecting the amount or the composition of free fatty acid and induced unfolded protein response (UPR), evidenced by increased expression of C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) mRNAs and splicing of Xbox-binding protein 1 (XBP1) mRNA. SCD1 knockdown-induced UPR was rescued by various unsaturated fatty acids and was enhanced by saturated fatty acid. Lysophosphatidylcholine acyltransferase 3 (LPCAT3), which incorporates preferentially polyunsaturated fatty acids into phosphatidylcholine, was up-regulated in SCD1 knockdown cells. Knockdown of LPCAT3 synergistically enhanced UPR with SCD1 knockdown. Finally we showed that palmitic acid-induced UPR was significantly enhanced by LPCAT3 knockdown as well as SCD1 knockdown. These results suggest that a decrease in membrane phospholipid unsaturation induces UPR.
doi:10.1074/jbc.M110.126870
PMCID: PMC2903364  PMID: 20489212
Fatty Acid; Fatty Acid Metabolism; Membrane; Membrane Lipids; Phospholipid; Phospholipid Metabolism; Lipotoxicity; Lysophospholipid Acyltransferase; Unfolded Protein Response
24.  Lysophosphatidylcholine and Carotid Intima-Media Thickness in Young Smokers: A Role for Oxidized LDL-Induced Expression of PBMC Lipoprotein-Associated Phospholipase A2? 
PLoS ONE  2013;8(12):e83092.
Background
Although cigarette smoking has been associated with carotid intima-media thickness (CIMT) the mechanisms are yet not completely known. Lysophosphatidylcholine (lysoPC), a main product of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, appears to be a major determinant of the pro-atherogenic properties of oxidized LDL (oxLDL) and to induce proteoglycan synthesis, a main player in intimal thickening. In this study we assessed whether cigarette smoking-induced oxidative stress may influence plasma Lp-PLA2 and lysoPC and Lp-PLA2 expression in peripheral blood mononuclear cells (PBMC), as well as the relationship between lysoPC and CIMT.
Methods/Results
45 healthy smokers and 45 age and sex-matched subjects participated in this study. Smokers, compared to non-smokers, showed increased plasma concentrations of oxLDL, Lp-PLA2 and lysoPC together with up-regulation of Lp-PLA2 (mRNA and protein) expression in PBMC (P<0.001). Plasma Lp-PLA2 positively correlated with both lysoPC (r=0.639, P<0.001) and PBMC mRNA Lp-PLA2 (r=0.484, P<0.001) in all subjects. Moreover CIMT that was higher in smokers (P<0.001), positively correlated with lysoPC (r=0.55, P<0.001). Then in in vitro study we demonstrated that both oxLDL (at concentrations similar to those found in smoker’s serum) and oxidized phospholipids contained in oxLDL, were able to up-regulate mRNA Lp-PLA2 in PBMC. This effect was likely due, at least in part, to the enrichment in oxidized phospholipids found in PBMC after exposure to oxLDL. Our results also showed that in human aortic smooth muscle cells lysoPC, at concentrations similar to those found in smokers, increased the expression of biglycan and versican, two main proteoglycans.
Conclusions
In smokers a further effect of raised oxidative stress is the up-regulation of Lp-PLA2 expression in PBMC with subsequent increase of plasma Lp-PLA2 and lysoPC. Moreover the correlation between lysoPC and CIMT together with the finding that lysoPC up-regulates proteoglycan synthesis suggests that lysoPC may be a link between smoking and intimal thickening.
doi:10.1371/journal.pone.0083092
PMCID: PMC3866188  PMID: 24358251
25.  Development of phospholipase A2 and lysophosphatidylcholine metabolising enzyme activities in the neonatal rat intestine. 
Gut  1987;28(7):822-828.
We have studied the development of phospholipase A2 (PLA2) and lysophosphatidylcholine (lysoPC)-metabolising enzyme activities in the neonatal rat intestine and its relation to the intestinal permeability of macromolecules. The permeability was determined by feeding young rats a mixture of bovine serum albumin, bovine immunoglobulin G and fluorescein-isothiocyanate-conjugated dextran 70,000, and analysing the serum concentrations after six hours. The animals were then killed and the intestinal mucosa was homogenised and assessed for PLA2 and lysoPC-metabolising enzyme activities. The intestine was 'open' to the macromolecules in 14 day old animals, but 'closed' in 22 and 32 day old animals and in 14 day old rats treated with cortisone acetate on day 10, 11, and 12 postpartum. The activity of PLA2 (at pH 6 and 2 mM Ca2+) was higher in 32, 22, and cortisone treated 14 day old animals, than in untreated, 14 day old animals. Incubation of 14C-acyl-lysoPC with mucosa from 14 day old rats did not change the radioactivity pattern as shown by thin layer chromatography, whereas after incubation with mucosa from 22 or 32 day old animals all the radiolabel was found in free 14C-fatty acid and in 14C-phosphatidylcholine. These findings indicate that mucosal PLA2 activity increases during intestinal maturation and that the mucosa acquires the ability to acylate and deacylate lysoPC when it is 'closed' to macromolecules.
PMCID: PMC1433081  PMID: 2443432

Results 1-25 (506898)