Identification of a novel noninflammatory biosynthetic pathway of platelet-activating factor

Platelet-activating factor (PAF) is a potent lipid mediator playing various inflammatory and physiological roles. PAF is biosynthesized through two independent pathways called the de novo and remodeling pathways. Lyso-PAF acetyltransferase (lyso-PAF AT) was believed to biosynthesize PAF under inflammatory conditions, through the remodeling pathway. The first isolated lyso-PAF AT (LysoPAFAT/LPCAT2) had consistent properties. However, we show in this study the finding of a second lyso-PAF AT working under noninflammatory conditions. We partially purified a Ca(2+)-independent lyso-PAF AT from mouse lung. Immunoreactivity for lysophosphatidylcholine acyltransferase 1 (LPCAT1) was detected in the active fraction. Lpcat1-transfected Chinese hamster ovary cells exhibited both LPCAT and lyso-PAF AT activities. We confirmed that LPCAT1 transfers acetate from acetyl-CoA to lyso-PAF by the identification of an acetyl-CoA (and other acyl-CoAs) interacting site in LPCAT1. We further showed that LPCAT1 activity and expression are independent of inflammatory signals. Therefore, these results suggest the molecular diversity of lyso-PAF ATs is as follows: one (LysoPAFAT/LPCAT2) is inducible and activated by inflammatory stimulation, and the other (LPCAT1) is constitutively expressed. Each lyso-PAF AT biosynthesizes inflammatory and physiological amounts of PAF, depending on the cell type. These findings provide important knowledge for the understanding of the diverse pathological and physiological roles of PAF.     

Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 at Ser34 Enhances Platelet-activating Factor Production in Endotoxin-stimulated Macrophages

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions under physiological and pathological conditions. We have recently identified two enzymes involved in PAF production: lysophosphatidylcholine acyltransferase-1 (LPCAT1) and LPCAT2. We found that LPCAT2 is highly expressed in inflammatory cells and is activated by lipopolysaccharide (LPS) treatment through Toll-like receptor 4. However, the molecular mechanism for the activation remains elusive. In this study, Phos-tag SDS-PAGE revealed the LPS-induced phosphorylation of LPCAT2. Furthermore, mass spectrometry and mutagenesis analyses identified Ser³⁴ of LPCAT2 as the phosphorylation site to enhance the catalytic activities. The experiments using inhibitors and siRNA against MAPK cascades demonstrated that LPCAT2 phosphorylation through LPS-TLR4 signaling may directly depend on MAPK-activated protein kinase 2 (MAPKAP kinase 2 or MK2). These findings develop a further understanding of both PAF production and phospholipid remodeling triggered by inflammatory stimuli. Specific inhibition of the PAF biosynthetic activity by phosphorylated LPCAT2 will provide a novel target for the regulation of inflammatory disorders.     

Lysophosphatidylcholine Acyltransferase1 Overexpression Promotes Oral Squamous Cell Carcinoma Progression via Enhanced Biosynthesis of Platelet-Activating Factor

Background

The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs.

Methods

We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression.

Results

LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression.

Conclusion

LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.     

Selective inhibitors of a PAF biosynthetic enzyme lysophosphatidylcholine acyltransferase 2

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.     

Identification and characterization of a major liver lysophosphatidylcholine acyltransferase

Phosphatidylcholine (PC) is synthesized through the Kennedy pathway, but more than 50% of PC is remodeled through the Lands cycle, i.e. the deacylation and reacylation of PC to attain the final and proper fatty acids within PC. The reacylation step is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), and we report here the identification of a novel LPCAT, which we named LPCAT3. LPCAT3 belongs to the membrane-bound O-acyltransferase (MBOAT) family and encodes a protein of 487 amino acids with a calculated molecular mass of 56 kDa. Membranes from HEK293 cells overexpressing LPCAT3 showed significantly increased LPCAT activity as assessed by thin layer chromatography analysis with substrate preference toward unsaturated fatty acids. LPCAT3 is localized within the endoplasmic reticulum and is primarily expressed in metabolic tissues including liver, adipose, and pancreas. In a human hepatoma Huh7 cells, RNA interference-mediated knockdown of LPCAT3 resulted in virtually complete loss of membrane LPCAT activity, suggesting that LPCAT3 is primarily responsible for hepatic LPCAT activity. Furthermore, peroxisome proliferator-activated receptor alpha agonists dose-dependently regulated LPCAT3 in liver in a peroxisome proliferator-activated receptor alpha-dependent fashion, implicating a role of LPCAT3 in lipid homeostasis. Our studies identify a long-sought enzyme that plays a critical role in PC remodeling in metabolic tissues and provide an invaluable tool for future investigations on how PC remodeling may potentially impact glucose and lipid homeostasis.     

Characterization of human lysophospholipid acyltransferase 3

Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K_0.5 (5 μM), a monounsaturated acyl-CoA having the highest apparent V_max (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.     

Rapid Production of Platelet-activating Factor Is Induced by Protein Kinase Cα-mediated Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 Protein

Platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is synthesized rapidly in response to extracellular stimuli by the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). We have reported previously that lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: rapid activation (30 s) after PAF stimulation and minutes to hours after LPS stimulation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) was later identified as a Ca²⁺-dependent lyso-PAFAT. However, the mechanism of rapid lyso-PAFAT activation within 30 s has not been elucidated. Here we show a new signaling pathway for rapid biosynthesis of PAF that is mediated by phosphorylation of LPCAT2 at Ser-34. Stimulation by either PAF or ATP resulted in PKCα-mediated phosphorylation of LPCAT2 to enhance lyso-PAFAT activity and rapid PAF production. Biochemical analyses showed that the phosphorylation of Ser-34 resulted in augmentation of V_max with minimal K_m change. Our results offer an answer for the previously unknown mechanism of rapid PAF production.     

Effects of platelet-activating factor, tumor necrosis factor, and interleukin-1alpha on the expression of apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL) in plasma, exclusively expressed in liver and in kidney. The function of apoM is yet unknown. The human apoM gene is located in the major histocompatibility complex class III region on chromosome 6. Because many genes located in this region are related to the immune response, we have investigated whether apoM might also be involved in the host inflammatory response. In this study we examined effects of the platelet-activating factor (PAF), tumor necrosis factor (TNF-alpha), and interleukin-1alpha (IL-1alpha) on apoM expression in a hepatoblastoma cell line, HepG2 cells. PAF significantly enhanced the apoM mRNA levels and the secretion of apoM in HepG2 cell cultures. The enhancement of apoM secretion is seen at a low concentration of PAF (2 ng/ml), whereas a high concentration of PAF increases both the apoM mRNA levels and apoM secretion. Neither TNF-alpha nor IL-1alpha influenced apoM mRNA level and secretion. Furthermore, Lexipafant, a PAF-receptor (PAF-R) antagonist significantly suppressed the mRNA level and the secretion of apoM in HepG2 cells in a dose-dependent manner. Neither PAF nor Lexipafant influenced the mRNA levels and the secretion of apoA-I, apoB and apoE in HepG2 cells, indicating that the effects of PAF or Lexipafant on the apoM production on hepatic cells are selective for apoM. The cellular mechanism of the effects of PAF or Lexipafant on apoM metabolism requires further investigations.     

Cross-talk between Remodeling and de Novo Pathways Maintains Phospholipid Balance through Ubiquitination

Phosphatidylcholine (PtdCho), the major phospholipid of animal membranes, is generated by its remodeling and de novo synthesis. Overexpression of the remodeling enzyme, LPCAT1 (acyl-CoA:lysophosphatidylcholine acyltransferase) in epithelia decreased de novo PtdCho synthesis without significantly altering cellular PtdCho mass. Overexpression of LPCAT1 increased degradation of CPT1 (cholinephosphotransferase), a resident Golgi enzyme that catalyzes the terminal step for de novo PtdCho synthesis. CPT1 degradation involved its multiubiquitination and processing via the lysosomal pathway. CPT1 mutants harboring arginine substitutions at multiple carboxyl-terminal lysines exhibited proteolytic resistance to effects of LPCAT1 overexpression in cells and restored de novo PtdCho synthesis. Thus, cross-talk between phospholipid remodeling and de novo pathways involves ubiquitin-lysosomal processing of a key molecular target that mechanistically provides homeostatic control of cellular PtdCho content.     

Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) Specifically Interacts with Phospholipid Transfer Protein StarD10 to Facilitate Surfactant Phospholipid Trafficking in Alveolar Type II Cells

Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79–271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.     

Correlation of MicroRNA-16, MicroRNA-21 and MicroRNA-101 Expression with Cyclooxygenase-2 Expression and Angiogenic Factors in Cirrhotic and Noncirrhotic Human Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is a classical example of inflammation-linked cancer and is characterized by hypervascularity suggesting rich angiogenesis. Cycloxygenase-2 (COX-2) is a potent mediator of inflammation and is considered to upregulate angiogenesis. The aims of the study are (1) to analyze expression of Cox-2 mRNA, Cox-2 protein, miR-16, miR-21 and miR-101 in HCC and adjacent liver parenchyma in cirrhotic and noncirrhotic liver, (2) to investigate the relation between COX-2 expression, miR-21 expression and angiogenic factors in these tissues and (3) to investigate the association between miR-16 and miR-101 and COX-2 expression.

Methods

Tissue samples of HCC and adjacent liver parenchyma of 21 noncirrhotic livers and 20 cirrhotic livers were analyzed for COX-2 expression at the mRNA level (qRT-PCR) and at the protein level by Western blot and immunohistochemistry. Gene expression of VEGFA, VEGFR1, VEGFR2, Ang-1, Ang-2 and Tie-2 were correlated with COX-2 levels. miR-16, miR-21 and miR-101 gene expression levels were quantified in HCC tumor tissue.

Results

COX-2 mRNA and protein levels were lower in HCC as compared to adjacent liver parenchyma both in cirrhotic and noncirrhotic liver. COX-2 protein localized mainly in vascular and sinusoidal endothelial cells and in Kupffer cells. At the mRNA level but not at the protein level, COX-2 correlated with mRNA levels of angiogenic factors VEGFR1, Ang-1, and Tie2. miR-21 expression was higher in cirrhotic tissues versus noncirrhotic tissues. MiR-101 expression was lower in cirrhotic versus noncirrhotic adjacent liver parenchyma. None of the miRNAs correlelated with COX-2 expression. miR-21 correlated negatively with Tie-2 receptor in adjacent liver parenchyma.

Conclusions

In human HCC, COX-2 mRNA but not COX-2 protein levels are associated with expression levels of angiogenic factors. MiR-21 levels are not associated with angiogenic molecules. MiR-16 and miR-101 levels do not correlate with COX-2 mRNA and protein levels.     

Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine accumulation promotes very low density lipoprotein production by enhancing microsomal triglyceride transfer protein expression

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.     

Lysophosphatidylcholine acyltransferase 3 deficiency impairs 3T3L1 cell adipogenesis through activating Wnt/β-catenin pathway

Levels of polyunsaturated phosphatidylcholine (PC) influence plasma membrane structure and function. Phosphatidylcholine (PC) is synthesized de novo in the Kennedy pathway and then undergoes extensive deacylation/reacylation remodeling via Lands' cycle (non-Kennedy pathway). The reacylation is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), which adds a polyunsaturated fatty acid at the sn-2 position. Four LPCAT isoforms have been described to date, among which we found LPCAT3 to be the major isoform in adipose tissue, but its exact role in adipogenesis is unclear. In this study, we aimed to investigate whether LPCAT3 activity affects 3T3L1 cell adipogenic differentiation potential and its underline mechanism. Lentivirus-mediated LPCAT3 shRNA expression stably knocked down LPCAT3 in 3T3L1 preadipocytes and LPCAT3 deficiency dramatically reduced the levels of cellular polyunsaturated PCs. Importantly, we found that this deficiency activated the β-catenin dependent Wnt signaling pathway, which suppressed the expression of adipogenesis-related genes, thereby inhibiting 3T3L1 preadipocyte differentiation and lipid accumulation. Moreover, three different Wnt/β-catenin pathway inhibitors reversed the effect of LPCAP3 deficiency, suggesting that Wnt/β-catenin pathway activation is one of the causes for the observed phenotypes. To the best of our knowledge, we show here for the first time that PC remodeling is an important regulator of adipocyte differentiation.     

The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats

The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma-type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis.     

Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases

Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A(2) (cPLA(2)) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA(2) activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA(2).     

Stimulation of platelet-activating factor synthesis in human endothelial cells by activation of the de novo pathway. Phorbol 12-myristate 13-acetate activates 1-alkyl-2-lyso-sn-glycero-3-phosphate:acetyl-CoA acetyltransferase and dithiothreitol-insensitive 1-alkyl-2-acetyl-sn-glycerol:CDP-choline cholinephosphotransferase.

Human umbilical vein endothelial cells (HUVEC) produce platelet-activating factor (PAF) by a remodeling pathway involving a phospholipase A2 followed by an acetyl-CoA-dependent acetyltransferase which acetylates a lyso-PAF intermediate to form PAF and is stimulated by a variety of agents that generate inflammatory and allergic responses. A second route for PAF synthesis in mammalian tissues is a de novo pathway, which requires the participation of three enzymes: 1-alkyl-2-lyso-sn-glycero-3-phosphate (alkyllyso-GP): acetyl-CoA acetyltransferase, 1-alkyl-2-acetyl-sn-glycero-3-phosphate phosphohydrolase, and dithiothreitol (DDT)-insensitive 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-cholinecholinephosphotransferase. In the present study we show that protein kinase C activation by phorbol 12-myristate 13-acetate (PMA) induces PAF production in HUVEC by an increase of both alkyllyso-GP:acetyl-CoA acetyltransferase and DTT-insensitive alkylacetyl-G:CDP-choline choline-phosphotransferase. PAF synthesis, labeled precursors [( 3H]acetate and [methyl-3H]choline) incorporation, and both enzyme activities of the de novo pathway increase concomitantly in response to different doses of PMA. PMA does not activate the enzymes of the remodeling pathway. We conclude that both remodeling and the de novo pathway for PAF synthesis are present in HUVEC and might be alternatively activated depending on the conditions of cell stimulation.     

Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine acyltransferase 1 (LPCAT1). Expression in alveolar type II cells and possible involvement in surfactant production

Phosphatidylcholine (1,2-diacyl-sn-glycero-3-phosphocholine, PC), is an important constituent of biological membranes. It is also the major component of serum lipoproteins and pulmonary surfactant. In the remodeling pathway of PC biosynthesis, 1-acyl-sn-glycero-3-phosphocholine (LPC) is converted to PC by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23). Whereas LPCAT activity has been detected in several tissues, the structure and detailed biochemical information on the enzyme have not yet been reported. Here, we present the cloning and characterization of a cDNA for mouse lung-type LPCAT (LPCAT1). The cDNA encodes an enzyme of 60 kDa, with three putative transmembrane domains. When expressed in Chinese hamster ovary cells, mouse LPCAT1 exhibited Ca(2+)-independent activity with a pH optimum between 7.4 and 10. LPCAT1 demonstrated a clear preference for saturated fatty acyl-CoAs, and 1-myristoyl- or 1-palmitoyl-LPC as acyl donors and acceptors, respectively. Furthermore, the enzyme was predominantly expressed in the lung, in particular in alveolar type II cells. Thus, the enzyme might synthesize phosphatidylcholine in pulmonary surfactant and play a pivotal role in respiratory physiology.