BBA - Molecular and Cell Biology of Lipids (v.1761, #7)
Editorial Board (ii).
Protein kinase C regulatory domains: The art of decoding many different signals in membranes by Senena Corbalán-García; Juan C. Gómez-Fernández (633-654).
Protein kinase C (PKC) is a member of a family of Ser/Thr phosphotransferases that are involved in many cellular signaling pathways. These enzymes possess two regulatory domains, C1 and C2, that are the targets of different second messengers. The purpose of this review is to describe in molecular terms the diverse mechanisms of activation of PKCs in the light of very significant advances made in this field over recent years. The role of some critical amino acid residues concerning activation of the enzymes and their location within known structures of isolated domains will be presented. For example, the recently deduced 3D structures of the C2 domains show that these domains can additionally act as PtdIns(4,5)P2-binding or phosphotyrosine-binding modules depending on the isoenzyme. All these capacities to play different roles in the cell wide web of signals underline the notion that we are dealing with a multifunctional family of enzymes which, after 30 years of investigation, we are just beginning to understand.
Keywords: PKC; C1 domain; C2 domain; Calcium; Diacylglycerol; Phospholipid;
Lipid efflux by the ATP-binding cassette transporters ABCA1 and ABCG1 by Clara Cavelier; Iris Lorenzi; Lucia Rohrer; Arnold von Eckardstein (655-666).
Plasma levels of high-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) are inversely correlated with the risk of cardiovascular disease. One major atheroprotective mechanism of HDL and apoA-I is their role in reverse cholesterol transport, i.e., the transport of excess cholesterol from foam cells to the liver for secretion. The ATP-binding cassette transporters ABCA1 and ABCG1 play a pivotal role in this process by effluxing lipids from foam cells to apoA-I and HDL, respectively. In the liver, ABCA1 activity is one rate-limiting step in the formation of HDL. In macrophages, ABCA1 and ABCG1 prevent the excessive accumulation of lipids and thereby protect the arteries from developing atherosclerotic lesions. However, the mechanisms by which ABCA1 and ABCG1 mediate lipid removal are still unclear. Particularly, three questions remain controversial and are discussed in this review: (1) Do apoA-I and HDL directly interact with ABCA1 and ABCG1, respectively? (2) Does cholesterol efflux involve retroendocytosis of apoA-I or HDL? (3) Which lipids are directly transported by ABCA1 and ABCG1?
Keywords: Cholesterol efflux; apoA-I; HDL; Retroendocytosis; ABCA1; ABCG1;
Analysis of phospholipid species in rat peritoneal surface layer by liquid chromatography/electrospray ionization ion-trap mass spectrometry by Fei Gao; Xinkui Tian; Dawei Wen; Jie Liao; Tao Wang; Huwei Liu (667-676).
The main phospholipids in rat peritoneal surface layer were analyzed by normal-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) ion-trap mass spectrometry (MS). By using a silica gel column and a gradient of hexane/isopropanol/water as mobile phase containing 5 mmol/L ammonium formate as modifiers, a baseline separation of glycerophosphoehtanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and lyso-phosphatidylcholine (LPC) was obtained and more than 90 phospholipid constituents in rat peritoneal surface were identified and determined by on-line ion-trap MS detection. The major ethanolamine glycerophospholipids in rat peritoneal surfaces were plasmalogens that were highly enriched in polyunsaturated fatty acids at the sn-2 position. In addition, the fragmentation patterns for each phospholipid class by the ion-trap MS were discussed.
Keywords: Phospholipids; Peritoneal surface; HPLC; Ion-trap mass spectrometry;
Sterol dependent regulation of human TM7SF2 gene expression: Role of the encoded 3β-hydroxysterol Δ14-reductase in human cholesterol biosynthesis by Anna Maria Bennati; Marilena Castelli; Maria Agnese Della Fazia; Tommaso Beccari; Donatella Caruso; Giuseppe Servillo; Rita Roberti (677-685).
3β-hydroxysterol Δ14-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3β-hydroxysterol Δ14-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3β-hydroxysterol Δ14-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3β-hydroxysterol Δ14-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Δ14-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3β-hydroxysterol Δ14-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4- and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3β-hydroxysterol Δ14-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.
Keywords: TM7SF2; delta14-sterol reductase; lamin B receptor; cholesterol biosynthesis; gene expression; Greenberg skeletal dysplasia;
Analysis of the 5-lipoxygenase promoter and characterization of a vitamin D receptor binding site by Bernd L. Sorg; Niko Klan; Sabine Seuter; David Dishart; Olof Rådmark; Andreas Habenicht; Carsten Carlberg; Oliver Werz; Dieter Steinhilber (686-697).
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor beta (TGFβ) potently induce 5-lipoxygenase (5-LO) in myeloid cells. We analyzed vitamin D receptor (VDR) binding to putative vitamin D response elements within the 5-LO promoter and analyzed its function by reporter gene analysis. Binding of VDR and retinoid X receptor to the promoter region was shown in DNase I footprinting, electrophoretic mobility shift and chromatin immunoprecipitation assays. However, the identified VDR binding region did not mediate induction of reporter gene activity by 1,25(OH)2D3/TGFβ, neither in the 5-LO promoter context nor with the thymidine kinase (tk) promoter. Insertion of the rat atrial natriuretic factor VDRE in reporter plasmids containing the 5-LO promoter diminished induction by 1,25(OH)2D3/TGFβ as compared with the tk promoter. Similarly, low inductions were obtained when cells were transiently or stably transfected with constructs containing various 5-LO promoter regions. Concerning basal promoter activity, we identified a positive regulatory region (−779 to −229), which includes the VDR binding region, in 5-LO-positive MonoMac6 cells. In summary, the VDR/RXR complex binds to putative VDREs in the 5-LO promoter, but other sequences outside the 5-LO promoter seem to be responsible or additionally required for the prominent induction of 5-LO mRNA expression by 1,25(OH)2D3 and TGFβ.
Keywords: 5-lipoxygenase; Promoter; Vitamin D; Transforming growth factor beta;
Overexpression of SND p102, a rat homologue of p100 coactivator, promotes the secretion of lipoprotein phospholipids in primary hepatocytes by Lourdes Palacios; Begoña Ochoa; María José Gómez-Lechón; José Vicente Castell; Olatz Fresnedo (698-708).
SND p102 belongs to an evolutionarily conserved family of proteins first described as transcriptional coactivators, whose biological function has not yet been defined. High expression levels of homologues of SND p102 in non-nuclear compartments of lipid secretory tissues and in murine liver endoplasmic reticulum suggest a role for SND p102 in lipoprotein secretion in hepatocytes. To address this issue, after ascribing by confocal microscopy and Western blotting a non-nuclear localization of SND p102 in rat hepatocytes, we cloned its full-length cDNA, developed adenoviral vectors encoding the cDNA or a specific antisense sequence, and characterized the lipoprotein particles created and released for 24 h by transfected rat hepatocytes. The cellular ability to secrete apoB and apoA-I was not affected by SND p102 differential expression, nor was that of lipoproteins-triglyceride, -cholesterol and -cholesteryl esters. However, cells overexpressing SND p102 secreted phospholipid-rich lipoproteins. Compared with hepatocytes with basal or attenuated SND p102 expression, they secreted ∼45% and 80% more phospholipid in d < 1.015 g/mL and 1.015 < d < 1.24 g/mL lipoproteins, respectively. Oversecretion affected all phospholipid classes and did not significantly disturb the cellular phospholipid homeostasis. Collectively, the results suggest a specific, positive association of SND p102 and phospholipid release in lipoprotein particles in hepatocytes.
Keywords: SND p102; Phospholipid secretion; VLDL production; Adenoviral transfection;
Adiponectin plays an important role in efficient energy usage under energy shortage by Kiyomi Saito; Satoru Arata; Tomohiko Hosono; Yoshihiro Sano; Katsuhiko Takahashi; Nam-Ho Choi-Miura; Yasuko Nakano; Takashi Tobe; Motowo Tomita (709-716).
Adiponectin is an adipose tissue-specific secretory protein known to be an insulin-sensitizing protein. In this study, we generated adiponectin sense and antisense transgenic (Tg) mice to investigate whether adiponectin plays a role in the regulation of energy homeostasis during the growth stage. Spontaneous motor activity of antisense Tg mice were markedly reduced during fasting, particularly in young female mice, compared with wild type (Wt) and sense Tg mice. Furthermore, both body weight and adipose tissue mass of the antisense female Tg mice drastically reduced during fasting. To examine the relationship between the collapse of abdominal white adipose tissue (WAT) and serum adiponectin level, we measured the expression of genes related to energy expenditure, such as uncoupling protein (UCP). Notably, the mRNA of UCP1 in the WAT of antisense Tg female mice was markedly less than that of Wt mice and the UCP1 mRNA was strongly increased during fasting. These findings suggest that the serum adiponectin is important to maintaining energy homeostasis under energy shortage conditions, such as over female pubertal development.
Keywords: Adiponectin; Adipose tissue; Fasting; UCP;
Fatty acids bound to human serum albumin and its structural variants modulate apolipoprotein B secretion in HepG2 cells by Ji-Sook Ha; Andre Theriault; Nadhipuram V. Bhagavan; Chung-Eun Ha (717-724).
Epidemiologic studies have shown an inverse relationship between human serum albumin (HSA) levels and coronary heart disease (CHD). However, no mechanisms have been identified to explain this relationship. We hypothesized that this relationship is due to differences in binding affinity of fatty acids to HSA and subsequent atherogenic lipoprotein synthesis and secretion from hepatocytes. To test our hypothesis we undertook the current study. Using HepG2 cells, we demonstrated that oleic acid (OA) bound to HSA in a molar ratio of 4:1 and after incubation for 24 h stimulated apolipoprotein B (apoB) secretion. We also tested whether mutant forms of HSA could alter the binding affinity for fatty acids and change the availability of substrate for lipoprotein secretion. Based on the results obtained in this study using 11 HSA mutant proteins complexed with OA, we were able to classify into three major mutant groups based on their effects on apoB secretion. One group in particular (R410Q/Y411W, R410A/Y411A, and W214L/Y411W) showed a significantly diminished effect on apoB secretion when compared to the wild type HSA/OA complex. Furthermore, the amount of free OA internalized in HepG2 cells in the presence of HSA mutant proteins was in good agreement with the effects seen on apoB secretion by the various HSA mutants. This suggests that some mutant forms of HSA might potentially bind fatty acids with a much higher binding affinity and thus deprive fatty acids available for lipoprotein assembly in hepatocytes. In conclusion, our data illustrate that certain HSA polymorphic forms may be protective against the development of CHD and warrants further investigation.
Keywords: Human serum albumin; In vitro protein expression; Site-directed mutagenesis; HepG2 cell; Fatty acid; Apolipoprotein B100;
Arabidopsis thaliana expresses two functional isoforms of Arvp, a protein involved in the regulation of cellular lipid homeostasis by Oriol Forés; Montserrat Arró; Albert Pahissa; Sergi Ferrero; Melody Germann; Joseph Stukey; Virginia McDonough; Joseph T. Nickels; Narciso Campos; Albert Ferrer (725-735).
Arv1p is involved in the regulation of cellular lipid homeostasis in the yeast Saccharomyces cerevisiae. Here, we report the characterization of the two Arabidopsis thaliana ARV genes and the encoded proteins, AtArv1p and AtArv2p. The functional identity of AtArv1p and AtArv2p was demonstrated by complementation of the thermosensitive phenotype of the arv1Δ yeast mutant strain YJN1756. Both A. thaliana proteins contain the bipartite Arv1 homology domain (AHD), which consists of an NH2-terminal cysteine-rich subdomain with a putative zinc-binding motif followed by a C-terminal subdomain of 33 amino acids. Removal of the cysteine-rich subdomain has no effect on Arvp activity, whereas the presence of the C-terminal subdomain of the AHD is critical for Arvp function. Localization experiments of AtArv1p and AtArv2p tagged with green fluorescent protein (GFP) and expressed in onion epidermal cells demonstrated that both proteins are exclusively targeted to the endoplasmic reticulum. Analysis of β-glucuronidase (GUS) activity in transgenic A. thaliana plants carrying chimeric ARV1::GUS and ARV2::GUS genes showed that ARV gene promoters direct largely overlapping patterns of expression that are restricted to tissues in which cells are actively dividing or expanding. The results of this study support the notion that plants, yeast and mammals share common molecular mechanisms regulating intracellular lipid homeostasis.
Keywords: Arabidopsis; Lipid homeostasis; Sterol; Sphingolipid; Plant; Yeast;
Human originated bacteria, Lactobacillus rhamnosus PL60, produce conjugated linoleic acid and show anti-obesity effects in diet-induced obese mice by Hui-Young Lee; Jong-Hwan Park; Seung-Hyeok Seok; Min-Won Baek; Dong-Jae Kim; Ki-Eun Lee; Kyung-Soo Paek; Yeonhee Lee; Jae-Hak Park (736-744).
Many previous studies have reported that conjugated linoleic acid could be produced by starter culture bacteria, but the effects of the bacteria were not investigated. Moreover, there was no evidence of the conjugated linoleic acid-producing bacteria having potential health or nutritional effects related to conjugated linoleic acid, including reducing body fat. Here, we investigated the anti-obesity effect of Lactobacillus rhamnosus PL60, a human originated bacterium that produces t10, c12-conjugated linoleic acid, on diet-induced obese mice. After 8 weeks of feeding, L. rhamnosus PL60 reduced body weight without reducing energy intake, and caused a significant, specific reduction of white adipose tissue (epididymal and perirenal). Although the size of epididymal adipocytes was not reduced by L. rhamnosus PL60, apoptotic signals and UCP-2 mRNA levels increased in adipose tissue. Liver steatosis, a well known side effect of CLA, was not observed by L. rhamnosus PL60 treatment; on the contrary it seemed to be normalized. Results showed that the amount of conjugated linoleic acid produced by Lactobacillus rhamnosus PL60 was enough to produce an anti-obesity effect.
Keywords: Conjugated linoleic acid; Lactobacillus rhamnosus; Diet induced obese; Anti-obesity; Mouse;
Analysis of expression of secreted phospholipases A2 in mouse tissues at protein and mRNA levels by Leena I. Eerola; Fanny Surrel; Timo J. Nevalainen; Michael H. Gelb; Gérard Lambeau; V. Jukka O. Laine (745-756).
Secreted phospholipases A2 (sPLA2) form a group of low-molecular weight enzymes that catalyze the hydrolysis of phospholipids. Some sPLA2s are likely to play a role in inflammation, cancer, and as antibacterial enzymes in innate immunity. We developed specific and sensitive time-resolved fluroimmunoassays (TR-FIA) for mouse group (G) IB, GIIA, GIID, GIIE, GIIF, GV and GX sPLA2s and measured their concentrations in mouse serum and tissues obtained from both Balb/c and C57BL/6J mice. We also analyzed the mRNA expression of the sPLA2s by quantitative real-time reverse transcriptase PCR (qPCR). In most tissues, the concentrations of sPLA2 proteins corresponded to the expression of sPLA2s at the mRNA level. With a few exceptions, the sPLA2 proteins were found in the gastrointestinal tract. The qPCR results showed that GIB sPLA2 is synthesized widely in the gastrointestinal tract, including esophagus and colon, in addition to stomach and pancreas. Our results also suggest that the loss of GIIA sPLA2 in the intestine of GIIA sPLA2-deficient C57BL/6J mice is not compensated by other sPLA2s under normal conditions. Outside the gastrointestinal tract, sPLA2s were expressed occasionally in a number of tissues. The TR-FIAs developed in the current study may serve as useful tools to measure the levels of sPLA2 proteins in mouse serum and tissues in various experimental settings.
Keywords: Mouse; mRNA; Protein concentration; Secreted phospholipase A2; Time-resolved fluoroimmunoassay; Tissue;
Microsomal preparations from plant and yeast acylate free fatty acids without prior activation to acyl-thioesters by Andrea Neal; Antoni Banaś; Walentyna Banaś; Ulf Ståhl; Anders S. Carlsson; Sten Stymne (757-764).
Acylation of fatty acids to hydroxy groups in cells generally require activation to a thioester (ACP or CoA) or transacylation from another oxygen ester. We now show that microsomal membranes from Arabidopsis leaves efficiently acylate free fatty acids to long chain alcohols with no activation of the fatty acids to thioesters prior to acylation. Studies of the fatty alcohol and fatty acids specificities of the reaction in membranes from Arabidopsis leaves revealed that long chain (C18–C24) unsaturated fatty alcohols and C18–C22 unsaturated fatty acids were preferred. Microsomal preparations from Arabidopsis roots and leaves and from yeast efficiently synthesized ethyl esters from ethanol and free fatty acids. This reaction also occurred without prior activation of the fatty acid to a thioester. The results presented strongly suggest that wax ester and ethyl ester formation are carried out by separate enzymes. The physiological significance of the reactions in plants is discussed in connection to suberin and cutin synthesis. The results also have implication regarding the interpretation of lipid metabolic experiments done with microsomal fraction.
Keywords: Wax ester; Ethyl ester; Free fatty acid; Acylation; Acyl-thioester; Alloxydim;
Lipid metabolism in human endothelial cells by Cécile Héliès-Toussaint; Ségolène Gambert; Pauline Roller; Sabine Tricot; Bernard Lacour; Alain Grynberg (765-774).
Although lipids are largely involved in cardiovascular physiopathology, the lipid metabolism in endothelial cells remains largely unknown. Human umbilical vein endothelial cells (HUVECs) were used to investigate the metabolism of complex lipids. The membrane phospholipid homeostasis results from both de novo synthesis and remodelling that ensures the fine tuning of the phospholipid fatty acid composition. Using [3H]-glycerol and phosphoderivatives we showed the efficiency of glycerolipid synthesis from glycerol (0.9 nmol h−1 mg proteins−1), but not from its phosphorylated form suggesting the requirement of a functional glycerol kinase in HUVECs. Conversely, the synthesis of triacylglycerols was very low (less than 5% of phospholipid synthesis). The incorporation rate of fatty acids into phospholipids showed that there is a specific fate for each fatty acid in respect to its chain length and saturation level. Moreover in steady state condition, increasing the long chain ω3 polyunsaturated fatty acids in the medium resulted in an increased polyunsaturated/saturated ratio in phospholipids (from 0.42 to 0.63). [14C]O2 was produced form either [14C]-glucose or [14C]-palmitate indicating the functionality of the oxidation pathways, although β-oxidation was less efficient than glucose oxidation. The endothelial cell lipid metabolism involves conventional pathways, with functional rates largely slower than in hepatocytes or in cardiomyocytes.
Keywords: HUVEC; Phospholipid; Glycerol; Oxidation;
A ten-residue domain (Y11–A20) in the NH2-terminus modulates membrane association of annexin A7 by Avinash Chander; Devendra G Naidu; Xiao-Liang Chen (775-784).
Annexin A7 (synexin, annexin VII) is postulated to promote membrane fusion during surfactant secretion in alveolar type II cells and catecholamine secretion in adrenal chromaffin cells. Recently, we demonstrated that the 1–29 residues in the NH2-terminus could, possibly by interaction with the COOH-terminus, influence the Ca2+-dependent membrane binding, aggregation, and fusion properties of annexin A7 (A7). In this study, we further investigated this 29-residue domain by evaluating several deletion and point mutations for membrane-associated functions of A7. In comparison to A7, the mutants lacking 1–29 residues (A7Δ1–29) or 1–21 residues (A7Δ1–21), but not those lacking 1–10 residues (A7Δ1–10) or 21–29 residues (A7Δ21–29), showed diminished membrane binding. Segmental deletion of 10–20 residues (A7Δ10–20) also decreased the protein binding to membranes. The Ca2+-dependent membrane aggregation of PLV with A7Δ1–29 was maximally diminished but less so with A7Δ10–20 or A7Δ1–21 in comparison to that with A7. However, phospholipid vesicle (PVL) aggregation was unaffected with A7Δ1–10 or A7Δ21–29. The Ca2+-dependent membrane fusion of PLV was also diminished with A7Δ10–20 and A7Δ1–29, but not with A7Δ1–10. Since the mode of annexin A7 association and function with biological membranes could be different, we also evaluated these proteins for functional changes with isolated lung lamellar bodies. In comparison to A7, the binding to lamellar bodies was diminished for A7Δ1–29 and A7Δ1–21 but not for A7Δ1–10. The Ca2+-dependent fusion of isolated lamellar bodies with PLV was also diminished with A7Δ1–29, but not with A7Δ10–20 or A7Δ1–21. Taken together, our studies suggest that the 10-residue domain (Y11–A20) in the NH2-terminus modifies the phospholipid binding and aggregation properties of annexin A7. For binding and fusion of biological membranes, the 10–29-residue domain may be required although the annexin A7 properties are primarily modulated through the Y11–A20 domain.
Keywords: Structure–function analysis; Membrane aggregation; Membrane fusion; Lung surfactant secretion; Lung lamellar body; Protein fluorescence; Protein unfolding; Biological membrane; Recombinant protein;
Lung phospholipid metabolism in transgenic mice overexpressing peroxiredoxin 6 by Aron B. Fisher; Chandra Dodia; Kevin Yu; Yefim Manevich; Sheldon I. Feinstein (785-792).
Previous studies with peroxiredoxin 6 (Prdx6) null mice demonstrated that the phospholipase A2 activity of this enzyme plays a major role in lung phospholipid metabolism. This study evaluated lung phospholipid metabolism in transgenic mice that over-express Prdx6. Lung lysosomal type PLA2 activity in transgenic mice was 222% of wild type in lung homogenate and 280% in isolated lamellar bodies. Total phospholipid, phosphatidylcholine (PC) and disaturated PC were decreased approximately 20–35% in bronchoalveolar lung fluid, lung homogenate, and lung lamellar bodies in transgenic mice although lung compliance and type 2 cell ultrastructure were unaltered. To study metabolism, unilamellar liposomes (3H-DPPC: PC: cholesterol: PG, 10: 5: 3: 2 mol fraction) were instilled endotracheally in anesthetized mice and lungs were removed for perfusion. Compared to wild type, transgenic mice showed similar net uptake of liposomes in 2 h, but significantly increased 3H-DPPC degradation (38.9 ± 1.1 vs. 29.0 ± 1.3% of recovered dpm). The PLA2 competitive inhibitor MJ33 decreased degradation to 15% of recovered dpm in both transgenic and wild type lungs. Incorporation of [14C] palmitate into DSPC at 24 h after its intravenous injection was markedly increased in both the lung surfactant (+100%) and lamellar bodies (+188%) while incorporation of [3H] choline was increased by only 10–20%. These results indicate increased DPPC degradation and synthesis by the reacylation pathway with Prdx6 overexpression and provide additional evidence that the PLA2 activity of Prdx6 has an important role in lung surfactant turnover.
Keywords: Phospholipase A2; Phospholipid remodeling; Phospholipid synthesis; Lung surfactant;
Cytokine-induced monocyte adhesion to endothelial cells involves platelet-activating factor: Suppression by conjugated linoleic acid by Alan A. Sneddon; Ewan McLeod; Klaus W.J. Wahle; John R. Arthur (793-801).
Monocyte–endothelium interaction is key to many acute and chronic inflammatory diseases. We have investigated the factors regulating monocyte attachment to cytokine-activated human umbilical vein endothelial cells (HUVEC) and the modulatory effect of the polyunsaturated fatty acid (PUFA), conjugated linoleic acid (CLA) in this process. Both TNF-α and IL-1β induced HUVEC platelet-activating factor (PAF) production and PAF was required for subsequent firm THP-1 monocyte adhesion since it was inhibited by both PAF receptor antagonists (BN-52021 or CV-6209) and a PAF synthesis inhibitor (sanguinarine). CLA inhibited the binding of both THP-1 and isolated human peripheral blood monocytes to HUVEC by up to 40% with the CLA t10,c12 isomer suppressing adhesion dose-dependently. Investigation into the mechanism involved demonstrated that with IL-1β, VCAM-1 and ICAM-1 levels and pro-inflammatory cytokine expression were largely unaffected by CLA. Through the use of PAF receptor antagonists and PAF synthesis inhibitors, CLA was shown to inhibit cytokine-induced binding by suppressing PAF production. Direct assay of PAF levels confirmed this result. We conclude that endothelial-generated PAF plays a central role in cytokine-induced monocyte adherence to endothelium and that the anti-inflammatory action of PUFAs such as CLA in suppressing monocyte–endothelial interaction is mediated through attenuation of pro-inflammatory phospholipids such as PAF.
Keywords: CLA; Inflammation; PUFA; Phospholipid; Cell adhesion; PAF antagonist;