BBA - Molecular and Cell Biology of Lipids (v.1635, #2-3)

The functional relationship between ganglioside GM3 and two tyrosine-kinase receptors, the normal protein p185c-neu and the mutant oncogenic protein p185neu, was examined in HC11 cells and in MG1361 cells, respectively. In the former, p185c-neu expression and activation are controlled by EGF addition to the culture medium and by epidermal growth factor receptor (EGFR) activity, whereas the latter express unchangingly high levels of constitutively activated p185neu. Studies were carried out using (±)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ganglioside biosynthesis resulting in ganglioside depletion, and addition of exogenous GM3 to the culture medium. In HC11 cells treated with only [D]-PDMP, p185c-neu levels remain similar to control cells, whereas levels of tyrosine-phosphorylated p185c-neu increase after treatment with [D]-PDMP in combination with EGF. When exogenous GM3 is added in combination with [D]-PDMP and EGF, the enhanced phosphorylated-p185c-neu returns to control levels. Interestingly, EGFR levels also vary and, analogously to phosphorylated-p185c-neu, the increase of EGFR content consequent to the [D]-PDMP and EGF addition is reversed by exogenous GM3. In contrast, the addition of neither [D]-PDMP nor exogenous GM3 modifies expression and tyrosine-phosphorylation levels of p185neu in MG1361 cells. These findings indicate that changes in GM3 content modulate the tyrosine-phosphorylated p185c-neu levels in a reversible manner, but this is not specific for p185c-neu because EGFR levels are also modified. Furthermore, these data suggest that GM3 may play a functional role by affecting the internalisation pathway of p185c-neu/EGFR heterodimers, but not of p185neu homodimers.
Keywords: neu; erbB2; EGFR; Ganglioside; HC11 cell line; MG1361 cell line;

l-α-Glycerylphosphorylcholine inhibits the transfer function of phosphatidylinositol transfer protein α by Hiroaki Komatsu; Jan Westerman; Gerry T. Snoek; Theodore F. Taraschi; Nathan Janes (67-74).
Phosphatidylinositol transfer protein α (PITP-α) is a bifunctional phospholipid transfer protein that is highly selective for phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho). Polar lipid metabolites, including l-α-glycerylphosphorylcholine (GroPCho), increasingly have been linked to changes in cellular function and to disease. In this study, polar lipid metabolites of PtdIns and PtdCho were tested for their ability to influence PITP-α activity. GroPCho inhibited the ability of PITP-α to transfer PtdIns or PtdCho between liposomes. The IC50 of both processes was dependent on membrane composition. d-myo-inositol 1-phosphate and glycerylphosphorylinositol modestly enhanced PITP-α-mediated phospholipid transfer. Choline, phosphorylcholine (PCho), CDP-choline, glyceryl-3-phosphate, myo-inositol and d-myo-inositol 1,4,5-trisphosphate had little effect. Membrane surface charge was a strong determinant of the GroPCho inhibition with the inhibition being greatest for highly anionic membranes. GroPCho was shown to enhance the binding of PITP-α to anionic vesicles. In membranes of low surface charge, phosphatidylethanolamine (PtdEtn) was a determinant enabling the GroPCho inhibition. Anionic charge and PtdEtn content appeared to increase the strength of PITP-α-membrane interactions. The GroPCho-enhanced PITP-α-membrane binding was sufficient to cause inhibition, but not sufficient to account for the extent of inhibition observed. Processes associated with strengthened PITP-α-membrane binding in the presence of GroPCho appeared to impair the phospholipid insertion/extraction process.
Keywords: Choline alfoscerate; Lipid transfer; Phosphatidylethanolamine; Polyphosphoinositide signaling; Signal transduction;

The effects of four conjugated linoleic acid (CLA) isomers on in vitro collagen-induced human platelet aggregation and thromboxane (TXB2, the inactive metabolite of the proaggregatory TXA2) production were examined. As the free fatty acid (FFA), 9t, 11t-CLA was the most effective inhibitor of these two processes (I50s of 2.2 and 4 μM, respectively) and the 9c, 11c-CLA was the least effective (I50s of 8.3 and 37 μM) of the isomers tested. When platelets were preesterified with either 25 μM 9t, 11t-CLA or 9c, 11c-CLA, CLA incorporation in total platelet lipids increased from 0.24% to 0.31% and 0.38%, and most of this increase was found to be in the phosphatidyl choline and phosphatidyl ethanolamine subclasses. The decrease in arachidonic acid (AA) content in total fatty acids or phospholipids was an order of magnitude greater. Furthermore, no significant differences between platelets prelabeled with either 9t, 11t- or 9c, 11c-CLA in the inhibition of collagen-induced aggregation and TXB2 formation were observed. However, platelets prelabeled with 9c, 11c-CLA stimulated basal TXB2 production (4-fold) which was not observed with platelets pretreated with either 9t, 11t-CLA, linoleic acid or stearic acid. This enhancement was associated with a 2.4–5-fold increase in the release of endogenous AA. Our results suggest that the presence of a conjugated cis, cis double bond appears to change the lipid environment sufficiently to stimulate the basal platelet phospholipase activity, which in turn increases the formation of TXB2.
Keywords: Linoleic acid isomer; Platelet function; Arachidonic acid;

Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium by Takashi Naka; Nagatoshi Fujiwara; Ikuya Yano; Shinji Maeda; Matsumi Doe; Miki Minamino; Norikazu Ikeda; Yoshiko Kato; Kazuhito Watabe; Yoshio Kumazawa; Ikuko Tomiyasu; Kazuo Kobayashi (83-92).
The unique feature of the genus Sphingobacterium is the presence of sphingophospholipids and ceramides, besides diacylglycerophospholipids. As major cellular lipid components, five kinds of sphingophospholipids were purified from Sphingobacterium spiritivorum ATCC 33861T, the type species of genus Sphingobacterium. They were identified as ceramide phosphorylethanolamines (CerPE-1 and CerPE-2), ceramide phosphoryl-myo-inositols (CerPI-1 and CerPI-2), and ceramide phosphorylmannose (CerPM-1). The ceramide of CerPE-1, CerPI-1, and CerPM-1 was composed of 15-methylhexadecasphinganine (isoheptadeca sphinganine, iso-C17:0) and 13-methyltetradecanoic acid (isopentadecanoic acid, iso-C15:0), whereas that of CerPE-2 and CerPI-2 was composed of isoheptadeca sphinganine and 2-hydroxy-13-methyltetradecanoic acid (2-hydroxy isopentadecanoic acid, 2-OH iso-C15:0). These sphingophospholipids were also found in cellular lipids of Sphingobacterium multivorum ATCC 33613T, Sphingobacterium mizutaii ATCC 33299T, Sphingobacterium faecium IFO 15299T, Sphingobacterium thalpophilum ATCC 43320T, and Sphingobacterium antarcticum ATCC 51969T. To our knowledge, the existence of CerPM-1 is a novel sphingophospholipid through eukaryotic and prokaryotic cells.
Keywords: Sphingobacterium; Sphingophospholipid; Ceramide; Bacteria; Taxonomy;

Inhibition of apolipoprotein B secretion by taurocholate is controlled by the N-terminal end of the protein in rat hepatoma McArdle-RH7777 cells by Baukje M. Elzinga; Julius F.W. Baller; Arjen R. Mensenkamp; Zemin Yao; Luis B. Agellon; Folkert Kuipers; Henkjan J. Verkade (93-103).
Bile salts (BS) inhibit the secretion of apolipoprotein B (apoB) and triacylglycerol (TG) in primary rat, mouse and human hepatocytes and in mice in vivo. We investigated whether lipidation of apoB into a lipoprotein particle is required for this inhibitory action of BS. The sodium/taurocholate co-transporting polypeptide (Ntcp) was co-expressed in McArdle-RH7777 (McA-RH7777) cells stably expressing the full-length human apoB100 (h-apoB100, secreted as TG-rich lipoprotein particles) or carboxyl-truncated human apoB18 (h-apoB18, secreted in lipid-free form). The doubly transfected cell lines (h-apoB/r-Ntcp) effectively accumulated taurocholic acid (TC). TC incubation decreased the secretion of endogenous rat apoB100 (−50%) and h-apoB18 (−35%), but did not affect secretion of rat apoA-I. Pulse-chase experiments (35S-methionine) indicated that the impaired secretion of radiolabeled h-apoB18 and h-apoB100 was associated with accelerated intracellular degradation. The calpain protease inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) partially inhibited intracellular apoB degradation but did not affect the amount of either h-apoB18 or h-apoB100 secreted into the medium, indicating that inhibition of apoB secretion by TC is not due to calpain-dependent proteasomal degradation. We conclude that TC does not inhibit apoB secretion by interference with its lipidation, but rather involves a mechanism dependent on the N-terminal end of apoB.
Keywords: Very-low-density lipoprotein; Apolipoprotein B; McA-RH7777; Bile salt; Taurocholate; Degradation;

Regulation of sphingosine kinase 1 gene expression by protein kinase C in a human leukemia cell line, MEG-O1 by Yusuke Nakade; Yoshiko Banno; Keiko T-Koizumi; Kazumi Hagiwara; Sayaka Sobue; Masahiro Koda; Motoshi Suzuki; Tetsuhito Kojima; Akira Takagi; Haruhiko Asano; Yoshinori Nozawa; Takashi Murate (104-116).
The prolonged treatment with phorbol 12-myristate 13-acetate (PMA) of a human megakaryoblastic leukemia cell line, MEG-O1, induced increase of sphingosine kinase (SPHK) enzyme activity and SPHK1 protein expression as well as SPHK1 message. Protein kinase C (PKC) inhibitor prevented the PMA-induced SPHK1 gene expression. To elucidate the regulatory mechanism of this gene expression, we examined the promoter area (distal to the first exon) and its binding proteins. Luciferase analyses showed that the area of 300 bp from the first exon was sufficient for PMA-responsiveness, and that specificity protein 1 (Sp1)- and two activator protein 2 (AP-2)-binding motifs within this area were necessary for responsiveness. Inhibitors for PKC™ and MEK1 decreased this PMA-induced promoter activity. Electrophoresis mobility shift assay (EMSA) showed that Sp1 protein was originally bound to the Sp1 site and that two additional bands bound to the two AP-2 motifs were observed only when stimulated with PMA in MEG-O1 cells. The appearance of these bands resulted from binding to an unknown protein rather than AP-2. These results indicated that PMA up-regulates SPHK1 gene expression through PMA-responsive elements of the 5′ promoter area of the gene, and suggested that PMA-mediated SPHK1 gene expression would be mediated via PKC- and ERK-dependent signal transduction pathway by binding the transcription factor to AP-2 motifs.
Keywords: SPHK1 gene expression; PMA; PKC; MEG-O1 cell; Promoter analysis; AP-2 site;

Low density lipoprotein induces eNOS translocation to membrane caveolae: the role of RhoA activation and stress fiber formation by Yi Zhu; Hai-Ling Liao; Xiao-Lin Niu; Yuan Yuan; Tong Lin; Lynne Verna; Michael B. Stemerman (117-126).
A decrease in the bioavailability of endothelium-derived nitric oxide (NO) is linked to hypercholesterolemia. However, the mechanism by which low density lipoprotein (LDL) mediates endothelial NO synthase (eNOS) dysfunction remains controversial. We investigate the effect of LDL on eNOS regulation in human endothelial cells (ECs). In cultured ECs, a high level of LDL increased the abundance of eNOS and caveolin-1 (Cav-1) in the membrane caveolae and the association of eNOS with Cav-1. Furthermore, it decreased the basal level of NO and blocked NO production stimulated by the calcium ionophore A23187. LDL exposure also increased the formation of stress fibers and the membrane translocation of eNOS. These effects can be blocked by cytochalasin D, an actin cytoskeleton disruptor. In revealing the mechanism underlying the translocation of eNOS, we found that a high level of LDL increased the level of membrane-associated and GTP-formed RhoA and activated the RhoA downstream kinase ROCK-1 activity. Y-27632, a specific inhibitor of ROCK-1, blocked LDL-induced stress fiber formation, eNOS translocation and NO production. In conclusion, a high level of LDL increases the movement of eNOS to membrane caveolae via the increased stress fibers. The RhoA-mediated pathway may play a crucial role in this process in vascular ECs.
Keywords: eNOS; Caveolin-1; RhoA; LDL; Endothelial cell;

Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C by Manasa V. Gudheti; Yamaira I. Gonzalez; Sum P. Lee; Steven P. Wrenn (127-141).
Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5–20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.
Keywords: Apo A-I; PLC; Bile; Vesicle; Cholesterol; Apo A-I complex;