BBA - Molecular and Cell Biology of Lipids (v.1735, #2)

Phosphatidylinositol biosynthesis: Biochemistry and regulation by Mary E. Gardocki; Niketa Jani; John M. Lopes (89-100).
Phosphatidylinositol (PI) is a ubiquitous membrane lipid in eukaryotes. It is becoming increasingly obvious that PI and its metabolites play a myriad of very diverse roles in eukaryotic cells. The Saccharomyces cerevisiae PIS1 gene is essential and encodes PI synthase, which is required for the synthesis of PI. Recently, PIS1 expression was found to be regulated in response to carbon source and oxygen availability. It is particularly significant that the promoter elements required for these responses are conserved evolutionarily throughout the Saccharomyces genus. In addition, several genome-wide strategies coupled with more traditional screens suggest that several other factors regulate PIS1 expression. The impact of regulating PIS1 expression on PI synthesis will be discussed along with the possible role(s) that this may have on diseases such as cancer.

Pulmonary surfactant protein A inhibits the lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria and microsomes by Ana M. Terrasa; Margarita H. Guajardo; Elizabeth de Armas Sanabria; Angel Catalá (101-110).
Reactive oxygen species play an important role in several acute lung injuries. The lung tissue contains polyunsaturated fatty acids (PUFAs) that are substrates of lipid peroxidation that may lead to loss of the functional integrity of the cell membranes. In this study, we compare the in vitro protective effect of pulmonary surfactant protein A (SP-A), purified from porcine surfactant, against ascorbate–Fe2+ lipid peroxidation stimulated by linoleic acid hydroperoxide (LHP) of the mitochondria and microsomes isolated from rat lung; deprived organelles of ascorbate and LHP were utilized as control. The process was measured simultaneously by chemiluminescence as well as by PUFA degradation of the total lipids isolated from these organelles. The addition of LHP to rat lung mitochondria or microsomes produces a marked increase in light emission; the highest value of activation was produced in microsomes (total chemiluminescence: 20.015 ± 1.735 × 105 cpm). The inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (2.5 to 5.0 μg) of SP-A in rat lung mitochondria and 2.5 to 7.5 μg of SP-A in rat lung microsomes. The inhibitory effect reaches the highest values in the mitochondria, thus, 5.0 μg of SP-A produces a 100% inhibition in this membranes whereas 7.5 μg of SP-A produces a 51.25 ± 3.48% inhibition in microsomes. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized membranes was found in the arachidonic acid content; this decreased from 9.68 ± 1.60% in the native group to 5.72 ± 1.64% in peroxidized mitochondria and from 7.39 ± 1.14% to 3.21 ± 0.77% in microsomes. These changes were less pronounced in SP-A treated membranes; as an example, in the presence of 5.0 μg of SP-A, we observed a total protection of 20:4 n-6 (9.41 ± 3.29%) in mitochondria, whereas 7.5 μg of SP-A produced a 65% protection in microsomes (5.95 ± 0.73%). Under these experimental conditions, SP-A produces a smaller inhibitory effect in microsomes than in mitochondria. Additional studies of lipid peroxidation of rat lung mitochondria or microsomes using equal amounts of albumin and even higher compared to SPA were carried out. Our results indicate that under our experimental conditions, BSA was unable to inhibit lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria or microsomes, thus indicating that this effect is specific to SP-A.
Keywords: Rat lung; Lipid peroxidation; Linoleic acid hydroperoxide; Pulmonary surfactant protein A;

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p by René G. Ott; Karin Athenstaedt; Claudia Hrastnik; Erich Leitner; Helmut Bergler; Günther Daum (111-118).
Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Δ mutation in erg3Δ background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Δ erg3Δ. This distribution is not result of the erg3Δ background, because in the erg3Δ strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Δ erg3Δ appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Δ, supplies sterol to the plasma membrane.
Keywords: Lanosterol; Ergosterol; Lanosterol C-14 demethylase; Yeast; Endoplasmic reticulum; Lipid particles;

P388D1 cells exposed to bacterial lipopolysaccharide (LPS) mobilize arachidonic acid (AA) for prostaglandin synthesis in two temporally distinct pathways. The “immediate pathway” is triggered within minutes by receptor agonists such as platelet-activating factor (PAF) but only if the cells have previously been primed with LPS for 1 h. The “delayed pathway” occurs in response to LPS alone over the course of several hours. We have now investigated the subcellular localization of both the Group IV cytosolic phospholipase A2 (cPLA2) and the Group V secreted PLA2 (sPLA2) during these two temporally distinct routes of AA release. We have prepared cells overexpressing fusion proteins of sPLA2-GFP and cPLA2-RFP. In the resting cells, cPLA2-RFP was uniformly located throughout the cytoplasm, and short-term treatment with LPS did not induce translocation to perinuclear and/or Golgi membranes. However, such a translocation occurred almost immediately after the addition of PAF to the cells. Long-term exposure of the cells to LPS led to the translocation of cPLA2-RFP to intracellular membranes after 3 h, and correlates with a significant release of AA in a cPLA2-dependent manner. At the same time period that the delayed association of cPLA2 with perinuclear membranes is detected, an intense fluorescence arising from the sPLA2-GFP was found around the nucleus in the sPLA2-GFP stably transfected cells. In parallel with these changes, significant AA release was detected from the sPLA2-GFP transfectants in a cPLA2-dependent manner, which may reflect cross-talk between sPLA2 and cPLA2. The subcellular localization of the Group VIA Ca2+-independent PLA2 (iPLA2) was also investigated. Cells overexpressing iPLA2-GFP showed no fluorescence changes under any activation condition. However, the iPLA2-GFP-expressing cells showed relatively high basal AA release, confirming a role for iPLA2 in basal deacylation reactions. These new data illustrate the subcellular localization changes that accompany the distinct roles that each of the three kinds of PLA2 present in P388D1 macrophages play in AA mobilization.
Keywords: Phospholipase A2; Arachidonic acid; Macrophage; Platelet-activating factor; Lipopolysaccharide; Prostaglandin;

The effect of dietary sphingolipids on plasma sphingomyelin metabolism and atherosclerosis by Zhiqiang Li; Maria J. Basterr; Tiruneh K. Hailemariam; Mohammad Reza Hojjati; Shendi Lu; Jin Liu; Ruijie Liu; Hongwen Zhou; Xian-Cheng Jiang (130-134).
Sphingomyelin (SM) plays a very important role in cell membrane formation and plasma lipoprotein metabolism. All these functions may have an impact on atherosclerotic development. To investigate the relationship between SM metabolism and atherosclerosis, we utilized a sphingolipid-rich diet to feed LDL receptor gene knockout (LDLr KO) mice and studied lipid metabolism and atherosclerosis in the mice. After 3 months of a sphingolipid-rich diet, we found a significant increase in SM, cholesterol, and SM/phosphatidylcholine (PC) ratio (50%, P  < 0.001; 62%, P  < 0.01; and 45%, P  < 0.01, respectively), compared to chow fed diet. HDL-lipids were not significantly altered. Non-HDL-SM, non-HDL-C, and non-HDL-SM/non-HDL-PC ratio were significantly increased (115%, P  < 0.001; 106%, P  < 0.001; and 106%, P  < 0.01, respectively). FPLC confirmed the results. SDS-PAGE showed an increase of apoB48 and apoB100, but no changes of apoAI. Moreover, we found that an SM-rich diet significantly increased atherosclerotic lesion area in both root assay and en face assay, compared to chow diet (58,210 ± 15,300 μm2 vs. 9670 ± 2370 μm2, P  < 0.001; 5.9 ± 3.1% vs. 1.1 ± 0.9%, P  < 0.001). These results indicate that the enrichment of sphingolipids in diet has proatherogenic properties.
Keywords: Plasma sphingomyelin; Lipoprotein; Low-density lipoprotein; High-density lipoprotein; Cholesterol; Atherosclerosis;

Activation of phospholipase A2 and MAP kinases by oxidized low-density lipoproteins in immortalized GP8.39 endothelial cells by Gabriella Lupo; Ambra Nicotra; Giovanni Giurdanella; Carmelina Daniela Anfuso; Loriana Romeo; Giulia Biondi; Cataldo Tirolo; Bianca Marchetti; Nicolò Ragusa; Mario Alberghina (135-150).
In immortalized rat brain endothelial cells (GP8.39), we have previously shown that oxidized LDL (oxLDL), after 24-h treatment, stimulates arachidonic acid release and phosphatidylcholine hydrolysis by activation of cytosolic phospholipase A2 (cPLA2). A putative role for MAPKs in this process has emerged. Here, we studied the contribution of Ca2+-independent phospholipase A2 (iPLA2), and the role of the MAP kinase family as well as both cPLA2 and iPLA2 mRNA expression by RT-PCR in oxLDL toxicity to GP8.39 cells in vitro. The activation of extracellular signal-regulated kinases ERK1/2, p38 and c-Jun NH2-terminal kinase (JNK) was assessed with Western blotting and kinase activity assays. iPLA2 activity, which was found as a membrane-associated enzyme, was more stimulated by oxLDL compared with native LDL. The phosphorylation of ERK1/2, p38 and JNKs was also significantly enhanced in a dose-dependent manner. PD98059, an ERK inhibitor, SB203580, a p38 inhibitor, and SP600125, an JNK inhibitor, abolished the stimulation of all three members of the MAPK family by oxLDL. Confocal microscopy analysis and subcellular fractionation confirmed either an increase in phosphorylated form of ERKs, p38 and JNKs, or their nuclear translocation upon activation. A strong inhibition of MAPK activation was also observed when endothelial cells were treated with GF109203X, a PKC inhibitor, indicating the important role of both PKC and all three MAPKs in mediating the maximal oxLDL response. Finally, compared with samples untreated or treated with native LDL, treatment with oxLDL (100 μM hydroperoxides) for 24 h significantly increased the levels of constitutively expressed iPLA2 protein (by 5.1-fold) and mRNA (by 3.1-fold), as well as cPLA2 protein (by 4.4-fold) and mRNA (by 1.5-fold). Together, these data link the stimulation of PKC–ERK–p38–JNK pathways and PLA2 activity by oxLDL to the prooxidant mechanism of the lipoprotein complex, which may initially stimulate the endothelial cell reaction against noxious stimuli as well as metabolic repair, such as during inflammation and atherosclerosis.
Keywords: Phospholipase A2; Low-density lipoprotein; MAP kinase; Endothelial cell; mRNA;