BBA - Molecular and Cell Biology of Lipids (v.1771, #7)

Diacylglycerol kinases: Why so many of them? by Fumio Sakane; Shin-ichi Imai; Masahiro Kai; Satoshi Yasuda; Hideo Kanoh (793-806).
Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. In addition to the C1 domains (protein kinase C-like zinc finger structures) conserved commonly in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile α motif domain and ankyrin repeats. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways conducting development, neural and immune responses, cytoskeleton reorganization and carcinogenesis. Moreover, there has been rapidly growing evidence indicating that individual DGK isoforms exert their specific roles through interactions with unique partner proteins such as protein kinase Cs, Ras guanyl nucleotide-releasing protein, chimaerins and phosphatidylinositol-4-phosphate 5-kinase. Therefore, an emerging paradigm for DGK is that the individual DGK isoforms assembled in their own signaling complexes should carry out spatio-temporally segregated tasks for a wide range of biological processes via regulating local, but not global, concentrations of DAG and/or PA.
Keywords: Diacylglycerol kinase; Phosphatidic acid; Isozyme; Protein kinase C; Signal transduction; C1 domain;

Scavenger receptor class B Type I (SR-BI) assembles into detergent-sensitive dimers and tetramers by Daisy Sahoo; Yolanda F. Darlington; Diana Pop; David L. Williams; Margery A. Connelly (807-817).
High density lipoproteins (HDL) are protective against cardiovascular disease due to their important role in the reverse cholesterol transport (RCT) pathway. The selective transfer of cholesteryl ester (CE) from the HDL core to cells, the last step in RCT, is mediated by scavenger receptor class B type I (SR-BI). SR-BI is a heavily glycosylated cell surface receptor that is highly expressed in the liver, ovaries, testes and adrenal glands, where selective uptake of HDL-CE is most prevalent. Previous studies have shown that SR-BI oligomerizes with itself in steroidogenic tissues as well as in diverse cell lines. In the present study, we provide further evidence for the homo-oligomerization of SR-BI. We show by FPLC and blue native PAGE that SR-BI forms complexes whose sizes suggest the formation of monomers, dimers, and tetramers. Interestingly, homo-oligomerization occurs even with the absence of SR-BI's C-terminal cytoplasmic domain. Finally, we report that an inhibitor of SR-BI-mediated cholesterol transport, BLT-1, and mutations in the putative leucine zipper region of SR-BI have profound effects on SR-BI function, however, they do not affect receptor self-association. These observations indicate that SR-BI homo-oligomerization occurs even when the receptor is non-functional.
Keywords: Scavenger receptor; SR-BI; Oligomerization; Tetramer; Selective uptake; Detergent;

Scavenger receptor class B, type I (SR-BI) homo-dimerizes via its C-terminal region: Fluorescence resonance energy transfer analysis by Daisy Sahoo; Yinan Peng; Jeffery R. Smith; Yolanda F. Darlington; Margery A. Connelly (818-829).
Expression of the scavenger receptor class B, type I (SR-BI) receptor facilitates high density lipoprotein cholesterol transport and correlates with protection against atherosclerosis. Studies have shown that SR-BI self-associates, but many of the techniques used to characterize SR-BI homo-oligomerization were wrought with the prospect of producing artifacts. Therefore, we employed fluorescence resonance energy transfer (FRET) to visualize SR-BI homo-oligomerization with the benefit of gaining information about its quaternary structure in the absence of typical membrane receptor artifacts. To this end, SR-BI was tagged at the N- or C-termini with either cyan (CFP) or yellow (YFP) fluorescent protein. To test whether SR-BI subunits oligomerize through N–N, N–C or C–C terminal interactions, we co-expressed the appropriate SR-BI fusion protein combinations in COS-7 cells and measured live-cell FRET following acceptor photobleaching. We did not observe FRET with co-transfection of SR-BI with CFP and YFP at the N-termini nor at the N- and C-termini, suggesting that the N-termini are not proximal to each other or to the C-termini. However, FRET was observed with co-transfection of SR-BI with CFP and YFP at the C-termini, suggesting that the C-terminal ends are within 10 nm of each other, consistent with SR-BI dimerization via its C-terminal region.
Keywords: Scavenger receptor class B, Type I; SR-BI; Fluorescence resonance energy transfer; FRET; Selective uptake; Oligomerization; Acceptor photobleaching;

Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV) by Magalí Pellon-Maison; Mauro A. Montanaro; Rosalind A. Coleman; María R. Gonzalez-Baró (830-838).
Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.
Keywords: Triacylglycerol synthesis; Protein targeting; Mitochondria-endoplasmic reticulum interaction;

Rifampicin-induced CYP3A4 activation in CTX patients cannot replace chenodeoxycholic acid treatment by Auryan Szalat; Pavel Gershkovich; Alon Ben-Ari; Aviv Shaish; Yael Liberman; Eti Boutboul; Marc Gotkine; Amnon Hoffman; Dror Harats; Eran Leitersdorf; Vardiella Meiner (839-844).
Cerebrotendinous xanthomatosis (CTX) is a rare neurodegenerative disorder with cholestanol accumulation resulting from mutations in the sterol 27-hydroxylase gene (CYP27A). Conventional treatment includes chenodeoxycholic acid and HMG-CoA reductase inhibitors. Mice with disrupted Cyp27A (Cyp27 KO) do not show elevated cholestanol levels nor develop CTX manifestations. This phenomenon was proposed to be due to murine CYP3A overexpression leading to an alternative pathway for degradation of bile alcohols including cholestanol. Our objective was to examine the influence of CYP3A4 induction on cholestanol elimination in CTX patients. Rifampicin (600 mg/day × 7 days), known to induce the PXR, and thereby to increase CYP3A activity, was used. The degree of CYP3A4 induction was assessed by comparing midazolam pharmacokinetics before and after rifampicin treatment. Cholestanol levels and cholestanol/cholesterol ratios were assayed during the experimental period and compared to a 3 weeks period without treatment. The results show that despite 60% increase in CYP3A4 activity following rifampicin treatment, there is no significant change in cholestanol levels. We conclude that up-regulated expression of CYP3A affects cholestanol elimination in mice differently as compared to its effect in CTX patients. Therefore, CYP3A4 inducers cannot replace chenodeoxycholic acid for the treatment of CTX.
Keywords: Cerebrotendinous Xanthomatosis; CYP3A4; Rifampicin; Cholestanol;

Probucol therapy overcomes the reproductive defect in CTP: phosphocholine cytidylyltransferase β2 knockout mice by Christopher Gunter; Matthew Frank; Yong Tian; K. Gopal Murti; Jerold E. Rehg; Suzanne Jackowski (845-852).
The synthesis of phosphatidylcholine (PtdCho), the major phospholipid in mammalian cells, is regulated by the CTP:phosphocholine cytidylyltransferase (CCT). Loss of the CCTβ2 isoform expression in mice results in gonadal dysfunction. CCTβ2−/− females exhibit ovarian tissue disorganization with progressive loss of follicle formation and oocyte maturation. Ultrastructure revealed a disrupted association between ova and granulosa cells and disorganized Golgi apparati in oocytes of CCTβ2−/− mice. Probucol is a cholesterol-lowering agent that stimulates the uptake and retention of lipids carried by lipoproteins in peripheral tissues. Probucol therapy significantly lowered both serum cholesterol and PtdCho levels. Probucol therapy increased fertility in the CCTβ2−/− females 100%, although it did not completely correct the phenotype, the morphological abnormalities in the knockout ovaries or itself stimulate CCT activity directly. These data indicated that a deficiency in de novo PtdCho synthesis could be complemented by altering the metabolism of serum lipoproteins, an alternative source for cellular phospholipid.
Keywords: Phosphatidylcholine; Cytidylyltransferase; Gene knockout; Lipoprotein; Ovary; Fertility;

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for a detailed analysis of cellular phospholipid and cholesterol efflux in free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages (HMDM) loaded with enzymatically modified LDL (E-LDL). Although both cell models differed significantly in their cellular lipid composition, a higher apoA-I specific efflux was found for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species in both cell types. Moreover, medium chain sphingomyelin (SPM) species SPM 14:0 and SPM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, HMDM displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL3 mediated lipid efflux from HMDM using D9-choline and 13C3-FC stable isotope labeling revealed significantly different D9-PC and D9-SPM species pattern for apoA-I and HDL3 specific efflux media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL3 mediated efflux. Together with a partial loading of fibroblasts and HMDM with HDL3-derived CE species, these data add further evidence for retroendocytosis of HDL. In summary, analysis of apoA-I/ABCA1 and HDL3 mediated lipid efflux by ESI-MS/MS demonstrated a preferential efflux of monounsaturated PC and medium chain SPM to apoA-I. Moreover, this is the first study, which provides evidence for distinct cellular phospholipid pools used for lipid transfer to apoA-I and HDL3 from the analysis of phospholipid species pattern in HMDM.
Keywords: Tandem mass spectrometry; Stable isotope labeling; Electrospray ionization; Quantitative lipid analysis; Lipidomics;

Functional characterization of vitamin D responding regions in the human 5-Lipoxygenase gene by Sabine Seuter; Sami Väisänen; Olof Rådmark; Carsten Carlberg; Dieter Steinhilber (864-872).
5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of proinflammatory leukotrienes. The 5-LO gene is a primary target of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and its expression is prominently increased during myeloid cell differentiation. Since no functional vitamin D response element (VDRE) has been reported for this gene so far, we performed in silico screening of the whole 5-LO gene area (84 kb, including 10 kb promoter region) and identified 22 putative VDREs. Both gelshift and reporter gene assays identified four of these candidates as functional VDREs. Their approximate positions are − 2,250 (promoter), + 21,400 (intron 2), + 42,000 (intron 4) and + 50,600 (intron 5) in relation to the transcription start site (TSS). Remarkably, the VDRE at position + 42,000 is one of the strongest known VDREs of the human genome. Chromatin immunoprecipitation (ChIP) assays demonstrated simultaneous association of vitamin D receptor (VDR), retinoid X receptor (RXR) and RNA polymerase II (Pol II) to the 5-LO gene regions containing two of these four putative VDREs. This indicates DNA looping of the TSS to even very distant gene regions. In summary, we suggest that the upregulation of the primary 1α,25(OH)2D3 target 5-LO is mediated in vivo by a prominent VDRE in intron 4.
Keywords: 5-LO; VDR; Chromatin; In silico screening; Vitamin D response elements;

Ganglioside GM3 is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2 by Simona Milani; Elena Sottocornola; Stefania Zava; Patrizia Berselli; Bruno Berra; Irma Colombo (873-878).
Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM3 in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM3 upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors co-immunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM3, but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM3 is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM3 on ErbB2 activation are mediated by EGFR.
Keywords: ErbB2; EGFR; GM3; Gangliosides; HC11 cell line;

Hyperglycemia down-regulates apolipoprotein M expression in vivo and in vitro by Xiaoying Zhang; Bo Jiang; Guanghua Luo; Peter Nilsson-Ehle; Ning Xu (879-882).
Diabetes is associated with low concentrations of apoM in plasma. In db/db mice, ob/ob mice as well as in the alloxan-induced diabetic mouse, the low apoM levels are paralleled by decreased expression of the apoM gene. In the latter model, insulin substitution tended to reverse the low apoM expression. It is not known whether the impairment in apoM expression can be ascribed to hyperglycemia, insulin deficiency or insulin resistance. In the present study, we investigated apoM levels and expression in rats rendered hyperglycemic by short-term glucose infusion. As expected, serum insulin concentrations rose moderately during the infusions. Serum apoM concentrations and hepatic apoM mRNA levels were significantly reduced in the hyperglycemic rats, indicating that the low expression of apoM in these diabetic models can be ascribed to hyperglycemia rather than to insulin deficiency or insulin resistance. However, in HepG2 cells both glucose and insulin markedly inhibited apoM expression these effects were additive. Thus, the possible effects of insulin in vivo seem to be mediated indirectly.
Keywords: Apolipoprotein M; Glucose; Insulin; Hyperglycemia; Diabetes;

Lysophosphatidic acid induces prostate cancer PC3 cell migration via activation of LPA1, p42 and p38α by Feng Hao; Mingqi Tan; Xuemin Xu; Jiahuai Han; Duane D. Miller; Gabor Tigyi; Mei-Zhen Cui (883-892).
Prostate cancer cell migration is an essential event both in the progression of prostate cancer and in the steps leading to metastasis. We report here that lysophosphatidic acid (LPA), a potent bioactive phospholipid, induces prostate cancer PC3 cell migration via the activation of the LPA1 receptor, which is linked to a PTX-sensitive activation mechanism of the mitogen-activated protein kinases (MAPK). Our results demonstrate that parallel activation of ERK1/2 and p38, but not JNK, is responsible for LPA-stimulated PC3 cell migration. Furthermore, using small interfering RNA (siRNA) technology, and overexpressing dominant-negative mutants of p38 MAPK isotypes of α, β, γ and δ, we have identified that the activation of ERK2 (p42) and p38α, but not of ERK1 and the other isoforms of p38 MAPK, is required for LPA-induced migration. Our study provides the first evidence for a functional role of p42 and p38α in LPA-induced mammalian cell migration, and also demonstrates, for the first time, that the receptor LPA1 mediates prostate cancer cell migration. The results of the present study suggest that LPA, the receptor LPA1, ERK2 and p38α are important regulators for prostate cancer cell invasion and thus could play a significant role in the development of metastasis.
Keywords: Lysophosphatidic acid; Receptors; Cell migration; Protein kinases and prostate cancer cells;

A role for high density lipoproteins in hepatic phosphatidylcholine homeostasis by Zhaoyu Li; Luis B. Agellon; Dennis E. Vance (893-900).
Choline is (95%) found largely in the biosphere as a component of phosphatidylcholine (PC) which is made from choline via the CDP-choline pathway. Animals obtain choline from both the diet and via endogenous biosynthesis that involves the conversion of phosphatidylethanolamine into PC by phosphatidylethanolamine N-methyltransferase (PEMT), followed by PC catabolism. We have uncovered a striking gender-specific conservation of choline in female mice that does not occur in male mice. Female Pemt / mice maintained hepatic PC/total choline levels during the first day of choline deprivation and escaped liver damage whereas male Pemt / mice did not. Plasma PC levels in high-density lipoproteins (HDLs) were higher in male Pemt / mice than those in females before choline deprivation. Interestingly, after choline deprivation for 1 day, female, but not male, Pemt / mice increased HDL-PC levels. Glybenclamide, an inhibitor of PC efflux mediated by ABC transporters, eliminated this response to choline deprivation in females. These data suggest that (i) increased PC efflux from extra-hepatic tissues to HDLs in the circulation provided sufficient choline for the liver and compensated for loss of hepatic PC during the initial stages of choline deprivation in female, but not male, Pemt / mice, and (ii) plasma HDL in female mice has an important function in maintenance of hepatic PC as an acute response to severe choline deprivation.
Keywords: Choline; Phosphatidylcholine; Phosphatidylethanolamine N-methyltransferase; High density lipoprotein; Choline deficiency; Glybenclamide;

Oxidation of chylomicron remnant-like particles inhibits their uptake by THP-1 macrophages by apolipoprotein E-dependent processes by Fatos Bejta; Elizabeth H. Moore; Michael Avella; Peter J. Gough; Keith E. Suckling; Kathleen M. Botham (901-910).
The influence of the oxidative state of chylomicron remnants (CMR) on the mechanisms of their uptake and induction of lipid accumulation by macrophages derived from the human monocyte cell line, THP-1, during foam cell formation was investigated using chylomicron-remnant-like particles (CRLPs) at 3 different levels of oxidation. The oxidative state of CRLPs was varied by exposure to CuSO4 (oxCRLPs) or incorporation of the antioxidant, probucol (pCRLPs) into the particles. oxCRLPs caused significantly less accumulation of triacylglycerol in the macrophages than CRLPs, and their rate of uptake was lower, while pCRLPs caused more lipid accumulation and were taken up faster. Uptake of all 3 types of particles was inhibited to a similar extent when entry via the low density lipoprotein (LDL) receptor related protein (80–90%), LDL receptor (− 30–40%), CD36 (− 40%) and phagocytosis (− 35–40%) was blocked using lactoferrin, excess LDL, anti-CD36 and cytochalasin D, respectively, but blocking scavenger receptors-A or -B1 using poly inosinic acid or excess HDL had no effect. These findings show that oxidation of CRLPs lowers their rate of uptake and induction of lipid accumulation in macrophages. However, oxidation does not change the main pathways of internalisation of CRLPs into THP-1 macrophages, which occur mainly via the LRP with some contribution from the LDLr, while CD36 and phagocytosis have only a minor role, regardless of the oxidative state of the particles. Thus, the effects of CMR oxidation on foam cell formation contrast sharply with those of LDL oxidation and this may be important in the role of dietary oxidized lipids and antioxidants in modulating atherosclerosis.
Keywords: Chylomicron remnants; Foam cells; Oxidized lipoproteins; Macrophages; Atherosclerosis;