Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol.

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.

Isolation of a somatic cell mutant resistant to the induction of apoptosis by oxidized low density lipoprotein.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 microg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OX(R)), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OX(R) showed no enhanced calcium uptake in response to 25-hydroxycholesterol.

Translocational pausing of apolipoprotein B can be regulated by membrane lipid composition.

One potential mechanism by which apolipoprotein (apo) B secretion is regulated is via transient pausing during translocation across the endoplasmic reticulum membrane. We have previously shown that translocation and secretion of full-length and truncated variants of apoB 100 are impaired in hepatocytes in which microsomal membranes are enriched in the phospholipid phosphatidylmonomethylethanolamine (PMME). We have now investigated whether or not the decreased translocation of apoB is the result of altered membrane lipid composition having an impact on translocational pausing. Our experiments showed that less in vitro translated apoB-15 (the N-terminal 15% of human apoB-100) was translocated into the lumen of PMME-enriched microsomes than of control microsomes. Proteinase K treatment of the translocation products yielded discrete N-terminal fragments of apoB indicating that both types of microsomal membranes contained translocationally paused nascent chains. Similarly, apoB generated from a truncated mRNA lacking a stop codon was also found to be translocationally paused. However, restarting of translocation after translocational pausing was impaired in PMME-enriched, but not in control, microsomes. These data suggest that secretion of apoB-containing lipoproteins can be regulated by membrane lipid composition at the level of translocational pausing.

In vitro reconstitution of assembly of apolipoprotein B48-containing lipoproteins.

Human apolipoprotein B48 (apoB48) and apoB15 (the NH2-terminal 48 and 15% of apoB100, respectively) were translated in vitro from their respective mRNAs using a rabbit reticulocyte lysate and microsomes derived from rat liver or dog pancreas. Synthesis of phosphatidylcholine and triacylglycerols was reconstituted in freshly isolated microsomes by the addition of precursors of these glycerolipids (acylcoenzyme A, glycerol 3-phosphate, and CDP-choline) before, during, or after translation. Assembly of apoB15 and apoB48 with newly synthesized phospholipids and triacylglycerols was favored by active, co-translational lipid synthesis. Moreover, translocation of apoB48 but not B15 into the microsomal lumen was increased in the presence of co-translational lipid synthesis. When apoB48 was translated in vitro, approximately 50% of apoB48 was buoyant at a density of <1.10 g/ml in the lumen of liver microsomes only when lipid synthesis was reconstituted during translation. Microsomal triacylglycerol transfer protein has been proposed to be essential for lipidation and/or translocation of apoB48. However, apoB48 was translocated into the lumen of dog pancreas microsomes in which the activity of the microsomal triacylglycerol transfer protein was not detectable. These data indicate that (i) apoB15 and apoB48 bind newly synthesized phosphatidylcholine during translocation; (ii) apoB48 but not apoB15 associates co-translationally with triacylglycerols; (iii) translocation of apoB48 but not apoB15 is stimulated by lipid synthesis; (iv) assembly of buoyant apoB48-containing lipoproteins can be reconstituted in vitro in the presence of active lipid synthesis; and (v) even in microsomes lacking microsomal triacylglycerol transfer protein activity, apoB48 is translocated into the lumen.

Role of lipoproteins in the delivery of lipids to axons during axonal regeneration.

Nerve fiber elongation involves the input of lipids to the growing axons. Since cell bodies are often a great distance from the regenerating tips, alternative sources of lipids have been proposed. We previously demonstrated that axonal synthesis of phosphatidylcholine is required for axonal growth (Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) J. Cell Biol. 128, 913-918; Posse de Chaves, E., Vance, D. E., Campenot, R. B. and Vance, J. E. (1995) Biochem. J. 312, 411-417). In contrast, cholesterol is not made in axons. We now show that when compartmented cultures of rat sympathetic neurons are incubated with pravastatin, in the absence of exogenously supplied lipids, cholesterol synthesis is inhibited and axonal growth is impaired. The addition of cholesterol to the axons or cell bodies of neurons treated with this inhibitor restores normal axonal elongation. Similarly, a supply of cholesterol via lipoproteins restores normal axonal growth. In contrast, lipoproteins do not provide axons with sufficient phosphatidylcholine for normal elongation when axonal phosphatidylcholine synthesis is inhibited. Thus, our studies support the idea that during axonal regeneration lipoproteins can be taken up by axons from the microenvironment and supply sufficient cholesterol, but not phosphatidylcholine, for growth. We also show that neither apoE nor apoA-I within the lipoproteins is essential for axonal growth.

Monomethylethanolamine reduces plasma triacylglycerols and apolipoprotein B and increases apolipoprotein A-I rats without induction of fatty liver.

Monomethylethanolamine (MME) inhibits very low density lipoprotein (VLDL) secretion from cultured rat hepatocytes by disruption of translocation of apolipoprotein (apo) B across the endoplasmic reticulum membrane (A. E. Rusiñol, E. Y. W. Chan and J. E. Vance. 1993. J. Biol. Chem. 268: 25168-25175). We have now investigated whether or not plasma levels of lipids and apoB are reduced by dietary supplementation of rats with MME. In rats fed MME for 5 to 7 days, the levels of triacylglycerols and apoB in VLDL were reduced by 66% and 45%, respectively. At the same time, MME feeding also increased plasma apoA-I by 80%. No significant differences were found in body or liver weights between control and MME-fed rats, nor did the reduction of plasma VLDL in MME-fed rats result in accumulation of triacylglycerols in the liver. When the dietary period was extended to 15 weeks, essentially the same results were obtained except that plasma cholesterol was increased by 31% in MME-treated animals, apparently because of increased amounts of apoA-I and high density lipoproteins. According to post-mortem and microscopic examination, rats fed MME for 15 weeks were anatomically normal with no indication of any lipid accumulation in the liver. The ability of MME to reduce VLDL secretion and at the same time to increase the level of high density lipoproteins are attractive properties of a therapeutic agent for treatment of atherosclerosis in humans.

Inhibition of secretion of truncated apolipoproteins B by monomethylethanolamine is independent of the length of the apolipoprotein.

Translocation of apolipoprotein (apo) B across the endoplasmic reticulum membrane is a likely site for regulation of secretion of very low density lipoproteins from the liver. When primary rat hepatocytes are enriched with the phospholipid phosphatidylmonomethylethanolamine, the secretion of apoB, but not other proteins such as apoprotein A1 and albumin, is disrupted (Vance, J. E. (1991) J. Lipid Res. 32, 1971-1982). Moreover, less apoB enters the microsomal lumen and the intracellular degradation of apoB is increased (Rusiñol, A. E., Chan, E. Y. W., and Vance, J. E. (1993a) J. Biol. Chem. 268, 25168-25175). In the present study we have used McArdle 7777 rat hepatoma cells stably transfected with carboxyl-terminal-truncated variants of human apoB100 and have demonstrated that the reduction in apoB secretion induced by phosphatidylmonomethylethanolamine is not a function of assembly of the apoB into a buoyant lipoprotein particle. In addition, inhibition of the intracellular degradation of the apoproteins B does not restore apoB secretion, suggesting that the effect of phosphatidylmonomethylethanolamine enrichment on apoB degradation is secondary to the effect on translocation of the protein into the endoplasmic reticulum lumen. Furthermore, supplementation of the culture medium with oleic acid does not increase apoB secretion, reduce the intracellular degradation of apoB or reverse the effects of phosphatidylmonomethylethanolamine enrichment on these processes. Our data support the hypothesis that translocation of apoB protein across the endoplasmic reticulum membrane, regardless of the association of the apoB with neutral lipids, may be a key regulatory step in very low density lipoprotein secretion.

A unique mitochondria-associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre-Golgi secretory proteins including nascent lipoproteins.

An endoplasmic reticulum-like membrane fraction, termed the "mitochondria-associated membrane" (MAM), that co-isolates with mitochondria from rat liver has been characterized. One potential function of the MAM is as a membrane bridge between the endoplasmic reticulum and mitochondria that may be involved in transfer of phospholipids between these two organelles (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256). A polyclonal antibody directed against a peptide corresponding to the C terminus of phosphatidylethanolamine N-methyltransferase-2, a specific marker protein of the MAM (Cui, Z., Vance, J. E., Chen, M. H., Voelker, D. R., and Vance, D. E. (1993) J. Biol. Chem. 268, 16655-16663), was used in immunofluorescence and immunogold electron microscopy localization studies in rat hepatocytes. Immunoreactive protein was clustered in regions of the cell that did not correspond to the bulk of endoplasmic reticulum. A second potential role for the MAM, as a component of the secretory pathway that supplies lipids for assembly into very low density lipoproteins, has been examined. The MAM contains enzymes of similar, or higher, specific activities to those enzymes in the endoplasmic reticulum for the synthesis of phospholipids, triacylglycerols, cholesterol, and cholesteryl esters. Specific activities of diacylglycerol acyltransferase, acyl-coenzyme A:cholesterol acyltransferase, and phosphatidylserine synthase (base exchange enzyme) are enriched 2.2-3.4-fold in the MAM compared with endoplasmic reticulum. In addition, the microsomal triacylglycerol transfer protein, which is required for the assembly/secretion of apolipoprotein B-containing lipoproteins, was present in the MAM. Nascent apolipoprotein B-containing lipoproteins were isolated from the lumen of the MAM. These lipoproteins had the same average density and composition as nascent apolipoprotein B-containing lipoproteins isolated from heavy and light endoplasmic reticulum fractions, from the Golgi, and lipoproteins newly secreted by cultured rat hepatocytes. The MAM is a pre-Golgi compartment of the secretory route, as shown by pulse-chase studies with apolipoprotein B and albumin, as well as the sensitivity of luminal apolipoprotein B to endoglycosidase H.

Impaired biosynthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum of rat liver.

We have investigated the mechanism by which inhibition of phosphatidylcholine biosynthesis in rat hepatocytes by choline deprivation causes a reduction in the secretion of very low density lipoprotein (VLDL) (Yao, Z., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004). Rats ingested a choline-deficient or control diet for 3 days, and subcellular fractions of liver were prepared. No change in the amount of apolipoprotein B in the lumina of the endoplasmic reticulum was observed, but there was a 40-50% decrease of apolipoprotein B in the lumina of the Golgi from choline-deficient compared with control rats. Incubation of microsomes, derived from choline-deficient and -supplemented hepatocytes, with trypsin showed similar degradation of apolipoprotein B, indicating similar quantities of this protein are present on the surface and within the lumina. The VLDL particles in the Golgi of liver cells and in plasma, on average, were larger in samples derived from choline-deficient compared with choline-supplemented animals. Incubation of plasma VLDL with proteases demonstrated that the apolipoprotein B of plasma VLDL particles from choline-deficient animals had a different susceptibility to digestion than did VLDL from choline-supplemented animals. These data indicate that the number of VLDL particles assembled in the endoplasmic reticulum of liver is similar in choline-deficient and -supplemented rats, but the number of particles is decreased in the Golgi from choline-deficient animals.

Movement of apolipoprotein B into the lumen of microsomes from hepatocytes is disrupted in membranes enriched in phosphatidylmonomethylethanolamine.

When monolayer cultures of rat hepatocytes are incubated with the ethanolamine/choline analogue, monomethylethanolamine, the secretion of apolipoproteins B100 and B48, as well as the lipid constituents, of very low density lipoprotein (VLDL) is inhibited by approximately 50% (Vance, J. E. (1991) J. Lipid Res. 32, 1971-1982). In the present study we have investigated the mechanism by which monomethylethanolamine disrupts VLDL secretion. Hepatocytes were treated with 400 microM monomethylethanolamine overnight, which resulted in an increase in the cellular content of the derived phospholipid, phosphatidylmonomethylethanolamine, from 0.32 +/- 0.15 to 2.92 +/- 0.74 nmol/mg of cell protein. The biosynthesis of apoproteins B100 and B48 was not impaired by treatment of cells with monomethylethanolamine. However, monomethylethanolamine decreased by approximately 50% the amount of apoproteins B, but not of the typical secretory protein, albumin, present in the luminal content subfraction of microsomes. The intracellular degradation of apoproteins B was also increased in phosphatidylmonomethylethanolamine-enriched, compared with control, cells. Moreover, the pool of apoprotein B present in intact microsomes from hepatocytes incubated with monomethylethanolamine was more accessible to exogenously added trypsin, presumably because a larger pool of the apoprotein B was exposed on the cytosolic surface of these microsomes. The data strongly suggest that an increase in the microsomal content of phosphatidylmonomethylethanolamine diminishes the ability of apoprotein B to translocate across the endoplasmic reticulum membrane into the luminal compartment. Consequently, the association of apoprotein B with lipids and/or the normal assembly of mature VLDL particles is impaired.

Estrogen treatment increases phospholipid transfer activities in chicken liver.

The effect of subcutaneous beta-estradiol injection on liver phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol transfer activity of immature chicken has been determined. The estrogen administration significantly enhanced the transfer activity of both phosphatidylcholine (100%), phosphatidylethanolamine (160%), and phosphatidylinositol (150%). In vivo experiments revealed that the hormone-induced changes in liver lipid transfer activity were sensitive to a protein synthesis inhibitor, cycloheximide. A partial characterization of liver protein transfer on Sephacryl S-200 showed that multiple transfer proteins are involved in the beta-estradiol effect. This is the first time that hormonal modulation of phospholipid transfer activities is described, and the results suggest that the hepatic phospholipid transfer activities might be involved in the biosynthesis of plasma lipoproteins in vivo.