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.

Mechanisms of oxysterol-induced apoptosis.

The rationale for the present review is that oxysterols found in oxidized LDL (oxLDL) play a role in atherogenesis. This perspective is based on studies that show that induction of apoptosis in vascular cells is an important process in atherogenesis, that apoptosis can be induced by oxLDL, and that the oxysterol component of oxLDL is responsible for its proapoptotic activity. The evidence for these concepts is reviewed, as are studies on the mechanisms by which oxysterols can induce apoptosis. An elevation in intracellular calcium appears to be an early signal transduction event that leads to apoptosis through both the extrinsic and intrinsic apoptotic pathways.

25-Hydroxycholesterol activates a cytochrome c release-mediated caspase cascade.

We have previously shown that 25-hydroxycholesterol (25-OHC) treated CHO-K1 cells could be used as a model to investigate the signaling pathway of apoptosis induced by oxidized LDL in vascular cells. In the present study, we examine the execution phase of the apoptotic pathway in CHO-K1 cell death induced by 25-OHC. Oxysterol-induced apoptosis in CHO-K1 was accompanied by caspase activation and was preceded by mitochondrial cytochrome c release. The addition of a competitive caspase-3 inhibitor, Ac-DEVD-CHO, prevented 25-OHC-induced apoptotic cell death. Furthermore, immunoblot analysis showed that 25-OHC treatment induced the degradation of poly(ADP-ribose) polymerase (PARP)-a substrate for caspase 3 and a key enzyme involved in genome surveillance and DNA repair. Thus, we could demonstrate in CHO-K1 cells that 25-OHC activates the apoptotic machinery through induction of the release of cytochrome c from mitochodria into the cytosol and activation of a typical caspase cascade.

Transcriptional homeostatic control of membrane lipid composition.

Plasma membranes have a structural property, commonly referred to as membrane fluidity, that is compositionally regulated. The two main features of plasma membrane lipid composition that determine membrane fluidity are the ratio of cholesterol to phospholipids and the ratio of saturated to unsaturated fatty acids that are incorporated into the phospholipids. These ratios are determined, at least in part, by regulation of membrane lipid biosynthesis-particularly that of cholesterol and oleate. It now appears that cholesterol and oleate biosynthesis are feedback regulated by a common transcriptional mechanism which is governed by the maturation of the SREBP transcription factors. In this article, we briefly review our current understanding of transcriptional regulation of plasma membrane lipid biosynthesis by sterols and oleate. We also discuss studies related to the mechanism by which the physical state of membrane lipids signals the transcriptional regulatory machinery to control the rates of synthesis of these structural components of the lipid bilayer.

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.