ABSTRACT
Peroxisomal 3-ketoacyl-CoA thiolase B (Thb) catalyzes the final step in the peroxisomal ß-oxidation of straight-chain acyl-CoAs and is under the transcription control of the nuclear hormone receptor PPARα. PPARα binds to and is activated by the synthetic compound Wy14,643 (Wy). Here, we show that the magnitude of Wy-mediated induction of peroxisomal ß-oxidation of radiolabeled (1-(14)C) palmitate was significantly reduced in mice deficient for Thb. In contrast, mitochondrial ß-oxidation was unaltered in Thb(-/-) mice. Given that Wy-treatment induced Acox1 and MFP-1/-2 activity at a similar level in both genotypes, we concluded that the thiolase step alone was responsible for the reduced peroxisomal ß-oxidation of fatty acids. Electron microscopic analysis and cytochemical localization of catalase indicated that peroxisome proliferation in the liver after Wy-treatment was normal in Thb(-/-) mice. Intriguingly, micro-array analysis revealed that mRNA levels of genes encoding cholesterol biosynthesis enzymes were upregulated by Wy in Wild-Type (WT) mice but not in Thb(-/-) mice, which was confirmed at the protein level for the selected genes. The non-induction of genes encoding cholesterol biosynthesis enzymes by Wy in Thb(-/-) mice appeared to be unrelated to defective SREBP-2 or PPARα signaling. No difference was observed in the plasma lathosterol/cholesterol ratio (a marker for de novo cholesterol biosynthesis) between Wy-treated WT and Thb(-/-) mice, suggesting functional compensation. Overall, we conclude that ThA and SCPx/SCP2 thiolases cannot fully compensate for the absence of ThB. In addition, our data indicate that ThB is involved in the regulation of genes encoding cholesterol biosynthesis enzymes in the liver, suggesting that the peroxisome could be a promising candidate for the correction of cholesterol imbalance in dyslipidemia.
Subject(s)
Acetyl-CoA C-Acyltransferase/metabolism , Liver/enzymology , PPAR alpha/metabolism , Sterol Regulatory Element Binding Protein 2/metabolism , Up-Regulation , Acetyl-CoA C-Acyltransferase/genetics , Animals , Cholesterol/genetics , Cholesterol/metabolism , Gene Deletion , Gene Expression Regulation , Hepatomegaly/genetics , Hepatomegaly/pathology , Humans , Lipid Metabolism/genetics , Liver/pathology , Male , Mice , Mice, Knockout , Mitochondria/metabolism , Oxidation-Reduction , Palmitates/metabolism , Peroxisome Proliferators/pharmacology , Peroxisomes/metabolism , Pyrimidines/pharmacology , Signal TransductionABSTRACT
Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike gamma-tocopherol, the alpha-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, alpha-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with alpha-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of alpha-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by alpha-tocopherol, but not gamma-tocopherol pretreatment.
Subject(s)
Cell Death/physiology , Cholesterol/metabolism , Ketocholesterols/metabolism , Membrane Microdomains/metabolism , Muscle, Smooth, Vascular/physiology , Sphingolipids/metabolism , Vitamin E/pharmacology , alpha-Tocopherol/metabolism , Aorta , Cell Line , Cell Membrane Permeability , Humans , Hydrogen Peroxide/metabolism , Membrane Potentials , Mitochondrial Membranes/physiology , Muscle, Smooth, Vascular/cytology , Oxidation-Reduction , Tocopherols/metabolismABSTRACT
Oxysterols found in oxidized low-density lipoproteins are probably involved in the appearance of atheroma; some are cytotoxic and some able to induce cytokine secretion. An oxysterol-induced interleukin-8 (IL-8) secretion in human monocytes/macrophages has been previously noticed, but the mechanisms remained unclear. In this paper, we investigated the signaling pathways leading to the induction of IL-8 secretion in monocytic THP-1 cells treated with 7beta-hydroxycholesterol, a cytototoxic oxysterol, or with 25-hydroxycholesterol, an oxysterol non-cytotoxic toward this cell line. The oxysterol-induced IL-8 secretion appears to be a calcium-dependent phenomenon as shown by the use of calcium channel blockers, which strongly decreased IL-8 secretion and IL-8 messenger RNA (mRNA) levels. Fluo-3 staining used in flow cytometry and video microscopy revealed an oxysterol-induced Ca(2+) influx, varying according to the oxysterol studied, leading to the activation of the MEK/ERK1/2 pathway as demonstrated by Western blot analysis. ERK activation led to an increase of c-fos mRNA and/or an activation of c-fos. Luciferase reporter gene assay using constructs of the human IL-8 gene promoter and Transam assay revealed the involvement of the AP-1 transcription factor in oxysterol-dependent IL-8 secretion. These results demonstrate that oxysterol-induced IL-8 secretion is a calcium-dependent phenomenon involving the MEK/ERK1/2 pathway leading to the activation of IL-8 gene via AP-1 (c-fos).
