Abstract
Membrane physiology, plasma lipid levels, and intracellular sterol homeostasis are regulated by both fatty acids and cholesterol. Sterols regulate gene expression of key enzymes of cholesterol and fatty acid metabolism through proteolysis of the sterol regulatory element-binding protein (SREBP), which binds to sterol regulatory elements (SRE) contained in promoters of these genes. We investigated the effect of fatty acids on SRE-dependent gene expression and SREBP. Consistent results were obtained in three different cell lines (HepG2, Chinese hamster ovary, and CV-1) transfected with SRE-containing promoters linked to the luciferase expression vector. We show that micromolar concentrations of oleate and other polyunsaturated fatty acids (C18:2-C22:6) dose-dependently (0.075-0.6 mmol) decreased transcription of SRE-regulated genes by 20-75%. Few or no effects were seen with saturated free fatty acids. Fatty acid effects on SRE-dependent gene expression were independent and additive to those of exogenous sterols. Oleate decreased levels of the mature sterol regulatory element-binding proteins SREBP-1 and -2 and HMG-CoA synthase mRNA. Oleate had no effect in sterol regulation defective Chinese hamster ovary cells or in cells transfected with mutant SRE-containing promoters. We hypothesize that unsaturated fatty acids increase intracellular regulatory pools of cholesterol and thus affect mature SREBP levels and expression of SRE-dependent genes.
Highlights
Membrane physiology, plasma lipid levels, and intracellular sterol homeostasis are regulated by both fatty acids and cholesterol
Oleate Inhibits the Transcription of sterol regulatory elements (SRE)-containing Promoters—To determine effects of oleate on SRE-dependent gene expression, HepG2, CV-1, and Chinese hamster ovary (CHO) cells, cells differing in cell cholesterol homeostasis, were transiently transfected with a plasmid originating from hamster HMG-CoA synthase, containing three SRE linked to the luciferase reporter gene
A second line of evidence that the fatty acid effect is mediated by sterol regulatory elementbinding protein (SREBP) interacting with a sterol-specific binding site is provided by experiments in which CHO cells were transiently transfected with JS-15, a pSyn SRE plasmid mutated in the SRE regions resulting in insensitivity to sterols [18]
Summary
Materials—[␣32-P]Deoxycytidine 5Ј-triphosphate (Ͼ 3000 Ci/mmol) was obtained from ICN Biochemicals, Inc. (Irvine, CA). Enzymes for reverse transcription-polymerase chain reactions, Trizol, Lipofectin, and cell culture reagents were purchased from Life Technologies, Inc. Ethanol, oleate, and fatty acid free bovine serum albumin (BSA) were from Sigma. Plasmids—The pSyn SRE plasmid contains a generic TATA and three SRE elements (Ϫ325 to Ϫ225) of the hamster HMG-CoA synthase promoter fused into the luciferase pGL2 Basic vector (Promega, Madison, WI) [17, 18]. The fatty acid synthase promoter plasmid linked to luciferase (FAS-150) was described before [10]. The -galactosidase plasmid (-gal) used for transfection control, consists of the lacZ gene driven by a CMV promoter. CDNA Probe—The cDNA probe for Northern hybridization of HMGCoA synthase was obtained by reverse transcription-polymerase chain reaction from human THP-1 macrophages mRNA using previously described primers [20]. Data Analysis—Statistical significance was calculated by paired t tests
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