Regulation of sterol regulatory element-binding proteins by cholesterol flux in CaCo-2 cells.
The regulation of sterol regulatory element-binding proteins (SREBP) by cholesterol flux was studied in the intestinal cell line CaCo-2. CaCo-2 cells were incubated for 18 h with micelles containing 5 mM taurocholate and 500 microM oleic acid or micelles containing either 200 microM cholesterol or 150 microM lysophosphatidylcholine. In some incubations, an ACAT inhibitor was added or 25-hydroxycholesterol was substituted for cholesterol. The SREBP-1a transcript was 2-fold more abundant than the SREBP-1c transcript. In cells incubated with micelles containing cholesterol, rates of cholesterol synthesis were decreased and rates of esterification were increased. Cholesterol synthesis was decreased further by ACAT inhibition. Cholesterol influx decreased mRNA levels of SREBP-2, HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. ACAT inhibition modestly suppressed gene expression further. Neither SREBP-1a nor SREBP-1c mRNA levels were altered by cholesterol. Despite decreases in gene expression of the sterol-responsive genes by cholesterol, the amounts of precursor and mature forms of SREBP-1 and SREBP-2 were not altered. In contrast, if 25-hydroxycholesterol was substituted for cholesterol, both the precursor and mature forms of SREBP-2 were decreased. The polar sterol decreased the mature form of SREBP-1 but the amount of the precursor form was unchanged. In cells incubated with micelles containing lysophosphatidylcholine, which causes cholesterol to efflux from cells, sterol-responsive gene expression was increased. The amounts of precursor and mature forms of SREBP-1 and SREBP-2, however, were not altered. In contrast, if the cells were depleted of cholesterol by incubating them with lovastatin and cyclodextrin, the mature forms of SREBP-1 and SREBP-2 were increased, as were mRNA levels for the sterol-responsive genes. The data would suggest that cholesterol influx/efflux regulates mRNA levels of sterol-responsive genes independently of changes in the amount of mature SREBP. In contrast, 25-hydroxycholesterol influx or cholesterol depletion alters the amount of mature SREBP, leading to the regulation of sterol-responsive gene expression.
Field
F J
FJ
Department of Internal Medicine, 4-522 JCP, 200 Hawkins Drive, and Veterans Administration, University of Iowa College of Medicine, Iowa City, IA 52242, USA. f-jeffrey-field@uiowa.edu
Born
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Murthy
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Mathur
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D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D018938:Caco-2 Cells; D002784:Cholesterol / Q000096:biosynthesis / Q000172:deficiency / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004951:Esterification; D064429:Fatty Acid Synthases / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D006904:Hydroxymethylglutaryl-CoA Synthase; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008823:Micelles; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D006888:Hydroxycholesterols; D008244:Lysophosphatidylcholines; D008823:Micelles; D012333:RNA, Messenger; C497387:SREBF1 protein, human; C497390:SREBF2 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D051782:Sterol Regulatory Element Binding Protein 2; D014157:Transcription Factors; C007997:25-hydroxycholesterol; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D064429:Fatty Acid Synthases; D006904:Hydroxymethylglutaryl-CoA Synthase; D003066:Coenzyme A Ligases