Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver (cholesterol biosynthesisylow density lipoprotein receptorsy3-hydroxy-3-methylglutaryl CoA synthase and reductaseytranscriptional regulation)

Cholesterol feeding reduces the mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway and the low density lipoprotein receptor in livers of hamsters. Here we show that cholesterol feeding also reduces the levels of the nuclear NH2-terminal domains of sterol regulatory element binding proteins (SREBPs), which activate transcription of sterol-regulated genes. We show that livers of hamsters, like those of mice and humans, predominantly produce SREBP-2 and the 1c isoform of SREBP-1. Both are produced as membrane-bound precursors that must be proteolyzed to release the transcriptionally active NH2terminal domains. Diets containing 0.1% to 1.0% cholesterol decreased the amount of nuclear SREBP-1c without affecting the amount of the membrane precursor or its mRNA, suggesting that cholesterol inhibits the proteolytic processing of SREBP-1 in liver as it does in cultured cells. Cholesterol also appeared to reduce the proteolytic processing of SREBP-2. In addition, at high levels of dietary cholesterol the mRNA encoding SREBP-2 declined and the amount of the precursor also fell, suggesting that cholesterol accumulation also may inhibit transcription of the SREBP-2 gene. The highcholesterol diets reduced the amount of low density lipoprotein receptor mRNA by 30% and produced a more profound 70 –90% reduction in mRNAs encoding 3-hydroxy-3methylglutaryl CoA synthase and reductase. Treatment with lovastatin and Colestipol, which increases hepatic demands for cholesterol, increased the amount of SREBP-2 mRNA as well as the precursor and nuclear forms of the protein. This treatment caused a reciprocal decline in SREBP-1c mRNA and protein. Considered together, these data suggest that SREBPs play important roles in controlling transcription of sterol-regulated genes in liver, as they do in cultured cells. Since the pioneering work of Gould, carried out more than 40 years ago (1, 2), scientists have known that high-cholesterol diets suppress cholesterol synthesis in the livers of experimental animals. More recently, the converse also was shown to be true, i.e., manipulations that deplete the liver of cholesterol lead to an increase in cholesterol synthesis (see ref. 3 for review). Much of this control is attributable to coordinate changes in the levels of mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway, including 3-hydroxy-3methylglutaryl CoA (HMG CoA) synthase, HMG CoA reductase, farnesyl diphosphate synthase, squalene synthase, and others (see ref. 4 for review). The mRNA for the low density lipoprotein (LDL) receptor also is reduced by cholesterol feeding and increased by cholesterol depletion, although the amplitude of these changes is not as profound as that of the cholesterol biosynthetic enzymes, and the changes do not necessarily occur in parallel (4, 5). The changes in hepatic LDL receptors contribute to the elevation in blood cholesterol levels induced by high-cholesterol diets and to the reduction that follows hepatic cholesterol depletion (6). A potential mechanism for this regulation was disclosed recently through studies of nonhepatic cells in tissue culture. In these cells the transcription of genes encoding cholesterol biosynthetic enzymes and the LDL receptor is controlled by a family of transcription factors designated SREBPs (sterol regulatory element binding proteins) (see ref. 7 for review). The SREBPs are proteins of '1,150 amino acids that are bound to membranes of the endoplasmic reticulum. In steroldepleted cells, proteases release the NH2-terminal domains of the SREBPs, which are transcription factors of the basic-helixloop-helix-leucine zipper (bHLH-Zip) family. These soluble domains, designated the mature forms of the SREBPs, enter the nucleus where they activate transcription by binding to 10-bp sterol regulatory elements in the enhancer regions of target genes. When cultured cells are overloaded with sterols, the proteolytic process is inhibited, the SREBPs remain bound to endoplasmic reticulum membranes, and transcription of the target genes declines (7). The three known members of the SREBP family are produced by two genes (7). The SREBP-1 gene gives rise to two transcripts designated SREBP-1a and SREBP-1c, which differ only in the first exon that encodes an acidic transcription activation domain. This domain is much longer in SREBP-1a than in SREBP-1c, and therefore SREBP-1a is a much stronger activator of transcription (8). The third member of the family, designated SREBP-2, has a long activation domain, and its action resembles that of SREBP-1a. Tissue culture cells produce predominantly SREBP-1a and SREBP-2 (9), and the proteolytic processing of the two proteins is regulated in parallel (7). In liver, the pattern of SREBP expression and regulation differs from that observed in cultured cells. In livers of mice and humans, the SREBP-1c mRNA is at least 9-fold more abundant than the SREBP-1a mRNA (9). The abundance of the SREBP-2 transcript appears to be intermediate between these extremes (9). In hamster liver, depletion of cholesterol by treatment with a bile acid binding resin (Colestipol) and an HMG CoA reductase inhibitor (lovastatin) caused a paradoxical decline in the amount of total SREBP-1 protein and in the efficiency of its processing to the mature form (7, 10). At the same time, the total amount of SREBP-2 increased, and its processing to the mature form increased. If hamster liver produces SREBP-1c as its predominant isoform, then the net The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9412354-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: HMG CoA, 3-hydroxy-3-methylglutaryl CoA; LDL, low density lipoprotein; SREBP, sterol regulatory element binding protein.