The synthesis of fatty acids and cholesterol, the building blocks of membranes, is regulated by three membrane-bound transcription factors: sterol regulatory element-binding proteins (SREBP)-1a, -1c, and -2. Their function in liver has been characterized in transgenic mice that overexpress each SREBP isoform and in mice that lack all three nuclear SREBPs as a result of gene knockout of SREBP cleavage-activating protein (SCAP), a protein required for nuclear localization of SREBPs. Here, we use oligonucleotide arrays hybridized with RNA from livers of three lines of mice (transgenic for SREBP-1a, transgenic for SREBP-2, and knockout for SCAP) to identify genes that are likely to be direct targets of SREBPs in liver. A total of 1,003 genes showed statistically significant increased expression in livers of transgenic SREBP-1a mice, 505 increased in livers of transgenic SREBP-2 mice, and 343 showed decreased expression in Scap ؊͞؊ livers. A subset of 33 genes met the stringent combinatorial criteria of induction in both SREBP transgenics and decreased expression in SCAP-deficient mice. Of these 33 genes, 13 were previously identified as direct targets of SREBP action. Of the remaining 20 genes, 13 encode enzymes or carrier proteins involved in cholesterol metabolism, 3 participate in fatty acid metabolism, and 4 have no known connection to lipid metabolism. Through application of stringent combinatorial criteria, the transgenic͞knockout approach allows identification of genes whose activities are likely to be controlled directly by one family of transcription factors, in this case the SREBPs.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilases that reduces the number of LDL receptors (LDLRs) in liver through an undefined posttranscriptional mechanism. We show that purified PCSK9 added to the medium of HepG2 cells reduces the number of cellsurface LDLRs in a dose-and time-dependent manner. This activity was approximately 10-fold greater for a gain-of-function mutant, PCSK9(D374Y), that causes hypercholesterolemia. Binding and uptake of PCSK9 were largely dependent on the presence of LDLRs. Coimmunoprecipitation and ligand blotting studies indicated that PCSK9 and LDLR directly associate; both proteins colocalized to late endocytic compartments. Purified PCSK9 had no effect on cell-surface LDLRs in hepatocytes lacking autosomal recessive hypercholesterolemia (ARH), an adaptor protein required for endocytosis of the receptor. Transgenic mice overexpressing human PCSK9 in liver secreted large amounts of the protein into plasma, which increased plasma LDL cholesterol concentrations to levels similar to those of LDLR-knockout mice. To determine whether PCSK9 was active in plasma, transgenic PCSK9 mice were parabiosed with wild-type littermates. After parabiosis, secreted PCSK9 was transferred to the circulation of wild-type mice and reduced the number of hepatic LDLRs to nearly undetectable levels. We conclude that secreted PCSK9 associates with the LDLR and reduces hepatic LDLR protein levels.

Lipid homeostasis is transcriptionally regulated by three DNA-binding proteins, designated sterol regulatory element-binding protein (SREBP)-1a, -1c, and -2. Oligonucleotide arrays hybridized with RNA made from livers of transgenic SREBP-1a, transgenic SREBP-2, and SREBP cleavage-activating protein knockout mice recently identified 33 genes regulated by SREBPs in liver, four of which had no known connection to lipid metabolism. One of the four genes was PCSK9, which encodes proprotein convertase subtilisin/kexin type 9a, a protein that belongs to the proteinase K subfamily of subtilases. Mutations in PCSK9 are associated with an autosomal dominant form of hypercholesterolemia. Here, we demonstrate that hepatic overexpression of either wild-type or mutant PCSK9 in mice results in hypercholesterolemia. The hypercholesterolemia is due to a post-transcriptional event causing a reduction in low density lipoprotein (LDL) receptor protein prior to the internalization and recycling of the receptor. Overexpression of PCSK9 in primary hepatocytes and in mice lacking the LDL receptor does not alter apolipoprotein B secretion. These data are consistent with PCSK9 affecting plasma LDL cholesterol levels by altering LDL receptor protein levels via a post-transcriptional mechanism.Plasma LDL 1 cholesterol concentrations are determined by the relative rates of VLDL and LDL production by the liver and the rate of LDL uptake via hepatic LDL receptors (LDLRs) (1, 2). VLDL secretion from hepatocytes is positively correlated with rates of hepatic lipid synthesis (3). Genes required for cholesterol and triglyceride biosynthesis and, thus, VLDL production are regulated by three sterol regulatory element-binding proteins (SREBPs), SREBP-1a, SREBP-1c, and SREBP-2 (4, 5). SREBPs also are the principal transcriptional regulators of the LDL receptor gene, which clears apoB-containing lipoproteins, such as VLDL and LDL, from the plasma (5).To identify genes regulated by SREBPs, we used oligonucleotide arrays hybridized with RNA from livers of mice that overexpressed SREBPs (transgenic for SREBP-1a or transgenic for SREBP-2) and that lacked all SREBPs as a result of deleting SCAP, an escort protein required for SREBP activation (5). With this physiologic filter, 33 genes were identified that were increased in the transgenic livers and decreased in the SCAPdeficient livers. Four of these 33 genes had no known function. One of these four genes was Pcsk9, which encodes the proprotein convertase subtilisin/kexin type 9a, also designated NARC-1 (neural apoptosis-regulated convertase 1). Seidah et al. (6) showed that PCSK9 belongs to the proteinase K subfamily of subtilases. PCSK9 is synthesized first as a soluble zymogen that undergoes autocatalytic intramolecular processing in the ER to produce a prosegment that remains associated with the secreted enzyme.A link between PCSK9 and cholesterol metabolism was established by Abifadel et al. (7), who showed that two missense mutations in PCSK9 were associated with an autosomal dominant form of hype...

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the subtilases that promotes the internalization and degradation of LDL receptor in liver and thereby controls the level of LDL cholesterol in plasma. Here, we show that the expression of PCSK9 in HepG2 cells is completely dependent on the absence or presence of sterols. The minimal promoter region of the PCSK9 gene contains a sterol-regulatory element (SRE), which makes the transcription of PCSK9 dependent on sterols. Expression of nuclear forms of sterol-regulatory element binding protein-1 (SREBP-1) and SREBP-2 dramatically increased the promoter activity of PCSK9. In vitro-translated nuclear forms of SREBPs showed interactions with SRE, whereas mutations in SRE abolished their binding. In vivo studies in mice showed that Pcsk9 protein and mRNA were decreased significantly by fasting and increased by refeeding. However, supplementation with 2% cholesterol in the diet prevented the increase in Pcsk9. The amounts of Pcsk9 mRNA in livers of refed mice showed correlated regulation by the changes in the nuclear form of Srebp-2. In summary, it is suggested that the expression of PCSK9 is regulated by sterol at the transcriptional level in HepG2 cells and that both SREBP-1 and SREBP-2 can transcriptionally activate PCSK9 via SRE in its proximal promoter region in vitro. However, in vivo, it is suggested that the sterol-dependent regulation of PCSK9 is mediated predominantly by SREBP-2. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proteinase K subfamily of subtilisin serine proteases of which the gain-of-function mutations cause hypercholesterolemia (1-3). PCSK9 was independently identified as one of the genes that are regulated by sterol-regulatory element binding proteins (SREBPs) (4, 5). The SREBPs are members of the basic helix-loop-helix leucine zipper family of transcription factors that regulate the expression of the target genes by binding to the sterolregulatory elements (SREs) in their promoter regions (6). Using microarrays hybridized with RNA from livers of mice that either overexpressed nuclear forms of human SREBPs (transgenic model) or lacked SREBP-activating protein (knockout model), PCSK9 was identified as a SREBP target gene. Soon after the first cloning of this gene, with its relationship to neural apoptosis and liver regeneration, studies focused on its relationship with the regulation of cholesterol in plasma. Subsequently, the loss-of-function mutations of PCSK9 have been reported to decrease LDL cholesterol level (7-9) and reduce the risk of coronary heart disease (10). The definite evidence for a role of PCSK9 in LDL metabolism was revealed by a set of in vivo animal experiments. Adenovirus-mediated overexpression of PCSK9 reduced the amount of low density lipoprotein receptor (LDLR) in livers posttranscriptionally (11,12), whereas the amount of LDLR increased significantly in livers of Pcsk9 knockout mice (13). The mechanism by which PCSK9 reduces LDLR suggests that secreted PCSK9 in plas...

This article is available online at http://www.jlr.org effect of statins in dyslipidemic hamsters. J. Lipid Res . 2010. 51: 1486-1495. Supplementary key words LDL receptor • rosuvastatin • berberineThe number of LDL receptors (LDLR) expressed on the surface of hepatocytes is a primary determinant for plasma LDL-cholesterol (LDL-C) levels ( 1 ). Hepatic LDLR mediates the uptake of LDL particles from the circulation and delivers the receptor-bound LDL to the endosomal system for degradation while the LDLR returns to the cell surface. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in the cellular cholesterol biosynthetic pathway. The inhibition of cholesterol de novo synthesis leads to increased numbers of cell surface LDLR by activation of LDLR gene transcription. Thus, statins are the most widely prescribed drugs to treat hypercholesterolemia and combined hyperlipidemia.Contrary to human studies in which statins effectively lower plasma total cholesterol (TC) and LDL-C, in animal studies, the LDL reducing effects of statins exhibit great species differences. Statins reduce plasma TC and LDL-C as the major drug action in rabbits, miniature pigs, dogs, monkeys, and guinea pigs ( 2-4 ) but not in rodents such as rats and hamsters ( 5-7 ). In rats and hamsters fed either a Abstract We investigated the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the resistance of dyslipidemic hamsters to statin-induced LDL-cholesterol (LDL-C) reduction and the molecular mechanism by which statins modulated PCSK9 gene expression in vivo. We utilized the fructose diet-induced dyslipidemic hamsters as an in vivo model and rosuvastatin to examine its effects on liver PCSK9 and LDL receptor (LDLR) expression and serum lipid levels. We showed that rosuvastatin induced PCSK9 mRNA to a greater extent than LDLR mRNA in the hamster liver. The net result was that hepatic LDLR protein level was reduced. This correlated closely with an increase in serum LDL-C with statin treatment. More importantly, we demonstrated that in addition to an increase in sterol response element binding protein 2 (SREBP2) expression, rosuvastatin treatment increased the liver expression of hepatocyte nuclear factor 1 ␣ (HNF1 ␣ ), the newly identifi ed key transactivator for PCSK9 gene expression. Our study suggests that the inducing effect of rosuvastatin on HNF1 ␣ is likely a underlying mechanism accounting for the higher induction of PCSK9 than LDLR because of the utilization of two transactivators (HNF1 ␣ and SREBP2) in PCSK9 transcription versus one (SREBP2) in LDLR transcription. Thus, the net balance is in favor of PCSK9-induced degradation of LDLR in the hamster liver, abrogating the effect of rosuvastatin on LDL-C lowering. Press, January 4, 2010 DOI 10.1194 Abbreviations: BBR, berberine; HNF1, hepatocyte nuclear factor 1; LDL-C, LDL-cholesterol; LDLR, LDL receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; SREBP, sterol response element binding protein; TC , total cholesterol; TG, triglyceri...

Expression of the HER2 oncogene is increased in ϳ30% of human breast carcinomas and is closely correlated with the expression of fatty acid synthase (FASN). In the present study, we determined the mechanism by which FASN and acetyl-CoA carboxylase ␣ (ACC␣) could be induced by HER2 overexpression. SK-BR-3 and BT-474 cells, breast cancer cells that overexpress HER2, expressed higher levels of FASN and ACC␣ compared with MCF-7 and MDA-MB-231 breast cancer cells in which HER2 expression is low. The induction of FASN and ACC␣ in BT474 cells were not mediated by the activation of SREBP-1. Exogenous HER2 expression in MDA-MB-231 cells induced the expression of FASN and ACC␣, and the HER2-mediated increase in ACC␣ and FASN was inhibited by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. In addition, the activation of mTOR by the overexpression of RHEB in MDA-MB-231 cells increased the synthetic rates of both FASN and ACC␣. On the other hand, FASN and ACC␣ were reduced in BT-474 cells by a blockade of the mTOR signaling pathway. These changes observed in their protein levels were not accompanied by changes in their mRNA levels. The 5-and 3-untranslated regions of both FASN and ACC␣ mRNAs were involved in selective translational induction that was mediated by mTOR signal transduction. These results strongly suggest that the major mechanism of HER2-mediated induction of FASN and ACC␣ in the breast cancer cells used in this study is translational regulation primarily through the mTOR signaling pathway.Because OA-519, a poor prognostic marker found in breast cancer cells, was shown to be a fatty acid synthase (FASN) 3 (1),a number of studies have demonstrated abnormally high levels of FASN in many human epithelial cancers and preneoplastic lesions (2, 3). FASN, a lipogenic enzyme, catalyzes the biosynthesis of palmitic acid that is used for the synthesis of triacylglycerol as a storage fuel molecule as well as membrane lipids including phospholipids and sphingolipids (4). In breast cancer cells, the expression of FASN is closely related to the aggressiveness of cancers as well as to the development, maintenance, and cell cycle progression of human cancers (5-7). Breast cancer cells that overexpress FASN undergo apoptosis when treated with small interfering RNAs (siRNAs) against FASN or FASN inhibitors, such as C75 and cerulenin (8 -11). Under physiological conditions, the activities of lipogenic enzymes, including FASN, are tightly regulated by nutritional and hormonal parameters at the transcription level. Sterol regulatory element-binding proteins (SREBP-1a, SREBP-1c, and SREBP-2) are the major transcription factors that mediate this regulation (12). SREBPs reside in endoplasmic reticulum membranes as inactive precursors. To become active, the NH 2 -terminal segments of SREBPs are released from the endoplasmic reticulum by proteolytic cleavage and enter the nucleus where they activate their target genes. SREBP-1 preferentially activates the genes in...

Acetyl-CoA carboxylase (ACC), the first committed enzyme in fatty acid (FA) synthesis, is regulated by phosphorylation/dephosphorylation, transcription, and an unusual mechanism of protein polymerization. Polymerization of ACC increases enzymatic activity and is induced in vitro by supraphysiological concentrations of citrate (>5 mM). Here, we show that MIG12, a 22 kDa cytosolic protein of previously unknown function, binds to ACC and lowers the threshold for citrate activation into the physiological range (<1 mM). In vitro, recombinant MIG12 induced polymerization of ACC (as determined by nondenaturing gels, FPLC, and electron microscopy) and increased ACC activity by >50-fold in the presence of 1 mM citrate. In vivo, overexpression of MIG12 in liver induced ACC polymerization, increased FA synthesis, and produced triglyceride accumulation and fatty liver. Thus, in addition to its regulation by phosphorylation and transcription, ACC is regulated at a tertiary level by MIG12, which facilitates ACC polymerization and enhances enzymatic activity.lipogenesis | SREBPs | steatosis F atty acids (FAs) are the essential components of phospholipids and provide the most important energy depot for the body in the form of triglycerides (TG). Newly synthesized FAs also serve as signaling molecules and modulators of transcription factor activity (1-3). Excess accumulation of FAs in tissues contributes to the pathogenesis of many common diseases, including insulin resistance, nonalcoholic fatty liver disease, diabetes, and cancer (4, 5). Given the central role of FAs in these diseases, the enzymes of FA biosynthesis are potential therapeutic targets.The first committed step in FA biosynthesis is carried out by acetyl-CoA carboxylase (ACC). ACC1 was isolated and characterized by Wakil et al. (6) and functions to carboxylate acetyl-CoA to form malonyl-CoA. Subsequently, a second ACC isoform, ACC2, was identified in mammals that is encoded by a separate gene but carries out the same enzymatic reaction (7,8).ACCs are members of a larger family of carboxylases that require biotin and ATP and use bicarbonate as a carbon donor. The malonyl-CoA produced by ACC can be used by FA synthase (FAS) for the sequential 2-carbon elongation reactions that generate palmitic acid (C16:0) in the cytosol. While both ACC1 and ACC2 produce malonyl-CoA, ACC1 is predominantly cytosolic and generates malonyl-CoA that is used by FAS to synthesize palmitic acid. ACC2 is associated with the mitochondrial membrane (9) and produces malonyl-CoA that serves to allosterically inhibit carnitine palmitoyl transferase I, the protein responsible for transport of long chain FAs into mitochondria for β-oxidation (10).Regulation of ACC1 and ACC2 occurs at multiple levels. ACC activity is acutely regulated by phosphorylation/dephosphorylation. AMP-activated protein kinase phosphorylates ACC and inhibits enzyme activity (11). In the fed state, excess glucose must be converted to FAs for energy storage in the form of TGs. Increased insulin signaling in the fed state res...