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Background & Aims: Excessive alcohol consumption can lead to alcohol-associated liver disease, a spectrum of conditions ranging from steatosis to fibrosis and cirrhosis. Bile acids regulate metabolic pathways by binding to cellular and nuclear receptors, and they also interact with the gut microbiome to control microbial overgrowth. Fibroblast growth factor 19 (FGF-19) is an ileum-derived hormone induced and released in response to bile acid activation of the nuclear receptor farnesoid X receptor. FGF-19 signaling is dysregulated with ethanol consumption and is increased in patients with alcoholic hepatitis. Here, we examined the effects of FGF-19 in a mouse model of chronic + binge ethanol feeding. Methods: After injection of adeno-associated virus-green fluorescent protein or AAV–FGF-19, female C57BL/6J mice were pair-fed a Lieber DeCarli liquid diet (5% v/v) or control diet for 10 days and were given a bolus gavage of 5% ethanol or maltose control to represent a binge drinking episode. Tissues were collected for analysis 9 hours after the binge. Results: Chronic + binge ethanol feeding induced steatosis regardless of FGF-19 expression. Interestingly, FGF-19 and ethanol resulted in significantly increased liver inflammation, as measured by Il6, Tgfβ, and Tnfα, compared with ethanol alone. Both ethanol and FGF-19 decreased bile acid synthesis, and FGF-19 significantly reduced secondary bile acids, leading to overgrowth of specific pathogenic bacteria including Enterococcus faecalis, Escherichia coli, and Clostridium perfringens. Conclusions: Dysregulation of FGF-19 and consequent changes in bile acid synthesis and composition during alcohol consumption may be a contributing factor to alcohol-induced liver disease and dysbiosis.

Background & Aims: Hepatocellular carcinoma (HCC) is a male-dominant disease, but targeted sex hormone therapies have not been successful. Bile acids are a potential liver carcinogen and are biomolecules with hormone-like effects. A few studies highlight their potential sex dimorphism in physiology and disease. We hypothesized that bile acids could be a potential molecular signature that explains sex disparity in HCC. Methods & Results: We used the farnesoid X receptor knockout (FxrKO) mouse model to study bile acid-dependent HCC. Temporal tracking of circulating bile acids determined more than 80% of FxrKO females developed spontaneous cholemia (ie, serum total bile acids ≥40 μmol/L) as early as 8 weeks old. Opposingly, FxrKO males were highly resistant to cholemia, with ∼23% incidence even when 26 weeks old. However, FxrKO males demonstrated higher levels of deoxycholate than females. Compared with males, FxrKO females had more severe cholestatic liver injury and further aberrancies in bile acid metabolism. Yet, FxrKO females expressed more detoxification transcripts and had greater renal excretion of bile acids. Intervention with CYP7A1 (rate limiting enzyme for bile acid biosynthesis) deficiency or taurine supplementation either completely or partially normalized bile acid levels and liver injury in FxrKO females. Despite higher cholemia prevalence in FxrKO females, their tumor burden was less compared with FxrKO males. An exception to this sex-dimorphic pattern was found in a subset of male and female FxrKO mice born with congenital cholemia due to portosystemic shunt, where both sexes had comparable robust HCC. Conclusions: Our study highlights bile acids as sex-dimorphic metabolites in HCC except in the case of portosystemic shunt.

Background and aims: Diet-induced obesity and metabolic syndrome can trigger the progression of fatty liver disease to non-alcoholic steatohepatitis and fibrosis, which is a major public health concern. Bile acids regulate metabolic homeostasis and inflammation in the liver and gut via the activation of nuclear farnesoid X receptor (Fxr) and the membrane receptor Takeda G protein-coupled receptor 5 (Tgr5). Tgr5 is highly expressed in the gut and skeletal muscle, and in cholangiocytes and Kupffer cells of the liver. Tgr5 is implicated in the mediation of liver and gut inflammation, as well as the maintenance of energy homeostasis. Here, we used a high fat, high fructose, and high sucrose (HFS) diet to determine how bile acid signaling through Tgr5 may regulate metabolism during the progression from fatty liver to non-alcoholic steatohepatitis and fibrosis. Materials and methods: Female C57BL/6J control wild type (WT) and Tgr5 knockout (Tgr5−/−) mice were fed HFS (high fat (40% kcal), high fructose, and 20% sucrose water) diet for 20 weeks. Metabolic phenotypes were characterized through examination of bile acid synthesis pathways, lipid and cholesterol metabolism pathways, and fibrosis and inflammation pathways. Results: Tgr5−/− mice were more glucose intolerant when fed HFS diet, despite gaining the same amount of weight as WT mice. Tgr5−/− mice accumulated significantly more hepatic cholesterol and triglycerides on HFS diet compared to WT mice, and gene expression of lipogenic genes was significantly upregulated. Hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) gene expression was consistently elevated in Tgr5−/− mice, while oxysterol 7alpha-hydroxylase (Cyp7b1), sterol 27-hydroxylase (Cyp27a1), Fxr, and small heterodimer partner (Shp) were downregulated by HFS diet. Surprisingly, hepatic inflammation and fibrosis were also significantly reduced in Tgr5−/− mice fed HFS diet, which may be due to altered serotonin signaling in the liver. Conclusions: Tgr5−/− mice may be protected from high fat, high sugar-induced hepatic inflammation and injury due to altered serotonin metabolism.

Bile acids are physiological detergents derived from cholesterol that aid in digestion and nutrient absorption, and they play roles in glucose, lipid, and energy metabolism and in gut microbiome and metabolic homeostasis. Bile acids mediate crosstalk between the liver and gut through bactericidal modulation of the gut microbiome, while gut microbes influence the composition of the circulating bile acid pool. Recent research indicates bile acids may also be important mediators of neurological disease by acting as peripheral signaling molecules that activate bile acid receptors in the blood-brain barrier and in the brain itself. This review highlights the role of bile acids in maintaining liver and gut microbe homeostasis, as well as their function as mediators of cellular signaling in the liver-gut-brain axis.

Cholesterol 7 alpha-hydroxylase (CYP7A1, EC1.14) is the first and rate-limiting enzyme in the classic bile acid synthesis pathway. Much progress has been made in understanding the transcriptional regulation of CYP7A1 gene expression and the underlying molecular mechanisms of bile acid feedback regulation of CYP7A1 and bile acid synthesis in the last three decades. Discovery of bile acid-activated receptors and their roles in the regulation of lipid, glucose and energy metabolism have been translated to the development of bile acid-based drug therapies for the treatment of liver-related metabolic diseases such as alcoholic and non-alcoholic fatty liver diseases, liver cirrhosis, diabetes, obesity and hepatocellular carcinoma. This review will provide an update on the advances in our understanding of the molecular biology and mechanistic insights of the regulation of CYP7A1 in bile acid synthesis in the last 40 years.

Bile acids play a critical role in the regulation of glucose, lipid, and energy metabolism through activation of the nuclear bile acid receptor farnesoid X receptor (FXR) and membrane G protein-coupled bile acid receptor-1 (Gpbar-1, aka TGR5). Agonist activation of FXR and TGR5 improves insulin and glucose sensitivity and stimulates energy metabolism to prevent diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD). Bile acids have both pro- and anti-inflammatory actions through FXR and TGR5 in the intestine and liver. In the intestine, bile acids activate FXR and TGR5 to stimulate fibroblast growth factor 15 and glucagon-like peptide-1 secretion. FXR and TGR5 agonists may have therapeutic potential for treating liver-related metabolic diseases, such as diabetes and NAFLD. Keywords: Bile acid metabolism, Cholestatic liver diseases, Metabolic diseases

Background & Aims: Hepatic cholesterol accumulation and autophagy defects contribute to hepatocyte injury in fatty liver disease. Bile acid synthesis is a major pathway for cholesterol catabolism in the liver. This study aims to understand the molecular link between cholesterol and bile acid metabolism and hepatic autophagy activity. Methods: The effects of cholesterol and cholesterol 7α-hydroxylase (CYP7A1) expression on autophagy and lysosome function were studied in cell models. The effects and mechanism of disrupting enterohepatic bile acid circulation on hepatic autophagy were studied in mice. Results: The results first showed differential regulation of hepatic autophagy by free cholesterol and cholesterol ester, whereby a modest increase of cellular free cholesterol, but not cholesterol ester, impaired lysosome function and caused marked autolysosome accumulation. We found that CYP7A1 induction, either by cholestyramine feeding in mice or adenovirus-mediated CYP7A1 expression in hepatocytes, caused strong autophagy induction. Mechanistically, we showed that CYP7A1 expression markedly attenuated growth factor/AKT signaling activation of mechanistic target of rapamycin (mTOR), but not amino acid signaling to mTOR in vitro and in vivo. Metabolomics analysis further found that CYP7A1 induction not only decreased hepatic cholesterol but also altered phospholipid and sphingolipid compositions. Collectively, these results suggest that CYP7A1 induction interferes with growth factor activation of AKT/mTOR signaling possibly by altering membrane lipid composition. Finally, we showed that cholestyramine feeding restored impaired hepatic autophagy and improved metabolic homeostasis in Western dietâfed mice. Conclusions: This study identified a novel CYP7A1âAKTâmTOR signaling axis that selectively induces hepatic autophagy, which helps improve hepatocellular integrity and metabolic homeostasis. Keywords: Cholesterol, Cholestyramine, Fatty Liver, Nuclear Receptor

Cholesterol 7α-hydroxylase (CYP7A1) is the first and rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. In addition to absorption and digestion of nutrients, bile acids play a critical role in the regulation of lipid, glucose, and energy homeostasis. We have backcrossed Cyp7a1−/− mice in a mixed B6/129Sv genetic background to C57BL/6J mice to generate Cyp7a1−/− mice in a near-pure C57BL/6J background. These mice survive well and have normal growth and a bile acid pool size ∼60% of WT mice. The expression of the genes in the alternative bile acid synthesis pathway are upregulated, resulting in a more hydrophilic bile acid composition with reduced cholic acid (CA). Surprisingly, Cyp7a1−/− mice have improved glucose sensitivity with reduced liver triglycerides and fecal bile acid excretion, but increased fecal fatty acid excretion and respiratory exchange ratio (RER) when fed a high-fat/high-cholesterol diet. Supplementing chow and Western diets with CA restored bile acid composition, reversed the glucose tolerant phenotype, and reduced the RER. Our current study points to a critical role of bile acid composition, rather than bile acid pool size, in regulation of glucose, lipid, and energy metabolism to improve glucose and insulin tolerance, maintain metabolic homeostasis, and prevent high-fat diet-induced metabolic disorders.

Background & Aims: Bile acids are physiologic detergents that also activate nuclear receptors to regulate glucose and lipid homeostasis. Cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme that converts cholesterol to bile acids, is transcriptionally regulated by bile acids and circadian rhythms. Fasting, nutrients, and the circadian clock critically control hepatic bile acid and lipid homeostasis, and circadian misalignment is associated with the metabolic syndrome in humans. To delineate these interactions, we employed a sleep disruption model to induce circadian disruption and examined hepatic metabolism with respect to bile acids, lipids, and clock gene expression. Methods: B6xC57 mice were maintained on chow or Western diet and were sleep disrupted for 6 hours/day for 5 days. Mice were sacrificed at 4-hour intervals over 24 hours. Hepatic metabolic genes were examined, and bile acid pool and lipid profiles were measured over 24 hours. Results: Sleep disruption significantly suppressed circadian expression of core clock genes, genes involved in lipid metabolism, and key regulators of Cyp7a1 as well as Cyp7a1 expression itself. Sleep disruption abolished the peak in serum cholesterol and increased liver and serum free fatty acids. Bile acid pool size was increased while liver bile acids were decreased. Chromatin immunoprecipitation assay revealed that hepatocyte nuclear factor 4α (HNF4α) and D-site binding protein (Dbp) occupancies were suppressed at the Cyp7a1 promoter in sleep-disrupted mice. When coupled with Western diet, sleep disruption abolished liver clock rhythms and elevated free fatty acids. Conclusions: Even short-term circadian disruption dramatically alters hepatic clock gene expression, bile acid metabolism, and lipid homeostasis to contribute to dyslipidemia. Keywords: Bile Acid Synthesis, Circadian Rhythm, Lipid Metabolism

Mounting research evidence demonstrates a significant negative impact of circadian disruption on human health. Shift work, chronic jet lag and sleep disturbances are associated with increased incidence of metabolic syndrome, and consequently result in obesity, type 2 diabetes and dyslipidemia. Here, these associations are reviewed with respect to liver metabolism and disease.

Hepatic VLDL overproduction is a characteristic feature of diabetes and an important contributor to diabetic dyslipidemia. Hepatic sortilin 1 (Sort1), a cellular trafficking receptor, is a novel regulator of plasma lipid metabolism and reduces plasma cholesterol and triglycerides by inhibiting hepatic apolipoprotein B production. Elevated circulating free fatty acids play key roles in hepatic VLDL overproduction and the development of dyslipidemia. This study investigated the regulation of hepatic Sort1 in obesity and diabetes and the potential implications in diabetic dyslipidemia. Results showed that hepatic Sort1 protein was markedly decreased in mouse models of type I and type II diabetes and in human individuals with obesity and liver steatosis, whereas increasing hepatic Sort1 expression reduced plasma cholesterol and triglycerides in mice. Mechanistic studies showed that the saturated fatty acid palmitate activated extracellular signal-regulated kinase (ERK) and inhibited Sort1 protein by mechanisms involving Sort1 protein ubiquitination and degradation. Consistently, hepatic ERK signaling was activated in diabetic mice, whereas blocking ERK signaling by an ERK inhibitor increased hepatic Sort1 protein in mice. These results suggest that increased saturated fatty acids downregulate liver Sort1 protein, which may contribute to the development of dyslipidemia in obesity and diabetes.

Aldo-keto reductase 1B7 (AKR1B7) is proposed to play a role in detoxification of by-products of lipid peroxidation. In this article, we show that activation of the nuclear receptor farnesoid X receptor (FXR) induces AKR1B7 expression in the liver and intestine, and reduces the levels of malondialdehyde (MDA), the end product of lipid peroxidation, in the intestine but not in the liver. To determine whether AKR1B7 regulates MDA levels in vivo, we overexpressed AKR1B7 in the liver. Overexpression of AKR1B7 in the liver had no effect on hepatic or plasma MDA levels. Interestingly, hepatic expression of AKR1B7 significantly lowered plasma glucose levels in both wild-type and diabetic db/db mice, which was associated with reduced hepatic gluconeogenesis. Hepatic expression of AKR1B7 also significantly lowered hepatic triglyceride and cholesterol levels in db/db mice. These data reveal a novel function for AKR1B7 in lipid and glucose metabolism and suggest that AKR1B7 may not play a role in detoxification of lipid peroxides in the liver. AKR1B7 may be a therapeutic target for treatment of fatty liver disease associated with diabetes mellitus.

Cholesterol 7α-hydroxylase (CYP7A1) plays a critical role in regulation of bile acid synthesis in the liver. CYP7A1 mRNAs have very short half-lives, and bile acids destabilize CYP7A1 mRNA via the 3′-untranslated region (3′-UTR). However, the underlying mechanism of translational regulation of CYP7A1 mRNA remains unknown. Screening of a human micro RNA (miRNA) microarray has identified five differentially expressed miRNAs in human primary hepatocytes treated with chenodeoxycholic acid, GW4064, or fibroblast growth factor (FGF)19. These compounds also significantly induced the expression of miR-122a, a liver-specific and the predominant miRNA in human hepatocytes. The putative recognition sequences for miR-122a and miR-422a were localized in the 3′-UTR of human CYP7A1 mRNA. The miR-122a and miR-422a mimics inhibited, whereas their inhibitors stimulated CYP7A1 mRNA expression. These miRNAs specifically inhibited the activity of the CYP7A1-3′-UTR reporter plasmids, and mutations of miRNA binding sites in 3′-UTR abrogated miRNA inhibition of reporter activity. These results suggest that miR-122a and miR-422a may destabilize CYP7A1 mRNA to inhibit CYP7A1 expression. However, these miRNAs did not play a role in mediating FGF19 inhibition of CYP7A1 transcription. Under certain conditions, miRNA may reduce CYP7A1 mRNA stability to inhibit bile acid synthesis, and the miR-122a antagomirs may stimulate bile acid synthesis to reduce serum cholesterol and triglycerides.

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7α-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4α (HNF4α) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4α to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Bile acids are physiological detergents that generate bile flow and facilitate intestinal absorption and transport of lipids, nutrients, and vitamins. Bile acids also are signaling molecules and inflammatory agents that rapidly activate nuclear receptors and cell signaling pathways that regulate lipid, glucose, and energy metabolism. The enterohepatic circulation of bile acids exerts important physiological functions not only in feedback inhibition of bile acid synthesis but also in control of whole-body lipid homeostasis. In the liver, bile acids activate a nuclear receptor, farnesoid X receptor (FXR), that induces an atypical nuclear receptor small heterodimer partner, which subsequently inhibits nuclear receptors, liver-related homolog-1, and hepatocyte nuclear factor 4α and results in inhibiting transcription of the critical regulatory gene in bile acid synthesis, cholesterol 7α-hydroxylase (CYP7A1). In the intestine, FXR induces an intestinal hormone, fibroblast growth factor 15 (FGF15; or FGF19 in human), which activates hepatic FGF receptor 4 (FGFR4) signaling to inhibit bile acid synthesis. However, the mechanism by which FXR/FGF19/FGFR4 signaling inhibits CYP7A1 remains unknown. Bile acids are able to induce FGF19 in human hepatocytes, and the FGF19 autocrine pathway may exist in the human livers. Bile acids and bile acid receptors are therapeutic targets for development of drugs for treatment of cholestatic liver diseases, fatty liver diseases, diabetes, obesity, and metabolic syndrome.

The TGFβ1/Smad pathway plays a critical role in cholestasis and liver fibrosis. Previous studies show that TGFβ1, TNFα, and insulin inhibit cholesterol 7α-hydroxylase (CYP7A1) gene transcription and bile acid synthesis in human hepatocytes. In this study, we investigated insulin, TGFβ1, and TNFα regulation of rat Cyp7a1 gene transcription. In contrast to inhibition of human CYP7A1 gene transcription, TGFβ1 stimulates rat Cyp7a1 reporter activity. Smad3, FoxO1, and HNF4α synergistically stimulated rat Cyp7a1 gene transcription. Mutations of the Smad3, FoxO1, or HNF4α binding site attenuated the rat Cyp7a1 promoter activity. Furthermore, TNFα and cJun attenuated TGFβ1 stimulation of rat Cyp7a1. Insulin or adenovirus-mediated expression of constitutively active AKT1 inhibited FoxO1 and Smad3 synergy. In streptozotocin-induced diabetic rats, Cyp7a1 mRNA expression levels were induced and insulin attenuated CYP7A1 mRNA levels. Chromatin immunoprecipitation assay showed that FoxO1 binding to Cyp7a1 chromatin was increased in diabetic rat livers and insulin reduced FoxO1 binding. These results suggest a mechanistic basis for induction of Cyp7a1 activity and bile acid synthesis in cholestatic rats and in diabetic rats. The crosstalk of insulin, TGFβ and TNFα signaling pathways may regulate bile acid synthesis and lipid homeostasis in diabetes, fatty liver disease, and liver fibrosis.

Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes oxidative cleavage of the sterol side chain in the bile acid biosynthetic pathway in the liver and 27-hydroxylation of cholesterol in most tissues. Recent studies suggest that 27-hydroxycholesterol (27-HOC) activates liver orphan receptor α (LXRα) and induces the cholesterol efflux transporters ABCA1 and ABCG1 in macrophages. The steroid- and bile acid-activated pregnane X receptor (PXR) plays critical roles in the detoxification of bile acids, cholesterol metabolites, and xenobiotics. The role of CYP27A1 in the intestine is not known. This study investigated PXR and CYP27A1 regulation of cholesterol metabolism in the human intestinal cell lines Caco2 and Ls174T. A human PXR ligand, rifampicin, induced CYP27A1 mRNA expression in intestine cells but not in liver cells. Rifampicin induced CYP27A1 gene transcription, increased intracellular 27-HOC levels, and induced ABCA1 and ABCG1 mRNA expression only in intestine cells. A functional PXR binding site was identified in the human CYP27A1 gene. Chromatin immunoprecipitation assays revealed that rifampicin induced the PXR recruitment of steroid receptor coactivator 1 to CYP27A1 chromatin. Cholesterol loading markedly increased intracellular 27-HOC levels in intestine cells. Rifampicin, 27-HOC, and a potent LXRα agonist, T0901317, induced ABCA1 and ABCG1 protein expression and stimulated cholesterol efflux from intestine cells to apolipoprotein A-I and HDL. This study suggests an intestine-specific PXR/CYP27A1/LXRα pathway that regulates intestine cholesterol efflux and HDL assembly.

The transcription of the cholesterol 7α-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. To determine whether the molecular structure of the promoter is responsible for this downregulation, we cloned the rabbit CYP7A1 promoter, identified the binding sites for α-fetoprotein transcription factor (FTF) and liver X receptor (LXRα), and studied the effects of FTF, LXRα, and SHP on its transcription. Adding LXRα/retinoid X receptor together with their ligands (L/R) to the promoter/reporter construct transfected into HepG2 cells greatly increased its activity. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Thus, downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXRα. Although FTF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.

Hydrophobic bile acids strongly repressed transcription of the human cholesterol 7α-hydroxylase gene (CYP7A1) in the bile acid biosynthetic pathway in the liver. Farnesoid X receptor (FXR) repressed CYP7A1/Luc reporter activity in a transfection assay in human liver-derived HepG2 cells, but not in human embryonic kidney (HEK) 293 cells. FXR-binding activity was required for bile acid repression of CYP7A1 transcription despite the fact that FXR did not bind to the CYP7A1 promoter. FXR-induced liver-specific factors must be required for mediating bile acid repression. Bile acids and FXR repressed endogenous CYP7A1 but stimulated α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) mRNA expression in HepG2 cells. Feeding of rats with chenodeoxycholic acid repressed CYP7A1, induced FTF, but had no effect on SHP mRNA expression in the liver. FTF strongly repressed CYP7A1 transcription in a dose-dependent manner, and SHP further inhibited CYP7A1 in HepG2 cells, but not in HEK 293 cells. FXR only moderately stimulated SHP transcription, whereas FTF strongly inhibited SHP transcription in HepG2 cells. Results revealed that FTF was a dominant negative factor that was induced by bile acid-activated FXR to inhibit both CYP7A1 and SHP transcription. Differential regulation of FTF and SHP expression by bile acids may explain the wide variation in CYP7A1 expression and the rate of bile acid synthesis and regulation in different species. —Chen, W., E. Owsley, Y. Yang, D. Stroup, and J. Y. L. Chiang. Nuclear receptor-mediated repression of human cholesterol 7α-hydroxylase gene transcription by bile acids.

Fibrates are widely used hypolipidemic drugs that regulate the expression of many genes involved in lipid metabolism by activating the peroxisome proliferator-activated receptor α (PPARα). The objective of this study was to investigate the mechanism of action of peroxisome proliferators and PPARα on the transcription of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver. When cotransfected with the expression vectors for PPARα and RXRα, Wy14,643 reduced human and rat cholesterol 7α-hydroxylase gene (CYP7A1)/luciferase reporter activities by 88% and 43%, respectively, in HepG2 cells, but not in CV-1 or CHO cells. We have mapped the peroxisome proliferator response element (PPRE) to a conserved sequence containing the canonical AGGTCA direct repeats separated by one nucleotide (DR1). This DR1 sequence was mapped previously as a binding site for the hepatocyte nuclear factor 4 (HNF-4) which stimulates CYP7A1 transcription. Electrophoretic mobility shift assay (EMSA) showed no direct binding of in vitro synthesized PPARα/RXRα heterodimer to the DR1 sequence. PPARα and Wy14,643 did not affect HNF-4 binding to the DR1. However, Wy14,643 and PPARα/RXRα significantly reduced HNF-4 expression in HepG2 cells. These results suggest that PPARα and agonist repress cholesterol 7α-hydroxylase activity by reducing the availability of HNF-4 for binding to the DR-1 sequence and therefore attenuates the transactivation of CYP7A1 by HNF-4.—Marrapodi, M., and J. Y. L. Chiang. Peroxisome proliferator-activated receptor α (PPARα) and agonist inhibit cholesterol 7α-hydroxylase gene (CYP7A1) transcription. J. Lipid Res. 2000. 41: 514–520.

The gene for cholesterol 7α-hydroxylase (CYP7A1) contains a sequence at nt −149 to −118 that was found to play a large role in determining the overall transcriptional activity and regulation of the promoter. Hepatocyte nuclear factor 4 (HNF4) and chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) synergistically activate transcription of the CYP7A1 promoter. Transactivation of CYP7A1 by HNF4 in the human hepatoma cell line, HepG2, was enhanced by cotransfection with COUP-TFII or the basal transcription element binding protein (BTEB). HNF4 prepared from rat liver nuclear extracts bound to oligomers homologous to the nt −146 to −134 sequences in electrophoretic mobility shift assays (EMSA), which corresponded to a conserved region containing a direct repeat of hormone response elements spaced by one nucleotide (DR1). The sequences surrounding this DR1 were found to be essential for the HNF4 transactivation. In vitro-translated COUP-TFII was found to bind the adjacent sequences from nt −139 to −128 (DR0), but COUP-TFII interacted with this region at a much lower affinity than to the COUP-TFII-site at nt −72 to −57 (DR4). Mutations at nt −139 to −128 or nt −72 to −57 reduced the COUP-TFII and HNF4 synergy; however, these COUP-TFII-binding sequences were not absolutely required for the cooperative effect of HNF4 and COUP-TFII on transactivation. These results indicated that the observed transactivation was the result of protein/protein interactions facilitated by the juxtaposition of the binding elements. —Stroup, D., and J. Y. L. Chiang. HNF4 and COUP-TFII interact to modulate transcription of the cholesterol 7α-hydroxylase gene (CYP7A1).

The gene encoding cholesterol 7α-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt −149/−118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (−146-TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (−139-AGTTCAaggccGGGTAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (−175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription. —Crestani, M., A. Sadeghpour, D. Stroup, G. Galli, and J. Y. L. Chiang. Transcriptional activation of the cholesterol 7α-hydroxylase gene (CYP7A) by nuclear hormone receptors. J. Lipid Res. 1998. 39: 2192–2200.