Correlation of Farnesoid X Receptor Coactivator Recruitment and Cholesterol 7α-Hydroxylase Gene Repression by Bile Acids

doi:10.1006/mgme.2000.3106

Molecular Genetics and Metabolism

Volume 71, Issue 4, December 2000, Pages 609-615

https://doi.org/10.1006/mgme.2000.3106 Get rights and content

Abstract

Cholesterol conversion to bile acids in the liver is regulated by the rate-limiting enzyme cholesterol 7α-hydroxylase (CYP7A1). CYP7A1 activity is regulated by feedback repression by bile acids at the transcriptional level. The farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, was recently demonstrated to function as the bile acid receptor and its high level of expression in the liver implicates it in the transcriptional regulation of CYP7A1. This study compares the potencies of various bile acids in their ability to mediate recruitment of the transcriptional coactivator protein, steroid receptor coactivator-1 (SRC-1), to the FXR ligand binding domain with their ability to repress CYP7A1 expression in HepG2 cells. A mammalian two-hybrid assay was utilized to assess the ability of FXR to recruit SRC-1 in a ligand-dependent manner. Chenodeoxycholic acid (CDCA) was the most potent and efficacious compound in the SRC-1 recruitment assay (EC₅₀ = 11.7 μM) followed by deoxycholic acid (DCA; EC₅₀ = 19.0 μM). Ursodeoxycholic acid (UDCA) displayed minimal activity while cholic acid (CA) was inactive. In order to directly compare the potencies of the bile acids in the coactivator recruitment assay to their ability to repress CYP7A1 expression, a branched DNA assay was developed to rapidly measure CYP7A1 mRNA levels from HepG2 cells cultured in 96-well plates. The rank order and absolute potency was conserved (CDCA IC₅₀ = 8.7 μM, DCA IC₅₀ = 27.2 μM, UDCA and CA inactive) consistent with bile acid repression of CYP7A1 being mediated by FXR.

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Cited by (44)

Discovery of farnesoid X receptor and its role in bile acid metabolism
2022, Molecular and Cellular Endocrinology
In 1995, the nuclear hormone orphan receptor farnesoid X receptor (FXR, NR1H4) was identified as a farnesol receptor expressed mainly in liver, kidney, and adrenal gland of rats. In 1999, bile acids were identified as endogenous FXR ligands. Subsequently, FXR target genes involved in the regulation of hepatic bile acid synthesis, secretion, and intestinal re-absorption were identified. FXR signaling was proposed as a mechanism of feedback regulation of the rate-limiting enzyme for bile acid synthesis, cholesterol 7⍺-hydroxylase (CYP7A1). The primary bile acids synthesized in the liver are transformed to secondary bile acids by the gut microbiota. The gut-to-liver axis plays a critical role in the regulation of bile acid synthesis, composition and circulating bile acid pool size, which in turn regulates glucose, lipid, and energy metabolism. Dysregulation of bile acid metabolism and FXR signaling in the gut-to-liver axis contributes to metabolic diseases including obesity, diabetes, and non-alcoholic fatty liver disease. This review will cover the discovery of FXR as a bile acid sensor in the regulation of bile acid metabolism and as a metabolic regulator of lipid, glucose, and energy homeostasis. It will also provide an update of FXR functions in the gut-to-liver axis and the drug therapies targeting bile acids and FXR for the treatment of liver metabolic diseases.
Effect of bile acid supplementation on endogenous lipid synthesis in patients with short bowel syndrome: A pilot study
2020, Clinical Nutrition
Short bowel syndrome patients (SBS) receiving parenteral nutrition (PN) often have dyslipidaemia and can develop intestinal failure-associated liver disease (IFALD). These patients demonstrate increased cholesterol synthesis and hepatic lipogenesis. These lipid disturbances may be due to a decreased concentration of the bile acid pool or malabsorption. The aim of this pilot study was to evaluate the effect of bile acid administration on lipid synthesis in patients with SBS.
The 24 h fractional synthesis rate (FSR) of cholesterol and triglycerides was measured by the isotopic method (deuterated water) before and after 4 months of ursodeoxycholic acid (UDCA) treatment (20 mg/kg/day). Five short bowel patients (age: 53.4 ± 19.2 years) who had normal liver function and lipid plasmatic profiles received 1920 ± 300 ml of PN for 151 ± 74 days (mean PN energy intake was 27.0 ± 6.0 kcal/kg body weight, composed with 3.87 ± 1.38 g/kg of carbohydrate, 0.72 ± 0.25 g/kg of fat and 1.10 ± 0.23 g/kg of amino acids). Plasma metabolites, liver enzymes, 7-α-OH-cholesterol and steatosis levels were also evaluated before and after treatment. Student's t-tests were performed, and the results were expressed in means (±SD).
After treatment, decreases in the absolute values of cholesterol synthesis (0.31 ± 0.12 mmol L⁻¹ to 0.24 ± 0.11 mmol L⁻¹; p < 0.05), FSR of cholesterol (31.6 ± 4.7% to 26.4 ± 4.7%; p = 0.06) and FSR of triglycerides (12.8 ± 5.8% to 9.2 ± 5.5%; p < 0.01) were observed. Cholesterol and alanine aminotransferase concentrations also decreased (ALT) (p < 0.05). The absolute values of triglyceride synthesis and triglyceride concentrations remained unchanged.
In SBS patients, UDCA decreases the hepatic synthesis of triglycerides and cholesterol. These results suggest that UDCA could prevent the onset of the IFALD.
Differential modulation of FXR activity by chlorophacinone and ivermectin analogs
2016, Toxicology and Applied Pharmacology
Citation Excerpt :
It is known that bile acids regulate the transcription of CYP7A1 by feedback repression through FXR binding. Studies have shown that the ability of various bile acids to recruit transcriptional coactivator protein (e.g., SRC-1) to the ligand binding domain of FXR is consistent with their ability to repress Cyp7A1 mRNA levels (Bramlett et al., 2000). On the other hand, in vitro pharmacological studies on liver cells demonstrated that theonellasterol antagonized the expression of FXR target genes including ABCC4 for the multidrug resistance-associated protein 4 (MRP-4) by abolishing the release of nuclear corepressor NCoR from the promoter of genes (Renga et al., 2012), and the in vivo studies using mice with obstructive cholestasis showed that theonellasterol increased MRP-4 expression in the liver and protected mice from liver injury.
Chemicals that alter normal function of farnesoid X receptor (FXR) have been shown to affect the homeostasis of bile acids, glucose, and lipids. Several structural classes of environmental chemicals and drugs that modulated FXR transactivation were previously identified by quantitative high-throughput screening (qHTS) of the Tox21 10 K chemical collection. In the present study, we validated the FXR antagonist activity of selected structural classes, including avermectin anthelmintics, dihydropyridine calcium channel blockers, 1,3-indandione rodenticides, and pyrethroid pesticides, using in vitro assay and quantitative structural-activity relationship (QSAR) analysis approaches. (Z)-Guggulsterone, chlorophacinone, ivermectin, and their analogs were profiled for their ability to alter CDCA-mediated FXR binding using a panel of 154 coregulator motifs and to induce or inhibit transactivation and coactivator recruitment activities of constitutive androstane receptor (CAR), liver X receptor alpha (LXRα), or pregnane X receptor (PXR). Our results showed that chlorophacinone and ivermectin had distinct modes of action (MOA) in modulating FXR-coregulator interactions and compound selectivity against the four aforementioned functionally-relevant nuclear receptors. These findings collectively provide mechanistic insights regarding compound activities against FXR and possible explanations for in vivo toxicological observations of chlorophacinone, ivermectin, and their analogs.
Cholesterol 7α-hydroxylase protects the liver from inflammation and fibrosis by maintaining cholesterol homeostasis
2016, Journal of Lipid Research
Citation Excerpt :
As shown in Fig. 4C, GW4064 significantly increased the interaction between FXR and p65 through the LBD of FXR, because the DBD and the hinge domain of FXR do not interact with p65. Ligand-activated FXR is known to recruit coactivators such as steroid receptor coactivator-1 (SRC-1) to activate target gene transcription (33). SRC-1 is also a coactivator of NF-κB (34).
Cholesterol 7α-hydroxylase (CYP7A1) plays a critical role in control of bile acid and cholesterol homeostasis. Bile acids activate farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) to regulate lipid, glucose, and energy metabolism. However, the role of bile acids in hepatic inflammation and fibrosis remains unclear. In this study, we showed that adenovirus-mediated overexpression of Cyp7a1 ameliorated lipopolysaccharide (LPS)-induced inflammatory cell infiltration and pro-inflammatory cytokine production in WT and TGR5-deficient (Tgr5^−/−) mice, but not in FXR-deficient (Fxr^−/−) mice, suggesting that bile acid signaling through FXR protects against hepatic inflammation. Nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-luciferase reporter assay showed that FXR agonists significantly inhibited TNF-α-induced NF-κB activity. Furthermore, chromatin immunoprecipitation and mammalian two-hybrid assays showed that ligand-activated FXR interacted with NF-κB and blocked recruitment of steroid receptor coactivator-1 to cytokine promoter and resulted in inhibition of NF-κB activity. Methionine/choline-deficient (MCD) diet increased hepatic inflammation, free cholesterol, oxidative stress, apoptosis, and fibrosis in CYP7A1-deficient (Cyp7a1^−/−) mice compared with WT mice. Remarkably, adenovirus-mediated overexpression of Cyp7a1 effectively reduced hepatic free cholesterol and oxidative stress and reversed hepatic inflammation and fibrosis in MCD diet-fed Cyp7a1^−/− mice. Current studies suggest that increased Cyp7a1 expression and bile acid synthesis ameliorate hepatic inflammation through activation of FXR, whereas reduced bile acid synthesis aggravates MCD diet-induced hepatic inflammation and fibrosis. Maintaining bile acid and cholesterol homeostasis is important for protecting against liver injury and nonalcoholic fatty liver disease.
Nuclear receptors as new perspective for the management of liver diseases
2011, Gastroenterology
Nuclear receptors (NRs) are ligand-activated transcription factors that act as sensors for a broad range of natural and synthetic ligands and regulate several key hepatic functions including bile acid homeostasis, bile secretion, lipid and glucose metabolism, as well as drug deposition. Moreover, NRs control hepatic inflammation, regeneration, fibrosis, and tumor formation. Therefore, NRs are key for understanding the pathogenesis and pathophysiology of a wide range of hepatic disorders. Finally, targeting NRs and their alterations offers exciting new perspectives for the treatment of liver diseases.
Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay
2010, Analytical Biochemistry
Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR–CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR–LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR–CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR–LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.

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¹: To whom correspondence should be addressed at Lilly Research Laboratories, Lilly Corporate Center, DC0434, Indianapolis, IN 46285. Fax: (317) 276-1414. E-mail: [email protected].

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Molecular Genetics and Metabolism

Abstract

References (25)

Nuclear orphan receptors control cholesterol catabolism

Cell

Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway

J Biol Chem

Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXRα

Cell

The LXRs: A new class of oxysterol receptors

Curr Opin Gen Devel

Endogenous bile acids are ligands for the nuclear receptor FXR/BAR

Mol Cell

Hepatocyte nuclear factor 1 binds to and transactivates the human but not the rat CYP7A1 promoter

Biochem Biophys Res Commun

Identification of a bile acid-responsive element in the human ilieal bile acid-binding protein gene

J Biol Chem

Farnesoid X receptor responds to bile acids and represses cholesterol 7α-hydroxylase gene (CYP7A1) transcription

J Biol Chem

Thyroid hormone response elements differentially modulate the interactions of thyroid hormone receptors with two receptor binding domains in the steroid receptor coactivator-1

J Biol Chem

PPARγ activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes

Biochem Biophys Res Commun

Steroid hormone receptors compete for factors that mediate their enhancer function

Cell

Regulation of cholesterol 7 α-hydroxylase gene expression in Hep-G2 cells

J Biol Chem

Cited by (44)

Discovery of farnesoid X receptor and its role in bile acid metabolism

Effect of bile acid supplementation on endogenous lipid synthesis in patients with short bowel syndrome: A pilot study

Differential modulation of FXR activity by chlorophacinone and ivermectin analogs

Cholesterol 7α-hydroxylase protects the liver from inflammation and fibrosis by maintaining cholesterol homeostasis

Nuclear receptors as new perspective for the management of liver diseases

Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay

Regular ArticleCorrelation of Farnesoid X Receptor Coactivator Recruitment and Cholesterol 7α-Hydroxylase Gene Repression by Bile Acids

Abstract

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Regular Article
Correlation of Farnesoid X Receptor Coactivator Recruitment and Cholesterol 7α-Hydroxylase Gene Repression by Bile Acids