The ability of the aryl hydrocarbon receptor (AHR) to alter hepatic expression of cholesterol synthesis genes in a DRE-independent manner in mice and humans has been reported. We have examined the influence of functionally distinct classes of AHR ligands on the levels of Niemann–Pick C1-like intracellular cholesterol transporter (NPC1L1) and enzymes involved in the cholesterol synthesis pathway. NPC1L1 is known to mediate the intestinal absorption of dietary cholesterol and is clinically targeted. AHR ligands were capable of attenuating cholesterol uptake through repression of NPC1L1 expression. Through mutagenesis experiments targeting the two DRE sequences present in the promoter region of the NPC1L1 gene, we provide evidence that the repression does not require functional DRE sequences; while knockdown experiments demonstrated that this regulation is dependent on AHR and sterol-regulatory element-binding protein-2 (SREBP-2). Furthermore, upon ligand activation of AHR, the human intestinal Caco-2 cell line revealed coordinate repression of both mRNA and protein levels for a number of the cholesterol biosynthetic enzymes. Transcription of NPC1L1 and genes of the cholesterol synthesis pathway is predominantly regulated by SREBP-2, especially after treatment with a statin. Immunoblot analyses revealed a significant decrease in transcriptionally active SREBP-2 levels upon ligand treatment, whereas the precursor form of SREBP-2 was modestly increased by AHR activation. Mechanistic insights indicate that AHR induces proteolytic degradation of mature SREBP-2 in a calcium-dependent manner, which correlates with the AHR ligand-mediated upregulation of the transient receptor potential cation channel subfamily V member 6 (TRPV6) gene encoding for a membrane calcium channel. These observations emphasize a role for AHR in the systemic homeostatic regulation of cholesterol synthesis and absorption, indicating the potential use of this receptor as a target for the treatment of hyperlipidosis-associated metabolic diseases.
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Sanchis-Gomar F, Perez-Quilis C, Leischik R, Lucia A. Epidemiology of coronary heart disease and acute coronary syndrome. Ann Transl Med. 2016;4:256–256.
Trapani L, Segatto M, Pallottini V. Regulation and deregulation of cholesterol homeostasis: the liver as a metabolic ‘power station’. World J Hepatol. 2012;4:184–90.
Stancu C, Sima A. Statins: mechanism of action and effects. J Cell Mol Med. 2001;5:378–87.
Clader JW, Burnett DA, Caplen MA, Domalski MS, Dugar S, Vaccaro W, et al. 2-azetidinone cholesterol absorption inhibitors: structure-activity relationships on the heterocyclic nucleus. J Med Chem. 1996;39:3684–93.
Hammersley D, Signy M. Ezetimibe: an update on its clinical usefulness in specific patient groups. Ther Adv Chronic Dis. 2017;8:4–11.
Garcia-Calvo M1, Lisnock J, Bull HG, Hawes BE, Burnett DA, Braun MP. The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc Natl Acad Sci USA. 2005;102:8132–7.
Altmann SW. Niemann–Pick C1 like 1 protein is critical for intestinal cholesterol absorption. Science. 2004;303:1201–4.
Chang TY, Chang C. Ezetimibe blocks internalization of the NPC1L1/cholesterol complex. Cell Metab. 2008;7:469–71.
Ge L, Wang J, Qi W, Miao H-H, Cao J, Qu Y-X, et al. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1. Cell Metab. 2008;7:508–19.
Temel RE, Tang W, Ma Y, Rudel LL, Willingham MC, Ioannou YA, et al. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe. J Clin Investig. 2007;117:1968–78.
Beischlag TV, Luis Morales J, Hollingshead BD, Perdew GH. The aryl hydrocarbon receptor complex and the control of gene expression. Crit Rev Eukaryot Gene Expr. 2008;18:207–50.
Birnbaum LS, Tuomisto J. Non-carcinogenic effects of TCDD in animals. Food Addit Contam. 2000;17:275–88.
Lo R, Celius T, Forgacs AL, Dere E, MacPherson L, Harper P, et al. Identification of aryl hydrocarbon receptor binding targets in mouse hepatic tissue treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol. 2011;257:38–47.
Sato S, Shirakawa H, Tomita S, Ohsaki Y, Haketa K, Tooi O, et al. Low-dose dioxins alter gene expression related to cholesterol biosynthesis, lipogenesis, and glucose metabolism through the aryl hydrocarbon receptor-mediated pathway in mouse liver. Toxicol Appl Pharmacol. 2008;229:10–9.
Patel RD, Murray IA, Flaveny CA, Kusnadi A, Perdew GH. Ah receptor represses acute-phase response gene expression without binding to its cognate response element. Lab Investig. 2009;89:695–707.
Tanos R, Patel RD, Murray IA, Smith PB, Patterson AD, Perdew GH. Aryl hydrocarbon receptor regulates the cholesterol biosynthetic pathway in a dioxin response element-independent manner. Hepatology. 2012;55:1994–2004.
Murray IA, Krishnegowda G, DiNatale BC, Flaveny C, Chiaro C, Lin J-M, et al. Development of a selective modulator of aryl hydrocarbon (Ah) receptor activity that exhibits anti-inflammatory properties. Chem Res Toxicol. 2010;23:955–66.
Murray IA, Flaveny CA, Chiaro CR, Sharma AK, Tanos RS, Schroeder JC, et al. Suppression of cytokine-mediated complement factor gene expression through selective activation of the Ah receptor with 3’,4’-dimethoxy-alpha-naphthoflavone. Mol Pharmacol. 2011;79:508–19.
Brown MS, Goldstein JL. Cholesterol feedback: from Schoenheimer’s bottle to Scap’s MELADL. J Lipid Res. 2009;50:S15–S27.
Girer NG, Murray IA, Omiecinski CJ, Perdew GH. Hepatic aryl hydrocarbon receptor attenuates fibroblast growth factor 21 expression. J Biol Chem. 2106;291:15378–87.
Alrefai WA, Annaba F, Sarwar Z, Dwivedi A, Saksena S, Singla A, et al. Modulation of human Niemann-Pick C1-like 1 gene expression by sterol: role of sterol regulatory element binding protein 2. Am J Physiol Gastrointest Liver Physiol. 2007;292:G369–G376.
Muku GE, Lahoti TS, Murray IA, Podolsky MA, Smith KJ, Hubbard TD, et al. Ligand-mediated cytoplasmic retention of the Ah receptor inhibits macrophage-mediated acute inflammatory responses. Lab Investig. 2017;97:1471–87.
Feng D, Ohlsson L, Duan RD. Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression. Lipids Health Dis. 2010;9:40.
Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25:1105–11.
Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11:R106.
Pramfalk C, Jiang Z-Y, Cai Q, Hu H, Zhang S-D, Han T-Q, et al. HNF1alpha and SREBP2 are important regulators of NPC1L1 in human liver. J Lipid Res. 2010;51:1354–62.
Mansi I, Mortensen E. The controversy of a wider statin utilization: why? Expert Opin Drug Saf. 2013;12:327–37.
Miettinen TA, Gylling H. Cholesterol absorption efficiency and sterol metabolism in obesity. Atherosclerosis. 2000;153:241–8.
Tremblay AJ, Lamarche B, Lemelin V, Hoos L, Benjannet S, Seidah NG, et al. Atorvastatin increases intestinal expression of NPC1L1 in hyperlipidemic men. J Lipid Res. 2011;52:558–65.
Sudhop T, Lütjohann D, Kodal A, Igel M, Tribble DL, Shah S, et al. Inhibition of intestinal cholesterol absorption by ezetimibe in humans. Circulation. 2002;106:1943–8.
Hegele RA, Guy J, Ban MR, Wang J. NPC1L1 haplotype is associated with inter-individual variation in plasma low-density lipoprotein response to ezetimibe. Lipids Health Dis. 2005;4:16.
Pisciotta L, Fasano T, Bellocchio A, Bocchi L, Sallo R, Fresa R, et al. Effect of ezetimibe coadministered with statins in genotype-confirmed heterozygous FH patients. Atherosclerosis. 2007;194:e116–22.
Davis HR, Veltri EP. Zetia: inhibition of Niemann-Pick C1 Like 1 (NPC1L1) to reduce intestinal cholesterol absorption and treat hyperlipidemia. J Atheroscler Thromb. 2007;14:99–108.
Pearson T, Denke M, McBride P, Battisti WP, E. Brady W, Palmisano JE. Effectiveness of the addition of ezetimibe to ongoing statin therapy in modifying lipid profiles and attaining low-density lipoprotein cholesterol goals in older and elderly patients: Subanalyses of data from a randomized, double-blind, placebo-controlled. Am J Geriatr Pharmacother. 2005;3:218–28.
Davis HR, Compton DS, Hoos L, Tetzloff G. Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in apoE knockout mice. Arterioscler Thromb Vasc Biol. 2001;21:2032–8.
Klett EL, Patel S. Genetic defenses against noncholesterol sterols. Curr Opin Lipidol. 2003;14:341–5.
Dere E, Lo R, Celius T, Matthews J, Zacharewski TR. Integration of genome-wide computation DRE search, AhR chIP-chip and gene expression analyses of TCDD-elicited responses in the mouse liver. BMC Genomics. 2011;12:365.
Nault R, Forgacs AL, Dere E, Zacharewski TR. Comparisons of differential gene expression elicited by TCDD, PCB126, βNF, or ICZ in mouse hepatoma Hepa1c1c7 cells and C57BL/6 mouse liver. Toxicol Lett. 2013;223:52–9.
Angrish MM, Dominici CY, Zacharewski TR. TCDD-elicited effects on liver, serum, and adipose lipid composition in C57BL/6 mice. Toxicol Sci. 2013;131:108–15.
Yao L, Wang C, Zhang X, Peng L, Liu W, Zhang X, et al. Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice. Hepatology. 2016;64:92–105.
Lee JH, Wada T, Febbraio M, He J, Matsubara T, Lee MJ, et al. A novel role for the dioxin receptor in fatty acid metabolism and hepatic steatosis. Gastroenterology. 2010;139:653–63.
Moyer BJ, Rojas IY, Kerley-Hamilton JS, Hazlett HF, Nemani KV, Trask HW, et al. Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFβ, and IDO1. Toxicol Appl Pharmacol. 2016;300:13–24.
Xu CX, Wang C, Zhang ZM, Jaeger CD, Krager SL, Bottum KM, et al. A aryl hydrocarbon receptor deficiency protects mice from diet-induced adiposity and metabolic disorders through increased energy expenditure. Int J Obes. 2015;39:1300–9.
Tanos R, Murray IA, Smith PB, Patterson A, Perdew GH. Role of the Ah receptor in homeostatic control of fatty acid synthesis in the liver. Toxicol Sci. 2012;129:372–9.
Ohtake F, Baba A, Takada I, Okada M, Iwasaki K, Miki H, et al. Dioxin receptor is a ligand-dependent E3 ubiquitin ligase. Nature. 2007;446:562–6.
Ohtake F, Fujii-Kuriyama Y, Kato S. AhR acts as an E3 ubiquitin ligase to modulate steroid receptor functions. Biochem Pharmacol. 2009;77:474–84.
Mounho BJ, Davila DR, Burchiel SW. Characterization of intracellular calcium responses produced by polycyclic aromatic hydrocarbons in surface marker-defined human peripheral blood mononuclear cells. Toxicol Appl Pharmacol. 1997;145:323–30.
Tannheimer SL, Barton SL, Introduction S, Ethier P, Burchiel SW. Carcinogenic polycyclic aromatic hydrocarbons increase intracellular Ca 2 and cell proliferation in primary human mammary epithelial cells. Carcinogenesis. 1997;18:1172–82.
Mayati A, Levoin N, Paris H, N’Diaye M, Courtois A, Uriac P, et al. Induction of intracellular calcium concentration by environmental benzo(a)pyrene involves a β2-adrenergic receptor/adenylyl cyclase/Epac-1/inositol 1,4,5-trisphosphate pathway in endothelial cells. J Biol Chem. 2012;287:4041–52.
N’Diaye M, Le Ferrec E, Lagadic-Gossmann D, Corre S, Gilot D, Lecureur V, et al. Aryl hydrocarbon receptor- and calcium-dependent induction of the chemokine CCL1 by the environmental contaminant benzo[a]pyrene. J Biol Chem. 2006;281:19906–15.
We thank the Penn State Genomics Core Facility—University Park, PA, USA for RNA sequencing. We thank Curt Omiecinski and Denise Coslo for advice and technical assistance with the procurement and maintenance of primary human hepatocyte cultures from the Liver Tissue Cell Distribution System at the University of Pittsburgh, Pittsburg, PA, which is funded by National Institutes of Health Contract HHSN276201200017C. We also thank Marcia H Perdew for excellent editorial assistance. This work was supported in part by the National Institutes of Health Grants ES028244 and ES004869 and United States Department of Agriculture (Project 4607, GHP).
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Muku, G.E., Kusnadi, A., Kuzu, G. et al. Selective Ah receptor modulators attenuate NPC1L1-mediated cholesterol uptake through repression of SREBP-2 transcriptional activity. Lab Invest (2019) doi:10.1038/s41374-019-0306-x