Aryl hydrocarbon receptor regulates the cholesterol biosynthetic pathway in a dioxin response element-independent manner

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor belonging to the basic helix-loop-helix (bHLH) – Per ARNT Sim (PAS) family of transcription factors. Ligands for the AHR include the planar, hydrophobic halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs), many of which are environmental contaminants. Activation of AHR by xenobiotic agonists such as TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a prototypic potent ligand, is known to have toxic consequences, illustrating its role as an exogenous chemical sensor. Atypical ligands include bilirubin and indirubin 1, 2. The presence of potent endogenous ligands for the human AHR exhibiting agonistic activities such as kynurenic acid 3 and 3-indoxyl sulfate 4 have been identified. Upon ligand binding, the AHR heterodimerizes with the AHR nuclear translocator protein (ARNT), another bHLH-PAS family member 5. The AHR/ARNT heterodimer represents a fully competent transcription factor capable of binding a consensus sequence known as dioxin or xenobiotic response element (DRE or XRE). This specific high affinity interaction stimulates transcription of xenobiotic target genes, including cytochrome P450s CYP1A1 and CYP1B1, which are involved in the metabolism of xenobiotics. Interestingly, the regulation of xenobiotic metabolism in tissues (e.g. intestinal tract) by the AHR is important in the clearance of endogenous and exogenous compounds 6. Ahr-null mice exhibit a defined set of physiological phenotypes comprising a reduction in peripheral lymphocytes, vascular abnormalities in the heart and liver, diminished fertility, and overall slower growth, all of which indicate a constitutive role for the receptor 5. A growing list of AHR target genes has been identified that clearly point to a physiological role for the AHR beyond regulating xenobiotic metabolism. AHR target genes that play a role in cell proliferation, cell cycle control, epithelial-mesenchymal transition and inflammation (e.g., slug, epiregulin) have been identified 7, 8. Microarray studies performed in mice have revealed that daily exposure to low levels of TCDD had a profound impact on the expression of genes involved in circadian rhythm, cholesterol biosynthesis, fatty acid synthesis, and glucose metabolism in the liver 9. A similar study performed in rats revealed that high levels of TCDD exposure were required to alter genes involved in cholesterol metabolism and bile acid synthesis and transport 10. This observation is also supported by a study indicating a disruption in lipid metabolism in male guinea pigs through changes in expression of cholesterol synthesis genes following TCDD treatment 11. These results are consistent with TCDD-induced anorexia and wasting syndrome characterized by weight loss, muscle atrophy and a loss of appetite observed in rats 12. Results from human exposure studies revealed a significant disruption in lipid metabolism and high cholesterol and triglyceride levels in blood of workers exposed to TCDD 13. Taken together, these results strongly suggest the involvement of AHR in the regulation of cholesterol homeostasis in rodents and humans. The essential roles for cholesterol and the human diseases caused by disorders in its metabolism prompted the study of its mode of regulation in order to control its levels in vivo 14. In the body, cholesterol is either derived from the diet or from de novo synthesis occurring mainly in the liver through the mevalonate pathway. This pathway comprises several enzymes such as 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), farnesyl-diphosphate farnesyltransferase (FDFT1), squalene epoxidase (SE) and oxidosqualene cyclase (OSC), all of which have been shown to be under the regulation of the transcription factor sterol element binding protein 2 (SREBP2) 15. Nuclear receptors such as the estrogen receptor and the glucocorticoid receptor have been shown to function through alternate mechanisms in the absence of DNA-binding. Our laboratory has already established that DRE-binding was not necessary for ligand activated AHR to directly attenuate acute-phase gene expression, namely Saa3 16. This result has led us to test the hypothesis that the AHR can regulate gene expression in the absence of DRE binding in the liver. Using a transgenic mouse model that expresses the DRE-binding mutant AHR-A78D and microarray analysis, we examined the genes that are altered by activation of this receptor. Upon injection with β-naphthoflavone (BNF), an AHR agonist, the major class of genes markedly repressed was directly involved in cholesterol metabolism. We found a similar change in primary human hepatocytes following receptor activation, demonstrating receptor involvement in regulating cholesterol synthesis both in vivo in mice, and in human cells. Absence of the AHR in mice and human cells correlated with an increased level of expression of those enzymes, further proving an endogenous role of the receptor in cholesterol homeostasis in the absence of any exogenous ligand. Finally, we demonstrated that repression of cholesterol synthesis gene expression was mirrored by a repression in the rate of cholesterol secretion in primary human hepatocytes.