Selective Ah receptor modulators attenuate NPC1L1-mediated cholesterol uptake through repression of SREBP-2 transcriptional activity

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Abstract

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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Acknowledgements

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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Correspondence to Gary H. Perdew.

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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

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