Ser100-Phosphorylated ROR α Orchestrates CAR and HNF4 α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6.

Autor:	Fashe M; Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina., Hashiguchi T; Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina., Negishi M; Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina., Sueyoshi T; Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina Sueyoshi@niehs.nih.gov.
Jazyk:	angličtina
Zdroj:	Molecular pharmacology [Mol Pharmacol] 2020 Mar; Vol. 97 (3), pp. 191-201. Date of Electronic Publication: 2020 Jan 10.
DOI:	10.1124/mol.119.118273
Abstrakt:	We have previously shown that the retinoid-related orphan receptor alpha (ROR α ) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated ROR α orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4 α ) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). ROR α knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of ROR α in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that ROR α in the form of phosphorylated (p-) S100 directly bound to a newly identified ROR α response element (ROR α response element on CYP2B6 promoter, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 ROR α was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4 α binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated ROR α at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4 α formed a complex with Ser 100-phosphorylated ROR α , as shown by supershifted complexes with anti-p-Ser100 ROR α and anti-HNF4 α antibodies. Altogether, the results established that p-Ser100 ROR α bridging the PBREM and OARE orchestrates CAR and HNF4 α to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT: CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics, and it is prone to induction by xenobiotics, including phenobarbital via constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4 α ). Here, we show that retinoid-related orphan receptor alpha (ROR α ), through phosphorylated S100 residue, orchestrated CAR-HNF4 α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role of ROR α in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation sites within the DNA-binding domain of the receptor. (U.S. Government work not protected by U.S. copyright.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu