Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping

Abstract

Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tertbutylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions 22201 and 21930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQinduced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes.

The gene expression profile of a drug metabolism system and signal transduction pathways in the liver of mice treated with tert-butylhydroquinone or 3-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate of sodium

“…Previously it was shown in human hepatocytes that upregulation of other phase I xenobiotic-metabolizing enzymes such as CYP2C19 and CYP2C9 is mediated by distinct activation of the AP-1 site in the promoters of these genes. It has been shown elsewhere, using specific kinase inhibitors, that extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) are essential for the tBHQ-induced expression, whereas Nrf2 has no effect [22]. We also observed induction of mRNA, protein expression, and activity of Cyp2b10, another CAR target gene (Table 1, Figs 3 and 4C).…”

“…For Cyp1a1 , Cyp1a2 , and Cyp2b10 , we showed that this activation was also manifested in protein levels and activities. P450 induction by synthetic phenolic antioxidants has been observed previously [20–22]. In some studies [20, 21], the researchers proposed that tBHQ induces Cyp1a1 as a ligand of AhR.…”

CYP2C8 Is a Novel Target of Peroxisome Proliferator-Activated Receptor α in Human Liver

“…Immunoblotting experiments were performed in microsomes to detect CYP2C proteins before and after treatment with BF or dimethylsulfoxide (DMSO) controls. Microsomes were prepared from cultured human hepatocytes as previously described (Makia et al, 2014). Briefly, cells were suspended in ice-cold buffer (0.1 M potassium phosphate, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA) and homogenized using a Potter-Elvehjem homogenizer.…”

“…Electrophoretic mobility shift assays (EMSAs) with the in vitro transcribed proteins were performed essentially as described (Makia et al, 2014) by incubating 2 ml of the in vitro translated PPARa and RXRa proteins with labeled double-stranded oligonucleotides containing the PPRE control and various DR1s. The sequences of the complementary oligonucleotides were as follows: PPRE control: forward, 59-CAGGGGACCAGGACA AAGGTCACGTTCGGGA-39, and reverse, 59-TCCCGAACGTGACCT TTGTCCTGGTCCCCTG-39; DR1-A: forward, 59-ACCCTATGTGAA CTTCGAACTTTGGTTGATG-39, and reverse, 59-CATCAACCAA AGTTCGAAGTTCACATAGGGT-39; DR1-B: forward, 59-AATTACTA CTTCCCTTTGCCCTGGATAAAGG-39, and reverse, 59-CCTTTATC CAGGGCAAAGGGAAGTAGTAATT-39; DR1BMut: forward, 59-AAT TACTACTTCGGTTTGCGGTGGATAAAGG-39, and reverse, 59-CCTT TATCCACCGCAAACCGAAGTAGTAATT-39; DR1-C: forward, TAAAA CCAAACACGTCTGACCCACATTTTAC-39, and reverse, 59-GTAAA ATGTGGGTCAGACGTGTTTGGTTTTA-39; and DR1-D: forward, 59-TAAAAAGAAAGGTCAAGGCAGGAGCCTCAGC-39, and reverse, 59-GCTGAGGCTCCTGCCTTGACCTTTCTTTTTA-39.…”

Activation of the c‐Jun N‐terminal kinase pathway aggravates proteotoxicity of hepatic mutant Z alpha1‐antitrypsin

“…PCR product was cloned into a pGL3‐Basic vector (Promega) upstream of the firefly luciferase. AP‐1 consensus sequences were mutagenized by QuickChange site‐directed mutagenesis as described . The complementary DNA of human c‐Jun was amplified by RT‐PCR from HuH7 cells and cloned into p3xFLAG‐CMV‐14 vector.…”