A Novel Promoter Element Containing Multiple Overlapping Xenobiotic and Hypoxia Response Elements Mediates Induction of Cytochrome P4502S1 by Both Dioxin and Hypoxia

Abstract

Cytochrome P4502S1 (CYP2S1) is expressed at high levels in epithelial tissues and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) via the aryl hydrocarbon receptor (AHR). Transcriptional initiation of mouse Cyp2s1 was found to occur at three regions, ϳ198, 102, and 22 nucleotides from the translational initiation codon. Approximately 400 nucleotides upstream of its translational initiation codon, mouse Cyp2s1 contains three overlapping xenobiotic-responsive element (XRE) sequences, which make a major contribution toward dioxin inducibility. Each XRE sequence in this trimeric XRE can bind the AHR/aryl hydrocarbon receptor nuclear translocator (ARNT) dimer in a dioxin-dependent fashion in vitro and can mediate dioxin-dependent transcription. Cyp2s1 is also markedly inducible by hypoxia. Induction is dependent on hypoxiainducible factor-1 (HIF-1) and is mediated in large part by three overlapping hypoxia response elements (HREs) embedded within the trimeric XRE segment. Although each HRE within this segment can bind HIF-1␣/ARNT in vitro, the most 3 HRE contributes the most toward hypoxia inducibility. AHR/ARNT and HIF-1␣/ARNT dimers bind to the region containing the trimeric XRE segment of the endogenous Cyp2s1 gene in vivo in a dioxin-dependent fashion and hypoxia-dependent fashion, respectively. These observations identify a novel regulatory cassette that mediates changes in Cyp2s1 expression.The cytochrome P450 superfamily consists of at least 57 genes in humans and 102 genes in mice (1). In general, mammalian cytochrome P450s in families 1-4 metabolize foreign compounds (xenobiotics), although they also frequently metabolize endogenous molecules, such as steroids and fatty acids. Families 1-4 are often also inducible by xenobiotics. Members of family 1 are inducible by the potent tumor promoter, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), 6 and carcinogenic polycyclic aromatic hydrocarbons via the aryl hydrocarbon receptor (AHR) (2). Rylander et al. (3) identified human CYP2S1 by performing a homology search in a sequence data base, whereas we cloned mouse Cyp2s1 as a dioxin-inducible transcript in the mouse hepatoma cell line, Hepa-1, in which cells it is maximally induced about 10-fold (4). CYP2S1 is unusual for a non-CYP1 family member in being inducible by dioxin (although another Cyp2 family member, Cyp2a5, has recently been shown also to be dioxin-inducible (5)). Human CYP2S1 has also been shown to be inducible in skin by coal tar, which contains high concentrations of polycyclic aromatic hydrocarbon ligands for AHR. Human CYP2S1 can convert alltrans-retinoic acid to the catabolic products, 4-hydroxyretinoic acid and 5,6-epoxyretinoic acid (6), and it has been reported that the enzyme can metabolize naphthalene to two products (7). Both mouse and human CYP2S1 are expressed robustly in most epithelial surfaces and tissues, including the lung and intestinal tract and at all stages of embryogenesis (3, 8 -10).The facts that all other previously well characterized dioxininducible cytochrome P45...

Characterization of a Human Keratinocyte HaCaT Cell Line Model to Study the Regulation of CYP2S1

“…Although CYP2S1 is similarly expressed (Saarikoski et al, 2005), several previous studies have failed to detect CYP2S1 activity toward a variety of xenobiotics, including cigarette smoke (Wang et al, 2005) or known P450 substrates including benz[a]pyrene . In contrast, others have shown CYP2S1 to be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) (Rivera et al, 2007) and other chemical agents such as ␣-naphthalene (Karlgren et al, 2005) and atRA (Smith et al, 2003b), suggesting a role for CYP2S1 in xenobiotic metabolism. A role for CYP2S1 in atRA metabolism is confirmed by our results in HaCaT cells, where atRA induces CYP2S1 mRNA expression at a dose previously shown to induce CYP1B1 (Choudhary et al, 2004) and CYP2S1-mediated atRA metabolism in HaCaT cells is very similar to that observed in a CHO-based cell line coexpressing CYP2S1 and CPR.…”

CYP2S1 and CYP2W1 Mediate 2-(3,4-Dimethoxyphenyl)-5-Fluorobenzothiazole (GW-610, NSC 721648) Sensitivity in Breast and Colorectal Cancer Cells

“…Recently, human CYP2S1 and CYP2W1 isoforms have also been shown to be transcriptionally regulated by AhR (14,27,28). As such, we investigated whether 5F-203 and GW-610 are able to Induction of CYP1A1 mRNA and protein was observed in all cell lines on treatment of cells with 5F-203 and GW-610, including KM12 and HCC2998 CRC cells which are inherently resistant to 5F-203 ( Fig.…”

High-throughput evaluation of aryl hydrocarbon receptor-binding sites selected via chromatin immunoprecipitation-based screening in Hepa-1c1c7 cells stimulated with 2,3,7,8-tetrachlorodibenzo-p-dioxin

“…GATCCGGCTCTTGTCACGCAACTCC GAGCTCA GATCTGAGCTCGGAGTTGCGTG AGAAGAGCCG (Denison et al, 1988;Fukuda et al, 2004) Cyp1B1 ACAGCTCCGCACGCAAGGGGGA GGCGACAGGAATTCAGATCTCC CGGGT CTGTCGCCTCCCCCTTGCGTGC GGAGCTGTGAATTCAGATCTCC CGGGT (Eltom et al, 1999) Cyp2S1 CACATGCACGCACGCACGCACA CCAAGGAATTCAGATCTCCCGG GT CTTGGTGTGCGTGCGTGCGTGC ATGTGGAATTCAGATCTCCCGG GT (Rivera et al, 2007) Cyp2S5 CAAAGCCCCTGCTCACTCACGC ACTCTGGAAGCCTGCGAATTCA GATCTCCCGGGT GCAGGCTTCCAGAGTGCGTGAG TGAGCAGGGGCTTTGGAATTCA GATCTCCCGGGT (Arpiainen et al, 2005) Cyp19A1 CGGGAGATCTGAATTCTGAGAG TGAACTGCAGGAAG CGGGAGATCTGAATTCACCTCAT GGCTAAGGCAATG (Baba et al, 2005) a Lengths (bp) of amplified PCR products are indicated. b Dioxin-response elements (DREs) are underlined.…”

“…Nuclear extract prepared from Hepa-1c1c7 cells stimulated with TCDD was used as the material containing activated AhR for DNA binding. First to be used were FITC-labeled probes designed for the promoter region of well-characterized AhR target genes, including Cyp1A1, Cyp1B1, Cyp2S1, Cyp2A5, and Cyp19A1 (Arpiainen et al, 2005;Baba et al, 2005;Denison et al, 1988;Eltom et al, 1999;Rivera et al, 2007). These probes gave AhR-binding scores ranging from 2.2 to 3.2 ( Fig.…”

“…To our knowledge, only a limited number of functional AhR-binding sites have been identified, mostly within the promoter regions of AhR-regulated genes (Sun et al, 2004;Tijet et al, 2006), including Cyp1A1 (Cytochrome P450, 1A1) (Denison et al, 1988;Lusska et al, 1993), Cyp1A2 (Cytochrome P450, 1A2) (Quattrochi et al, 1998;Sogawa et al, 2004), Cyp1B1 (Cytochrome P450, 1B1) (Eltom et al, 1999), Nqo1 (NAD(P)H: quinone oxidoreductase 1) (Favreau and Pickett, 1991), Ugt1A1 (UDPglucuronosyltransferase 1A1) (Emi et al, 1996), Gsta1 (Glutathione S-transferase Ya) (Pimental et al, 1993), Aldh3A1 (Aldehyde dehydrogenase 3A1) (Boesch et al, 1999), Nrf2 (NF-E2 p45-related factor) (Miao et al, 2005), Pon1 (Paraoxonase 1) (Gouedard et al, 2004), Cyp19A1 (P450 aromatase) (Baba et al, 2005), c-myc (Yang et al, 2005), Cyp2S1 (Cytochrome P450, 2S1) (Rivera et al, 2007), and Cyp2A5 (Cytochrome P450, 2A5) (Arpiainen et al, 2005); Cyp1A1 and Cyp1B1 being studied most extensively as model AhR-induced genes. Previous gene expression profiling by the DNA microarray technique in mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD revealed that at least 285 genes exhibited obvious changes in their expression (Dere et al, 2006), but experimental genome-wide research for direct AhR-target genes has not been carried out so far.…”