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, ∼198, 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-1α/ARNT in vitro, the most 3′ HRE contributes the most toward hypoxia inducibility. AHR/ARNT and HIF-1α/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. 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, ∼198, 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-1α/ARNT in vitro, the most 3′ HRE contributes the most toward hypoxia inducibility. AHR/ARNT and HIF-1α/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 (1Nelson D.R. Zeldin D.C. Hoffman S.M. Maltais L.J. Wain H.M. Nebert D.W. Pharmacogenetics. 2004; 14: 1-18Crossref PubMed Scopus (778) Google Scholar). 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), 6The abbreviations used are: RACE, rapid amplification of cDNA ends; AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; XRE, xenobiotic-responsive element; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia response element; EMSA, electrophoretic mobility shift assay; CREB, cAMP-responsive element-binding protein. and carcinogenic polycyclic aromatic hydrocarbons via the aryl hydrocarbon receptor (AHR) (2Lewis D.F. Curr. Med. Chem. 2003; 10: 1955-1972Crossref PubMed Scopus (110) Google Scholar). Rylander et al. (3Rylander T. Neve E.P. Ingelman-Sundberg M. Oscarson M. Biochem. Biophys. Res. Commun. 2001; 281: 529-535Crossref PubMed Scopus (104) Google Scholar) 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 (4Rivera S.P. Saarikoski S.T. Hankinson O. Mol. Pharmacol. 2002; 61: 255-259Crossref PubMed Scopus (129) Google Scholar). 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 (5Arpiainen S. Raffalli-Mathieu F. Lang M.A. Pelkonen O. Hakkola J. Mol. Pharmacol. 2005; 67: 1325-1333Crossref PubMed Scopus (54) Google Scholar)). 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 all-trans-retinoic acid to the catabolic products, 4-hydroxyretinoic acid and 5,6-epoxyretinoic acid (6Smith G. Wolf C.R. Deeni Y.Y. Dawe R.S. Evans A.T. Comrie M.M. Ferguson J. Ibbotson S.H. Lancet. 2003; 361: 1336-1343Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and it has been reported that the enzyme can metabolize naphthalene to two products (7Karlgren M. Miura S. Ingelman-Sundberg M. Toxicol. Appl. Pharmacol. 2005; 207: 57-61Crossref PubMed Scopus (96) Google Scholar). 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 (3Rylander T. Neve E.P. Ingelman-Sundberg M. Oscarson M. Biochem. Biophys. Res. Commun. 2001; 281: 529-535Crossref PubMed Scopus (104) Google Scholar, 8Choudhary D. Jansson I. Stoilov I. Sarfarazi M. Schenkman J.B. Arch. Biochem. Biophys. 2005; 436: 50-61Crossref PubMed Scopus (217) Google Scholar, 9Saarikoski S.T. Rivera S.P. Hankinson O. Husgafvel-Pursiainen K. Toxicol. Appl. Pharmacol. 2005; 207: 62-69Crossref PubMed Scopus (93) Google Scholar, 10Saarikoski S.T. Wikman H.A. Smith G. Wolff C.H. Husgafvel-Pursiainen K. J. Histochem. Cytochem. 2005; 53: 549-556Crossref PubMed Scopus (65) Google Scholar). The facts that all other previously well characterized dioxin-inducible cytochrome P450s (CYP1A1, CYP1A2, and CYP1B1) contribute significantly to the activation of a variety of procarcinogens in mammals, that CYP2S1 is highly expressed in epithelial tissues exposed to the environment, that its expression pattern is similar to that of the procarcinogen-metabolizing enzyme CYP1B1 (8Choudhary D. Jansson I. Stoilov I. Sarfarazi M. Schenkman J.B. Arch. Biochem. Biophys. 2005; 436: 50-61Crossref PubMed Scopus (217) Google Scholar, 11Choudhary D. Jansson I. Schenkman J.B. Sarfarazi M. Stoilov I. Arch. Biochem. Biophys. 2003; 414: 91-100Crossref PubMed Scopus (208) Google Scholar), and that the Cyp2s1 gene is located in a cluster with other CYP2 family members that metabolize xenobiotics, including procarcinogens, suggest that Cyp2s1 may play a role in metabolic activation or deactivation of procarcinogens present in the environment. The enzyme may also play a role in the metabolism of endogenous compounds (such as retinoic acid) involved in the differentiation and/or maintenance of epithelial issues and in the metabolism of of the of which is the of this is and polycyclic aromatic hydrocarbon of gene is mediated by AHR. the and with the aryl hydrocarbon nuclear translocator (ARNT) protein. dimer to regulatory elements of transcription. The regulatory element involved is the xenobiotic-responsive element The sequence for a XRE is the XRE sequence can the of the XRE, and of to make with the AHR/ARNT dimer O. Pharmacol. Toxicol. PubMed Scopus Google Scholar). and the response to in part on in for a of genes involved in and and cell Transcriptional is mediated by hypoxia-inducible 1 which is a dimer of and the HIF-1α/ARNT dimer to hypoxia response elements (HREs) located in the regulatory region of transcription. The sequence of the HRE, that of the XRE G. 2005; Scholar). ARNT, which the of the XRE S. Hankinson O. Mol. Pharmacol. Google Scholar, J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), also this sequence in the the response is by a of These are of the with to its and the of the from Curr. 2004; PubMed Scopus (96) Google Scholar, Mol. 2004; PubMed Scopus Google Scholar). in the aromatic hydrocarbon response and the hypoxia that dioxin of mouse Cyp2s1 is mediated in large part by a novel regulatory of three overlapping located at nucleotides upstream of the Cyp2s1 translational Each of the of the trimeric XRE has the to bind the AHR/ARNT dimer and mediate dioxin-dependent transcription. In we that Cyp2s1 is inducible by and is mediated in part by three overlapping within the trimeric XRE segment. and cell was from the The cell is of the mouse hepatoma cell previously in this Nebert D.W. Hankinson O. J. Chem. Full Text PDF PubMed Google Scholar, O. S. PubMed Scopus Google Scholar). in with and in or was used at or The was in a hypoxia with a containing and The cells exposed to hypoxia at to and been previously S. Hankinson O. Mol. Pharmacol. Google Scholar). The was from to the Cyp2s1 sequence to in mouse Cyp2s1 and in human CYP2S1 was The was and high by the The was to by the in the which is to The was with and was from the by a the the of cDNA to the of the region within the The was by the as (4Rivera S.P. Saarikoski S.T. Hankinson O. Mol. Pharmacol. 2002; 61: 255-259Crossref PubMed Scopus (129) Google Scholar). was the to the The products of the cDNA cloned and on in the and of was the to the was to of the was by cDNA for mouse Cyp2s1 was used as of the expression was by the In as levels of by in for or and with hypoxia and or as was to the was the to the was on the The and as mouse Cyp2s1 is 3′ mouse 3′ or cells for 1 or with or and cells in and by at for at The in and for three of each with the was by in the of by a and with to Cyp2s1 at a with The of to in was used at with the and of Cyp2s1 for mouse Cyp2s1 was used to search the mouse data at the was that of sequence from mouse this as a a was with and as the was as the The was in and with The was and to of the region and the for the used to the as the the sequence was for the the sequence was and for the the sequence was The that the for was the the sequence was the the sequence was for the the sequence was for the the sequence was for the the sequence was and for the the sequence was the and the was used as and the was which is located in the the The including the are shown in The and The for The and by sequence of of the XRE on Cyp2s1 by used to three to within the XRE sequence to and in in to or of the xenobiotic-responsive elements within the mouse trimeric XRE segment containing the and the at with the used in all which was located 3′ to the The in was the as 1 1 1 The products with and in and as The of two in the trimeric XRE segment of the mouse Cyp2s1 gene or by two at the to as in In containing the and the at with the used in all which was located 3′ to the The in was the as The products the in and of the and of of and trimeric Cyp2s1 XRE and with and or cells at in of of the and of or of was with the of to the in the of of the was the the of the to the with cells exposed to or dioxin and and/or for with and to by at for 1 and to The in a for and the and the containing the trimeric XRE sequence from the mouse Cyp2s1 and containing XRE sequence from the mouse with by and used as a of or cells as M.M. Res. PubMed Scopus Google Scholar). each of the nuclear in a containing 1 1 and of in a of at for in the or of 1 of for of was with or a of the XRE sequence or of the sequence The for at and to in a at to this shift was as for a containing 1 and of in a of from or cells by for with dioxin and the cells for and the dioxin for and for was 1 the of hypoxia for from was used for the containing a HRE sequence from the human was used as a of cells at cells in a The the cells with for to endogenous gene or with or for in the or of D. was from the cells by the to the was on the to the to the Cyp2s1 and the genes on the and cells in with and in that they be the The cells with at dioxin for 1 or was by the of at for with and in 1 of at for at in of cell and and on for at high for on and a cell to to and was by for at at The was in was by the of of a of in and on a at for 1 and in a The with of the AHR, ARNT, or on a at The with of a of in and for 1 at on a The and in and and two in 1 1 by the of two of was by in a The with for 1 at was by and to used to the Cyp2s1 at and at from the translational The the trimeric in the region of for and at for for for by a for was as of the Transcriptional of used to the of Cyp2s1 a of we used cells with dioxin for of at about 102, and 22 nucleotides upstream of the translational initiation codon, The of is with the of in the Cyp2s1 of of Cyp2s1 by of of nucleotides upstream of the translational initiation of of nucleotides upstream of the translational initiation codon. in a of in the Cyp2s1 elements to the XRE sequence occur within to the of overlapping occur and and are at and of the Cyp2s1 upstream region the contains and in to identify elements that to 1 to the translational initiation codon. concentrations of each with the the mouse hepatoma cell line, Hepa-1, or the human hepatoma cell line, cells with or and a they for the dioxin was used for the human cell in with the of the human for ligands Toxicol. PubMed Scopus Google Scholar)). The contains the gene of the and as a for from the as to for each of a in cells are shown in containing of the region of Cyp2s1 similar dioxin of about containing of sequence the expressed by the These that is by the segment and upstream of the translational the three overlapping and that the XRE elements at and make contribution toward dioxin inducibility. Induction of the with a containing of the upstream sequence from the of the was in in cells is shown in The and inducible by The similar as the and was by the with the that is by the segment and the three overlapping of the was that of the of the upstream region of the of the of Each of the to each of the overlapping in the containing the of in all and was by the to in each XRE is to the XRE PubMed Scopus (126) Google Scholar). Each or two nucleotides of the in the XRE segment. from to at the on the of in the mouse gene O. Pharmacol. Toxicol. PubMed Scopus Google Scholar). to the dioxin of in of the was in cells in the mouse The and from three are in These are as the of the to the Cyp2s1 to the to the for each dioxin in the to the of the two XRE in the with this in of each XRE dioxin of two or to of all three In to the role of the XRE of the XRE we of this segment upstream of the gene by the containing each of the in each of the of the and all of also These with the and The and from three are shown in The similar to with the in the was two and all three the role of each of the three in AHR/ARNT by shift to the segment containing the three overlapping was used as in was nuclear from and cells with dioxin with from cells and and was by with a of a to XRE from the mouse gene The in the as the with the and was with to and also These observations identify the as to containing the or XRE used for the gene used as in the shift at concentrations the The to with the for to the AHR/ARNT whereas all of the other The in the and with the in which all three are with another in that of all the response to dioxin and also to the AHR/ARNT The of of with the XRE are also with each dioxin and to with the sequence for to the AHR/ARNT The and 1 although similar in the as the as as the 1 that the may in to is with the that at a to the with to AHR/ARNT to the XRE sequence in EMSA, that they can bind the XRE although with In the gene and electrophoretic mobility shift that each XRE in the trimeric XRE segment is of the AHR/ARNT dimer and dioxin-dependent transcription. of the of the in the XRE in to to of the nucleotides the sequence can the of the The sequence of the three in the trimeric XRE segment to the sequence for at the at the upstream of the sequence D. Jansson I. Schenkman J.B. Sarfarazi M. Stoilov I. Arch. Biochem. Biophys. 2003; 414: 91-100Crossref PubMed Scopus (208) Google Scholar). In each XRE in the trimeric this is gene that this to be a in for the XRE to to dioxin in cells J. Chem. Full Text PDF PubMed Google Scholar, J. Chem. Full Text PDF PubMed Google Scholar). in similar it was that with in this are in another mouse hepatoma cell and in cells S. J. M. J. Biochem. PubMed Scopus Google Scholar). to this in to the dioxin of Cyp2s1 in cells is by the of at this of the in which the was to in or two In the of the most XRE was from to the sequence of of the other two or the sequence of of the and the response of this to dioxin be with the In the sequence of the XRE is whereas the XRE is The response of this be with in which the XRE is In the sequence of the most 3′ XRE is whereas the XRE is In the and 3′ are whereas the XRE is The of and be with that of which the XRE is The for dioxin inducibility. The and from three are in of to in the was found to on dioxin inducibility. the that with at this to dioxin as as containing at this in cells and are at with the in J. Chem. Full Text PDF PubMed Google and J. Chem. Full Text PDF PubMed Google to for the dioxin of Cyp2s1 in Induction of found that of cells with to a in Cyp2s1 of hypoxia also induced the Cyp2s1 and was with that of dioxin of the Cyp2s1 occur in the of that previously that dioxin of Cyp2s1 on and (4Rivera S.P. Saarikoski S.T. Hankinson O. Mol. Pharmacol. 2002; 61: 255-259Crossref PubMed Scopus (129) Google and Induction of Cyp2s1 on Cyp2s1 cells for with the exposed for a to or in the of and for Cyp2s1 expression. cells or hypoxia in the of D. of Cyp2s1 by dioxin and hypoxia These that of Cyp2s1 by two via of Cyp2s1 by Cyp2s1 at least be to the that contributes to of of Induction of of HRE sequences, identified within to the translational of used a of the that we used to identify the of the gene to to search for elements to hypoxia. The and from three are in These that the segment a high of hypoxia inducibility. was as in the was in the that the hypoxia in the upstream region is to a to a segment and hypoxia in the and was that in the or that hypoxia may in the region and The region and contains the trimeric XRE this segment hypoxia the was inducible by hypoxia in and The and from three are in and with the dioxin hypoxia was in the cells in the overlapping HRE sequences, are within the trimeric XRE elements are all embedded in that are to the sequence for the HRE, I. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), for the most HRE, which from this at the most the hypoxia of 1 1 and 1 of the and each a HRE sequence The with in and cells with The the HRE on the of The the HRE The in the 3′ HRE These that the response mediated by this segment is to the 3′ In changes the sequence of the most 3′ HRE from that of the it is that the of this is to its on this HRE the HRE and that is to the 3′ with the XRE that we used for of AHR/ARNT from and cells used in and similar and was with from cells 1 and was or with to and and and was by of a of to the HRE from the human gene a at this was from cells from cells with the HRE and this was or with to These observations all identify this as to the The of this was with with which with the of the in the cell The HIF-1α/ARNT was a to the of to the containing 1 1 or in the the was a of 1 was used that each HRE in the trimeric XRE segment is of HIF-1α/ARNT in vitro and that all to be to with the from the gene which that the and HRE hypoxia inducibility. for this is that the and bind the HIF-1α/ARNT dimer with the 3′ HRE and with to of the the the of the gene and the that of Cyp2s1 is mediated to a by the embedded within the trimeric XRE sequence and that the 3′ HRE is the most in this it is of that the of the HIF-1α/ARNT dimer to the trimeric XRE in to of the AHR/ARNT dimer with nuclear from cells with dioxin and hypoxia that the HIF-1α/ARNT dimer has for the trimeric XRE sequence the AHR/ARNT of and of to the XRE in as a of of the AHR/ARNT and HIF-1α/ARNT dimers to the Cyp2s1 gene in of dioxin to of and to the Cyp2s1 whereas such in of the cell of hypoxia to to the Cyp2s1 gene in with dioxin and hypoxia to of and are shown in and of of the are shown in the hypoxia-dependent of to Cyp2s1 to be of hypoxia to a of being for the cells to hypoxia the to the containing we also of to hypoxia and dioxin and In this we of induced of AHR, and levels about 1 of In of was 1 with dioxin and hypoxia induced of and we used the trimeric XRE for amplification in the that dioxin AHR/ARNT the trimeric XRE region of the Cyp2s1 gene and that hypoxia HIF-1α/ARNT this These on the endogenous Cyp2s1 gene in its the of the gene and in a role for the trimeric XRE sequence in dioxin and hypoxia identified a regulatory segment in the Cyp2s1 gene containing three overlapping and three overlapping that to make a major contribution to dioxin and hypoxia of the as we are containing or containing overlapping of been reported previously for is of that two overlapping and two overlapping are located at upstream of the human CYP2S1 dioxin and hypoxia can CYP2S1 in human cells (4Rivera S.P. Saarikoski S.T. Hankinson O. Mol. Pharmacol. 2002; 61: 255-259Crossref PubMed Scopus (129) Google Scholar). T. S. and O. that previously been used to the that nucleotides the XRE and HRE may play in or gene including of XRE and HRE in gene and in the or of or and amplification of AHR/ARNT and in vivo suggest that the AHR/ARNT and dimers nucleotides the XRE and HRE sequences, respectively. These that the XRE sequence by the AHR/ARNT dimer may be to nucleotides in whereas that by the dimer may nucleotides O. Pharmacol. Toxicol. PubMed Scopus Google Scholar, J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus (126) Google Scholar, K. M. K. 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Biophys. 2003; 414: 91-100Crossref PubMed Scopus (208) Google Scholar). the stages of the mouse is in a and of Cyp2s1 the enzyme is expressed at high levels in the has been that by dioxin may from in the and expression of cytochrome P450s for I. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is that the of dioxin at least in on its of Cyp2s1 and changes in concentrations of retinoic acid or other endogenous The of Cyp2s1 in is that of Cyp2s1 or to in the metabolism of is in they are in a or of of the by in in the tumor cells 2005; PubMed Scopus Google Scholar). of Cyp2s1 may be the for the in Cyp2s1 and expression in human S.T. Wikman H.A. Smith G. Wolff C.H. Husgafvel-Pursiainen K. J. Histochem. Cytochem. 2005; 53: 549-556Crossref PubMed Scopus (65) Google Scholar, M. S. Res. 2005; PubMed Scopus Google Scholar). is that this expression of Cyp2s1 the response of to The of Cyp2s1 by dioxin also polycyclic aromatic in epithelial tissues exposed to the the response of tissues to procarcinogens that are for the These issues all for