An Electrophile Responsive Element (EpRE) Regulates β-Naphthoflavone Induction of the Human γ-Glutamylcysteine Synthetase Regulatory Subunit Gene

Abstract

Exposure of HepG2 cells to β-naphthoflavone (β-NF) results in time- and dose-dependent increase in the steady-state mRNA levels for both the catalytic (GCSh) and regulatory (GCS1) subunits of γ-glutamylcysteine synthetase (GCS) which catalyzes the rate-limiting step in the de novo synthesis of the cellular antioxidant glutathione (GSH) (Mulcahy, R. T., Wartman, M. A., Bailey, H. B., and Gipp, J. J. (1997) J. Biol. Chem. 272, 7445–7454). Cloning and sequencing of the GCS1promoter region is reported. Regulatory sequences mediating basal and β-NF induced expression of the GCSl gene were identified using a series of promoter/reporter fusion genes transfected into HepG2 cells. Sequences directing basal and β-NF induced expression were localized between nucleotides −344 and −242 (numbered relative to the translation start site).Mutational analyses indicate that basal expression of the GCSl gene is directed by a consensus AP-1-binding site located 33 base pairs upstream of a consensus electrophile responsive element (EpRE) sequence; both cis -elements are capable of supporting β-NF inducibility. Elimination of the inducible response requires simultaneous mutation of both sequences, however, in the presence of an intact EpRE the upstream AP-1 site is irrelevant to induction. Regulation of expression of both human GCS subunit genes in response to β-NF is therefore mediated by cis -elements satisfying the consensus core EpRE motif. Exposure of HepG2 cells to β-naphthoflavone (β-NF) results in time- and dose-dependent increase in the steady-state mRNA levels for both the catalytic (GCSh) and regulatory (GCS1) subunits of γ-glutamylcysteine synthetase (GCS) which catalyzes the rate-limiting step in the de novo synthesis of the cellular antioxidant glutathione (GSH) (Mulcahy, R. T., Wartman, M. A., Bailey, H. B., and Gipp, J. J. (1997) J. Biol. Chem. 272, 7445–7454). Cloning and sequencing of the GCS1promoter region is reported. Regulatory sequences mediating basal and β-NF induced expression of the GCSl gene were identified using a series of promoter/reporter fusion genes transfected into HepG2 cells. Sequences directing basal and β-NF induced expression were localized between nucleotides −344 and −242 (numbered relative to the translation start site). Mutational analyses indicate that basal expression of the GCSl gene is directed by a consensus AP-1-binding site located 33 base pairs upstream of a consensus electrophile responsive element (EpRE) sequence; both cis -elements are capable of supporting β-NF inducibility. Elimination of the inducible response requires simultaneous mutation of both sequences, however, in the presence of an intact EpRE the upstream AP-1 site is irrelevant to induction. Regulation of expression of both human GCS subunit genes in response to β-NF is therefore mediated by cis -elements satisfying the consensus core EpRE motif. Glutathione (l-γ-glutamyl-cysteinyl-glycine; GSH) 1The abbreviations used are: GSH, glutathione; AhRE, aromatic hydrocarbon responsive element; AP-1, activation protein-1; β-NF, β-naphthoflavone; EpRE, electrophile responsive element; GCS, γ-glutamylcysteine synthetase; GCSh, GCS heavy subunit; GCSl, GCS light subunit; bp, base pair(s); kb, kilobase pair(s); NQO1, human NAD(P)H quinone oxidoreductase. 1The abbreviations used are: GSH, glutathione; AhRE, aromatic hydrocarbon responsive element; AP-1, activation protein-1; β-NF, β-naphthoflavone; EpRE, electrophile responsive element; GCS, γ-glutamylcysteine synthetase; GCSh, GCS heavy subunit; GCSl, GCS light subunit; bp, base pair(s); kb, kilobase pair(s); NQO1, human NAD(P)H quinone oxidoreductase. is the prominent cellular non-protein thiol, typically present in millimolar concentrations in most cell types (1Meister A. Dolphin D. Poulson R. Avramovic O. Coenzymes and Cofactors. Glutathione: Chemical, Biochemical and Medical Aspects. Vol. III. John Wiley and Sons, New York1989: 367-474Google Scholar, 2Reed D.J. Ann. Rev. Pharmacol. Toxicol. 1990; 30: 603-631Crossref PubMed Google Scholar). GSH is a predominant cellular antioxidant and as such serves critical functions in the maintenance of cellular redox balance, provides protection against reactive oxygen species, and is involved in the detoxification of xenobiotics either through direct conjugation with reactive species or through enzymatic reactions catalyzed by glutathione S -transferases (3Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3234) Google Scholar). GSH is frequently elevated in normal cells upon exposure to xenobiotics and has been implicated in tumor cell resistance to alkylating agents, Pt compounds, and anthracyclines (4Schroder C.P. Godwin A.K. O'Dwyer P.J. Tew K.D. Hamilton T.C. Ozols R.F. Cancer Invest. 1996; 14: 158-168Crossref PubMed Scopus (77) Google Scholar, 5O'Brien M.L. Tew K.D. Eur. J. 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Methods Enzymol. 1995; 252: 26-30Crossref PubMed Scopus (139) Google Scholar) have demonstrated that all the catalytic activity and the site of GSH feedback inhibition reside with the heavy subunit. However, the kinetic properties of the heavy subunit under physiological conditions are greatly influenced by association with the light subunit, presumably mediated by a redox-sensitive disulfide bond between the two subunits (16Huang C.-S. Chang L.-S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar). The influence of the regulatory subunit on the kinetic properties of the catalytic subunit is so profound that Huang et al. (16Huang C.-S. Chang L.-S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 19675-19680Abstract Full Text PDF PubMed Google Scholar) hypothesized that the monomeric catalytic subunit would be nonfunctional at the substrate (glutamate) and inhibitor (GSH) concentrations typically existing in cells. Our laboratory has cloned the cDNAs for the human liver GCSh (17Gipp J.J. Chang C. Mulcahy R.T. Biochem. Biophys. Res. Commun. 1992; 185: 29-35Crossref PubMed Scopus (122) Google Scholar) and GCSl (18Gipp J.J. Bailey H.H. Mulcahy R.T. Biochem. Biophys. Res. Commun. 1995; 206: 584-589Crossref PubMed Scopus (110) Google Scholar) subunits and recently reported the cloning and sequencing of the GCSh gene (19Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). We now report cloning and sequencing of the promoter and 5′-flanking sequence of the GCS light subunit gene. Steady-state levels of mRNA corresponding to the heavy and the light subunits of GCS have been reported to be elevated after exposure of cells to various xenobiotics such as β-NF (19Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), methyl mercury (20Woods J.S. Ellis M.E. Biochem. Pharmacol. 1995; 50: 1719-1724Crossref PubMed Scopus (86) Google Scholar), tert -butyl hydroquinone (11Liu R.M. Hu H.P. Robison T.W. Forman H.J. Am. J. Respir. Cell Mol. Biol. 1996; 14: 186-191Crossref PubMed Scopus (57) Google Scholar), and butylated hydroxyanisole (13Borroz K.I. Buetler T.M. Eaton D.L. Toxicol. Appl. Pharmacol. 1994; 126: 150-155Crossref PubMed Scopus (76) Google Scholar). Since several of these treatments also induce the expression of key Phase II detoxifying enzymes (21Prestera T. Holtzclaw W.D. Zhang Y. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2965-2969Crossref PubMed Scopus (404) Google Scholar), we hypothesized that the transcriptional up-regulation of the GCS subunit genes and genes of the Phase II battery may be mediated by common regulatory elements. Regulation of several Phase II enzymes in response to a wide variety of inducing agents, including several of those which induce GCS expression, is mediated, at least in part, by the presence of electrophile responsive elements (EpRE) 2In this report we use the designation EpRE to indicate elements matching the consensus sequence 5′-(A/G)TGACNNNGCA-3′. These elements have also been referred to as antioxidant responsive elements (AREs). 2In this report we use the designation EpRE to indicate elements matching the consensus sequence 5′-(A/G)TGACNNNGCA-3′. These elements have also been referred to as antioxidant responsive elements (AREs). within the 5′-flanking region of the gene (21Prestera T. Holtzclaw W.D. Zhang Y. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2965-2969Crossref PubMed Scopus (404) Google Scholar, 22Jaiswal A.K. Biochem. Pharmacol. 1994; 48: 439-444Crossref PubMed Scopus (224) Google Scholar, 23Daniel V. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 173-207Crossref PubMed Scopus (269) Google Scholar, 24Prestera T. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8965-8969Crossref PubMed Scopus (218) Google Scholar, 25Rushmore T.H. Morton M.R. Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar, 26Li Y. Jaiswal A.K. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, Pickett C.B. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, M. A. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Our laboratory has recently demonstrated that basal and expression of the GCSh gene is mediated by a consensus EpRE sequence located in the of the promoter of the GCS heavy subunit gene (19Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). We have also identified regulatory including a EpRE in the 5′-flanking sequence of the GCSl the that the expression of the regulatory subunit gene may be mediated by or EpRE or We have also demonstrated that exposure of HepG2 cells to β-NF, a aromatic capable of inducing gene expression results in increased expression of the GCSl as as the GCSh subunit gene (19Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). this is with the the of elements in induced expression of the GCSl gene has been in this we a to the regulatory elements for β-NF inducible expression of the GCSl gene. a series of promoter transfected into HepG2 we have been to that the EpRE sequence identified in the GCSl promoter is for in response to β-NF and that a AP-1 site located 33 upstream of the EpRE expression of the gene and is also capable of directing increased expression β-NF exposure the core EpRE sequence be sequence corresponding to the light subunit of human GCS and its 5′-flanking region were from a human by as R.T. Gipp J.J. Biochem. Biophys. Res. Commun. 1995; PubMed Scopus Google Scholar), corresponding to sequences present in (18Gipp J.J. Bailey H.H. Mulcahy R.T. Biochem. Biophys. Res. Commun. 1995; 206: 584-589Crossref PubMed Scopus (110) Google Scholar). nucleotides of GCSl sequence and of 5′-flanking sequence into and by the using S. Biochemical and corresponding to The sequence by sequencing were by cloning from the 5′-flanking sequence of the GCSl gene into for of the promoter activity or by GCSl or into for of from 5′-flanking region of and cloned into site of the to with enzymes to a series of as in series of promoter/reporter in of within the GCSl promoter were by The sequence of the were by of and in on and β-NF induced the sequence of to of the and to used to the relative of the EpRE its AP-1 within the and the upstream AP-1 site at in of the basal and β-NF induced expression of the GCSl subunit gene. The were into The of on the cis -elements is by the presence or of the corresponding the of the various on basal and β-NF induced expression are is intact cis -elements are as elements are as The and are of the corresponding sequences in The and fusion genes were with the into HepG2 cells by were as in the to the for activity and from to The to the of the the relative of on basal and induced expression of the The sequence of the a consensus AP-1 In to of this element several of the and were cloned into a in which the AP-1 site been S. and M. were therefore results with the two were of of the sequence were and cloned into the in were by to to and by using These were using with and with and in for into the HepG2 cells were in with and were transfected with using a HepG2 cells were at on with to the cells were transfected by the of expression of were used to for in for of the Res. PubMed Scopus Google Scholar) the gene under the of the human with after of cells were by the of for at and at for an in the of this the with the or at in cells were and for of and cell were with and and at for in of were from the and the at the in a for at The were to and on activity as by Methods Enzymol. PubMed Scopus Google Scholar). this and as = in at and to for of cell to the and in an of of in as relative light activity for on the of activity and of the a of light of using the M.M. Biochem. PubMed Scopus Google Scholar) with as a between were by of using least the human GCSl gene were by of a human using a of a region of the of the from identified by using to sequences in the of the from and into that this corresponding to the of the and of 5′-flanking of the of the The 5′-flanking region of the GCS light subunit gene several of including a of and several consensus AP-1, as as several were also and core sequence corresponding to the were however, a core EpRE at to The 5′-flanking sequence of the GCS light subunit gene also a consensus responsive which of such as in response to exposure to heavy and Since the gene start which cell and which are with consensus sequences, we have a which the to the base of the translation start In we have demonstrated that the steady-state mRNA levels for both the GCSh and GCSl genes are increased in HepG2 cells after with as as β-NF (19Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), levels at increase with the after the of of the GCSl 5′-flanking region basal and β-NF inducible expression of the GCSl a series of fusion genes were by cloning various from the GCSl into the These were transfected into HepG2 cells and activity in the presence and of In of basal expression in cells transfected with the that in HepG2 cells transfected with the transfected with the a basal expression the the presence of regulatory between and −344 that influence expression of the GCSl gene. expression in cells transfected with the of an sequence from to increase expression from to These that the sequences expression of the are localized within the upstream of the translation start that are at least two or a of basal expression, in the and in the Exposure to β-NF in increased expression in HepG2 cells transfected with the fusion gene. However, expression induced the basal were with The of β-NF increased by the of sequences upstream of the −344 for regulatory expression are located between nucleotides and of the sequence between and −344 of the GCSl promoter the presence of a consensus EpRE core sequence between and Since have been demonstrated to and expression of Phase II enzymes and we demonstrated that a EpRE for these in the of the human GCSh subunit gene (19Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar), we this element a of GCSl gene In to this responsive element to basal and β-NF induced expression of the GCSl an nucleotides to of the GCSl promoter and into the site of a in which expression is under of the the functional EpRE sequence Methods Enzymol. PubMed Scopus Google Scholar) from the human gene into the and used as a transfected into HepG2 the GCSl EpRE sequence in either directed a increase in basal activity relative to that in cells transfected with The of the increase to that in HepG2 cells transfected with the β-NF of HepG2 cells transfected with these in a of The to β-NF the GCSl EpRE directing both increased basal and inducible expression of the gene in response to β-NF that this EpRE response to β-NF, as as basal expression of the an a mutation in the EpRE core sequence to and cloned into the by of the in this is for both basal and expression T.H. Morton M.R. Pickett C.B. J. Biol. Chem. 1991; 266: 11632-11639Abstract Full Text PDF PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). in this mutation both the increased basal expression and by β-NF in cells transfected with the The results of these were therefore with the hypothesized of the EpRE core in the basal and β-NF induced expression of the GCSl gene. In an to that the GCSl EpRE GCSl gene expression in vivo the GCSl EpRE mutation into by the The the and 5′-flanking sequences were transfected into HepG2 cells and on basal and expression of the gene were The of the EpRE mutation into the sequence to expression in to that in HepG2 cells transfected with the the sequence expression in cells transfected with the = induced with β-NF, the of expression of that in cells transfected with the elements within the region of the the core EpRE, to β-NF inducibility. The GCSl EpRE sequence an core AP-1 sequence which also by the to mutation to the AP-1 site has been to influence basal and β-NF induced expression of the GCSh subunit gene. C. and R. T. In this region of the GCSl promoter also a AP-1 site located 33 upstream of the EpRE and the that these AP-1 to the inducible activity in a series of were of the EpRE sequence were The the core EpRE the AP-1 site The on the a consensus EpRE the AP-1 site is of the upstream AP-1 site were to and to the upstream AP-1 site at were and to be in of on the of the results with these two were and are referred to as a series of and were also in mutation of the AP-1 site activity relative to the intact AP-1 sequence These that maintenance of the AP-1 sequence is for either basal or induced expression of the gene. However, this of the upstream AP-1-binding expression in cells transfected with of the a mutation of the upstream AP-1 site and of the of the core EpRE or AP-1 were in basal expression these various that the upstream AP-1 site is for the increased basal expression localized to the region of the of the with intact upstream AP-1 basal expression by to the Exposure of these to β-NF in increased expression expression of that in cells transfected with intact EpRE sequences however, cells were transfected with the or in with of the upstream AP-1 site β-NF the of is as a of activity in cells to that in the by the and are to those induced by upstream and These that the upstream AP-1 can increased expression in response to β-NF in the of the EpRE for the in the using the the that the EpRE sequence is that of the upstream AP-1 is the of in cells transfected with an intact EpRE and upstream AP-1 site to that in cells transfected with sequences intact upstream AP-1 expression of the human GCSl gene is mediated by at least two elements or of within the and a within the The a consensus EpRE GCSl EpRE, at and a consensus AP-1 at The upstream AP-1 site at identified as the within the for the basal properties to this analyses are to the of elements for the properties with the These elements are to for the basal activity in cells transfected with the of the AP-1 site all series The of a functional for AP-1 in of the expression of the GCSl subunit gene is with evidence from which that AP-1 is an in the of GCS gene of the the GCS has been to be mediated by Mol. Biol. 1994; 14: PubMed Google Scholar), the of In basal and induced expression of the gene has been to be D.J. Mol. 1995; PubMed Scopus Google Scholar). et al. M.L. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar) recently a AP-1 for expression of the human GCS heavy subunit gene on the that the expression of the GCSh gene and steady-state GSH levels were in from by to et al. Godwin Ozols R.F. O'Dwyer P.J. Hamilton T.C. Cancer Res. 1995; Google Scholar) reported that of the GCS heavy subunit mRNA in a cell to of and a increase in AP-1 analyses and in laboratory also a for AP-1 in of basal expression of both GCS is therefore that the basal and induced expression of both GCS subunit genes AP-1 or in with of basal to expression the of the to those elements which to β-NF induced expression of the GCSl subunit gene. to the GCSh subunit the EpRE at of the GCSl 5′-flanking sequence a for this by the which demonstrated that the EpRE sequence β-NF However, mutation of this EpRE sequence in the from the GCSl gene in a in β-NF as in the of the EpRE in the heavy subunit promoter (19Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). The use of and demonstrated that the of the GCSl and to the presence of the upstream AP-1 of a mutation to the upstream AP-1 site to these the β-NF of the β-NF therefore simultaneous mutation of both of elements. in either the GCSl EpRE or the upstream AP-1 site is to direct the induced and of is The of β-NF exposure in cells transfected with either the or to that in cells transfected with fusion genes both intact elements that the presence of the EpRE sequence is and to the induced response of the of the upstream AP-1 is therefore to the that the upstream AP-1 element in β-NF in the presence of an intact However, the regulatory by this of cis elements may an against the of to the regulatory region of this gene. of expression to be the for the two GCS subunit genes indicate that the of gene expression by β-NF exposure is mediated by cis -elements present in the promoter of the two subunit its with the core EpRE consensus sequence and its functional properties in is that the GCSl EpRE, its as an EpRE in is by the that this element is involved in the of expression of the GCSl gene cloned into the promoter/reporter as of a of activity is as a functional of in In this is to that the GCSl EpRE sequence is to the recently T. 1994; Google Scholar, M. M. Mol. Biol. 1994; 14: PubMed Google Scholar) element of and of The sequence also an site which is by the of including and V. L. P. Res. PubMed Scopus Google Scholar, H. P. 1993; PubMed Scopus Google Scholar). The which regulatory to sequence H. M. J.D. Res. 1997; PubMed Scopus Google Scholar, V. Biol. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar). the also to of gene expression by with including of the AP-1 as as and T. 1994; Google M. M. Mol. Biol. 1994; 14: PubMed Google Scholar, H. M. J.D. Res. 1997; PubMed Scopus Google Scholar, V. Biol. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar). is that of the of and to the sequence we have referred to as the GCSl In we have been to the increased of to the GCSl EpRE sequence in response to β-NF R. and R. T. this have recently been for genes sequences, including the human R. Jaiswal A.K. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and genes J. Biochem. Biophys. Res. Commun. 1996; PubMed Scopus Google Scholar). et al. T. S. T. Y. T. M. Y. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar) recently demonstrated that of several in the is on the and expression and of to the EpRE the most direct evidence that these to the and induced expression of genes has been referred to as are in or of the or of In basal expression of the GCSl gene is influenced by a consensus AP-1-binding site located 33 upstream of a consensus EpRE sequence in the region of the The element is for basal of -elements are capable of supporting β-NF the EpRE element to the be of the Elimination of the inducible response requires simultaneous mutation of both The a to to involved in of GCS subunit gene expression and the for is that of the and AP-1 of are involved in of these