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Originally published In Press as doi:10.1074/jbc.M000405200 on May 18, 2000

J. Biol. Chem., Vol. 275, Issue 35, 27366-27376, September 1, 2000
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A CCAAT/Enhancer-binding Protein Site within Antioxidant/Electrophile Response Element Along with CREB-binding Protein Participate in the Negative Regulation of Rat GST-Ya Gene in Vascular Smooth Muscle Cells*

Yun-Houng Chen and Kenneth S. RamosDagger

From the Department of Physiology and Pharmacology, College of Veterinary Medicine, and Center for Environmental and Rural Health, Texas A & M University, College Station, Texas 77843 4466

Received for publication, January 19, 2000, and in revised form, April 12, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Studies were conducted to evaluate the negative regulatory function of rat (r)GST-Ya antioxidant/electrophile response element (ARE/EpRE) in vascular smooth muscle cells (vSMCs). We report that CCAAT/enhancer-binding protein (C/EBP)-beta interacts with ARE/EpRE in the rGST-Ya promoter and that aryl hydrocarbon receptor (AhR) is present within the protein complex binding to the C/EBP site. Overexpression of C/EBP-beta or C/EBP-alpha repressed, whereas AhR enhanced, 1.6CAT reporter activity in cells treated with benzo(a)pyrene (BaP). Overexpression of CREB-binding protein (CBP) nullified repression of rGST-Ya transcription. Human adenovirus E1A protein abrogated cotransactivation by CBP but an E1A mutant did not. Overexpression of C/EBPs abrogated stimulation of 1.6CAT by CBP or AhR alone, or in combination, regardless of BaP treatment. Similar profiles were observed using an AhRECAT construct. The C/EBP site within the ARE/EpRE inhibited chemical inducibility of the AhRE. The pattern of mouse GST-Ya regulation by BaP was similar to that of rGST-Ya. We conclude that multiple mechanisms mediate negative regulation of GST-Ya gene in vSMCs, most significant of which are that C/EBP-beta inhibits AhRE or ARE/EpRE inducibility of GST-Ya, limiting CBP levels compromise gene induction, functional interference exists between AhRE and ARE/EpRE, and AhR alone, or in combination with C/EBP-beta , functions as a repressor of the ARE/EpRE.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glutathione S-transferases (GSTs)1 have been regarded as a "triple threat" in the detoxification of xenobiotics (1). These enzymes catalyze conjugation of glutathione with substrates bearing an electrophilic moiety (2). They are also capable of binding xenobiotics on the enzyme surface preventing interactions with critical cellular macromolecules, such as proteins and nucleic acids (3). Furthermore, GSTs can form covalent bonds between reactive xenobiotics and the active site of the enzyme. Such binding inactivates the enzyme but also renders the xenobiotic inactive and represents an additional detoxification mechanism (4). Multiple GST isoforms have been characterized, with the GST-Ya subunit known to be the most abundant in rat liver (5). GST-Ya subunit is barely detectable in rat aorta (6), but its cell-specific expression in the mouse aorta, especially in vascular smooth muscle cells (vSMCs), has not been described. Pessah-Rasmussen and co-workers (7) reported that there is a positive correlation between the level of GST-trans-stilbene oxide (tSBO) in blood and in "healthy" arterial and venous tissue. Lower levels of GST-tSBO have been found in atherosclerotic segments of human arteries relative to healthy segments from the same artery. GST-Yk (GST8-8), a member of the GST Alpha family, plays a key role in protecting blood vessels against oxidative stress (8). As such, GSTs are believed to participate in the protection of blood vessels from alpha ,beta -unsaturated carbonyl toxins involved in atherosclerotic lesion formation (9).

Previous studies have reported that CCAAT/enhancer-binding protein-alpha (C/EBP-alpha ) is a member of the protein complex interacting with the AhRE in the rGST-Ya promoter (10). In this promoter, a C/EBP site (TTGCG) overlaps with the AhRE (TTGCGTG) (10). C/EBP-alpha increases the xenobiotic-induced response of a reporter vector containing three rGST-Ya AhRE sequences in HepG2 cells and binds to AhRE3 in the murine Cyp1a1 promoter (10, 11). The C/EBP high affinity sequence (ATTGCGCAAT) competes with the protein complex binding to the ARE/EpRE in the mouse GST-Ya promoter (12). These findings have led to the hypothesis that AhR (or AhR-related proteins) is an integral part of the ARE·EpRE-binding protein complex. We have recently demonstrated that a C/EBP-like site (ATTGCTAAT) that partially overlaps with the distal AP-1-like site (TGGCATTGC) within the ARE/EpRE in the rGST-Ya promoter mediates negative regulation of this gene in vSMCs (13). On the basis of these findings, we hypothesized that C/EBP-alpha , or other members of the C/EBP family, binds to the ARE/EpRE of rGST-Ya gene in vSMCs to negatively regulate its expression.

C/EBPs belong to the basic region-leucine zipper class of transcription factors (14). This family consists of C/EBP-alpha , -beta , -gamma , -delta , -epsilon , and -zeta (15). Among the six C/EBP family members, C/EBP-alpha , -beta , -delta , and -epsilon are activators of transcription of several genes (16-19). Negative regulation of gene expression by the C/EBP family has also been described. For example, the attenuation domain of C/EBP-alpha may diminish transactivation of the serum albumin gene (16, 18). In addition, its leucine zipper domain may exert a strong repressor effect on the albumin promoter in HeLa cells (18) and repress beta 2-adrenergic receptor gene expression in rat hepatocytes (20). C/EBP-alpha forms a heterodimer with ATF that binds the C/EBP site to repress transcription of a reporter vector containing two copies of the C/EBP consensus sequence (21). C/EBP-beta exists as two isoforms, liver activator protein and liver inhibitory protein. The liver activator protein functions as an activator, whereas the liver inhibitory protein lacks a transactivation domain and represses liver activator protein-inducible transcription of albumin (22). The liver activator protein also contains a negative regulatory domain functioning in HeLa and L (fibroblastic) cells but not in HepG2 cells (23). C/EBP-beta can form a repressor protein complex with NF-kappa B and estrogen receptor in the interleukin-6 promoter (24). C/EBP-gamma does not have an activation domain and functions as a trans-dominant repressor of transcription (25). C/EBP-delta is the main mediator of interleukin-1 suppression (26). C/EBP-epsilon also contains a repression domain (19), whereas C/EBP-zeta can form a heterodimer with other members in this family to inhibit gene transcription (27-28).

CREB-binding protein (CBP) is involved in the activation of a large variety of transcriptional enhancer elements through various transcription factors, including CREB, c-Jun, c-Myb, c-Fos, MyoD, the Stat proteins, and the nuclear receptor superfamily (29). CBP possesses intrinsic histone acetyltransferase activity and may cause localized changes in chromatin structure of genes targeted via interaction with specific transcription factors (30). Janknecht and Hunter (29) were among the first to suggest that activation of different transcription factors by diverse signaling pathways may interfere with each other by competing for limiting cofactors (i.e. CBP/p300). Arias and co-workers (31) also reported that CREB and c-Jun interfere with, or "squelch," one another by competing for limiting intracellular CBP levels. Therefore, negative cross-talk between multiple signaling pathways may be accounted for by limiting amounts of common coactivators. Similar patterns have been described between liganded nuclear hormone receptors and AP-1 (32). Furthermore, E1A was shown to abrogate the CBP-induced stimulation of c-Fos activity (33).

In the present studies, we present the following evidence. 1) The C/EBP site within the ARE/EpRE inhibits chemical inducibility through the AhRE, the major BaP-responsive sequence in the rGST-Ya promoter. Conversely, the AhRE reduces inducible activity of ARE/EpRE by BaP. 2) C/EBP-beta functions as a repressor by competing for binding to the C/EBP site within the AhRE or ARE/EpRE. 3) Limiting intracellular CBP protein levels diminish the inducibility of rat or mouse GST-Ya gene by BaP. 4) Liganded AhR alone, or in combination with C/EBP-beta , functions as a repressor through the ARE/EpRE. These mechanisms mediate, at least in part, negative regulation of GST-Ya in vSMCs.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Murine vSMCs were prepared as described previously (13).

Plasmids-- Four CAT constructs kindly provided by Dr. C. B. Pickett (Schering Plow Research Institute, Lafayette, NJ) were employed to evaluate xenobiotic inducibility profiles of rGST-Ya. The 1.6CAT construct contained the rGST-Ya promoter region from bases -1651 to +66, including AhRE and ARE/EpRE sites at -914 to -882 and -722 to -682, respectively, whereas -164CAT contains the minimal promoter region of the gene. ARE/EpRECAT contained the ARE/EpRE (from bases -722 to -682) linked to the minimal promoter region, whereas AhRECAT contained the AhRE (from bases -914 to -882) linked to the minimal promoter region. mAhR/pBK-CMV and mArnt/pBK-CMV expression vectors were kindly provided by Dr. J. P. Whitlock, Jr. (Stanford University) (34-35). pcDNA3-E1A and pcDNA3-E1ADelta 2/36 expression vectors were kindly provided by Dr. D. W. Hum (CHUL Research Center and Laval University, Canada) (36). pRc/RSV-mCBP expression vector was kindly provided by Dr. R. H. Goodman (Vollum Institute, OR) (37). The control vector for the CBP (CBP-CV) was constructed by removing full-length mouse CBP with XbaI and SpeI and religation of the vector with T4 DNA ligase. pcDNA3.1-mC/EBP-alpha and pcDNA3-mC/EBP-beta expression vectors were kindly provided by Dr. O. A. MacDougald (University of Michigan). All cotransfections were normalized by addition of CBP-CV and pcDNA3 expression plasmid as indicated (Invitrogen, CA). mGSTA1-ARE/EpRE luciferase reporter construct (abbreviated as mouse ARE/EpRE-luc) was kindly provided by Dr. J. A. Johnson (University of Wisconsin). pGL2-basic luciferase reporter construct was from Sigma and used as control plasmid for mouse ARE/EpRE-luc reporter activity.

Transfection, Chemical Treatments, and CAT/Luciferase/beta -Galactosidase Measurements-- vSMCs were transfected as described previously (13). Cells were treated with BaP at a final concentration of 3 µM, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at 1 nM, or H2O2 at 400 µM for 24 h. CAT and beta -galactosidase activities were determined as described previously (13), and CAT activities are expressed as percentage of control. Luciferase activity was determined by Luciferase Assay System from Promega (Madison, MI) following the manufacturer's instructions.

Site-directed Mutagenesis-- Site-directed mutagenesis was performed using a MorphTM site-specific plasmid DNA mutagenesis Kit from 5 Prime right-arrow 3 Prime, Inc. (Boulder, CO), according to the manufacturer's instructions. All mutations were confirmed by DNA sequencing.

Oligonucleotides-- Oligonucleotides were synthesized by the DNA Technologies Laboratory of the Center for Environmental and Rural Health at Texas A & M University. Complementary oligonucleotides were annealed and end-labeled as described previously (38). Rat or mouse GST-Ya ARE/EpREs and rGST-Ya AhRE were prepared as described (39). Random sequences for ARE/EpRE and AhRE were prepared as described previously (38, 40).

Antibodies-- AhR antibody was purchased from Affinity Bioreagents (Golden, CO) or kindly provided by Dr. Gary Perdew, Pennsylvania State University (College Park, PA). Normal IgG, c-Jun, C/EBP-alpha , C/EBP-beta , and C/EBP-delta antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse GST-Ya antibody was purchased from Biotrin (Dublin, Ireland).

Nuclear Extract Preparation and EMSA-- Nuclear extracts were prepared, and EMSA reactions were carried out as described previously (38), except that cells were treated with 0.3 µM BaP. In competition experiments, 50- or 100-fold molar excess of unlabeled cold DNA was preincubated with the reaction mixture for 5 min before addition of labeled probe. For supershift experiments, 1-2 µl of antibody was added after addition of the labeled probe and then incubated for 20 min.

Western Blot-- Fifty µg of total protein was obtained from cells treated with 3 µM BaP for 1, 2, 4, 8, 16, and 24 h. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) at constant voltage (10 V) overnight in the cold room. The ECL Western blotting detection system from Amersham Pharmacia Biotech was used in these experiments. Briefly, the membrane was blocked with phosphate-buffered saline/Tween containing 5% nonfat milk. After washing, the membrane was incubated with the primary antibody for 1 h according to manufacturer's instructions. The horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology) at a dilution of 1:30,000 was then incubated with the membrane for 1 h. The membrane was detected with ECL Western blotting reagents for 1 min and exposed to X-Omat film (Eastman Kodak Co.).

Statistics-- Data were expressed as means of relative CAT/luciferase activity ± S.E. Three determinations were carried out for each measurement.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

C/EBP-beta and AhR Interact with the ARE/EpRE and AhRE in vSMCs-- Previous studies in our laboratory have shown that a C/EBP-like site in the rGST-Ya promoter is responsible for negative regulation of the gene in vSMCs (13). The present studies were conducted to identify the protein(s) interacting with the C/EBP-like site (ATTGCTAAT). Supershift experiments using antibodies against C/EBP-alpha , -beta , -delta , and c-Jun were performed using vSMC nuclear extracts and the rGST-Ya ARE/EpRE as probe. Fig. 1A shows that an antibody to C/EBP-beta , but not C/EBP-alpha , -delta , or c-Jun, supershifted a specific ARE·EpRE protein complex. ARE/EpRE mutant competition experiments showed that ARE/EpRE wild type, m3, and m5 competed away the top band, whereas m4, m7, and m9, which contain mutations in the C/EBP-like site, exhibit different competition profiles (Fig. 1B). Direct comparison of the density of the top band after competition with m4, m7, and m9 shows weaker competition for m4 with mutation on GC and m7 with mutation on ATT. These results suggest that the ATTGC sequence in the ARE/EpRE is the region directly interacting with C/EBP-beta . Puga and co-workers (12) have reported that an antibody to AhR blocks the formation of mouse GST-Ya ARE·EpRE protein complex using mouse hepatoma cell nuclear extracts. Therefore, we conducted studies to determine if AhR is a component of the rGST-Ya ARE·EpRE protein complex in vSMCs. Our data showed that AhR antibody supershifted the top ARE·EpRE protein complex, the same complex affected by the C/EBP-beta antibody (Fig. 1C). We also found that AhR antibody supershifted two bands, results consistent with the presence of two AhR isoforms, 95 and 104 kDa, in C57BL6 mouse vSMCs (38).


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  		Fig. 1.   C/EBP-beta and AhR interact with ARE/EpRE in vSMCs. Nuclear extracts were prepared, and EMSA reactions were carried out as described under "Experimental Procedures." In competition experiments, 50- or 100-fold molar excess of unlabeled cold DNA was preincubated with the reaction mixture for 5 min before addition of labeled probe. For supershift experiments, 1-2 µl of antibody was added after addition of the labeled probe and then incubated for an additional 20 min. A, presence of C/EBP-beta in ARE·EpRE-binding protein complexes. An enlargement of the region with supershifted complexes is shown in the top panel. Lane 1, reaction without nuclear extract. Lane 2, cells treated with Me2SO (DMSO). Lane 3, cells treated with 0.3 µM BaP. Lanes 4 and 5, cells treated with 0.3 µM BaP and subsequently competed by 100× competitors as shown. Lanes 6-9, cells treated with 0.3 µM BaP, followed by addition of an antibody against C/EBP-alpha , C/EBP-beta , C/EBP-delta , or c-Jun, respectively. S, shifted band; SS, supershifted band. B, competition of ARE/EpRE with itself or ARE/EpRE mutants. Lane 1, reaction without nuclear extract. Lane 2, cells treated with 0.3 µM BaP. Lanes 3-8, cells treated with 0.3 µM BaP and subsequently competed with 50× cold competitors as indicated. Arrow indicates specific complex. Only 50× cold ARE/EpRE wild type was used as competitor to depict clearer competition patterns for the top complex. C, presence of AhR in ARE·EpRE-binding protein complexes. An enlargement of the region with supershifted complexes is shown in the top panel. Lane 1, reaction without nuclear extract. Lane 2, cells treated with Me2SO (DMSO). Lanes 3-6, cells treated with 0.3 µM BaP and subsequently competed as shown. Lanes 7 and 8, cells treated with 0.3 µM BaP, followed by addition of an antibody against AhR or IgG.

As suggested by Paulson and co-workers (10), C/EBP-alpha and AhR may interact with TTGCGTG within the AhRE in HepG2 cell nuclear extracts. In view of these results, supershift experiments were performed to determine if C/EBP family members and AhR interact with AhRE in vSMCs. C/EBP-beta and AhR, but not C/EBP-alpha , -delta , c-Jun, or IgG, supershifted the same complex (Fig. 2, A and B). This same protein complex was competed away by AhRE wild type, m2, and m6 but not AhRE m1, m3, m4, and m5 (Fig. 2C), suggesting that TTGCG is the target region for both C/EBP-beta and AhR.


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  		Fig. 2.   C/EBP-beta and AhR interact with AhRE in vSMCs. Nuclear extracts were prepared and EMSA reactions carried out as described under "Experimental Procedures." In competition experiments, 50- or 100-fold molar excess of unlabeled cold DNA was preincubated with reaction mixture for 5 min before addition of labeled probe. For supershift experiments, 1-2 µl of antibody was added after addition of the labeled probe and incubated an additional 20 min. A, presence of C/EBP-beta in AhRE-binding protein complexes. An enlargement of the region with supershifted complexes is shown in the top panel. Lane 1, reaction without nuclear extracts. Lane 2, cells treated with Me2SO. Lane 3, cells treated with 0.3 µM BaP. Lanes 4 and 5, cells treated with 0.3 µM BaP and subsequently competed by 100× cold competitors as shown. Lanes 6-9, cells treated with 0.3 µM BaP, followed by addition of an antibody against C/EBP-alpha , C/EBP-beta , C/EBP-delta or c-Jun, respectively. S, shifted band; SS, supershifted band. B, presence of AhR in AhRE-binding protein complexes. An enlargement of the region with supershifted complexes is shown in the top panel. Lane 1, reaction without nuclear extracts. Lane 2, cells treated with Me2SO. Lanes 3-6, cells treated with 0.3 µM BaP and subsequently competed as shown. Lanes 7 and 8, cells treated with 0.3 µM BaP, followed by addition of an antibody against AhR or IgG. C, competition of AhRE with itself or AhRE mutants. Lane 1, reaction without nuclear extracts. Lane 2, cells treated with 0.3 µM BaP. Lanes 3-8, cells treated with 0.3 µM BaP, and subsequently competed with 50× cold competitors as indicated. Arrow indicates the specific complex shifted by AhR or C/EBP-beta antibodies. w.t., wild type.

Overexpression of C/EBP-beta or C/EBP-alpha Down-regulate the Activity of 1.6CAT in vSMCs in Response to BaP, Whereas AhR Has the Opposite Effect-- To determine if C/EBP-beta or AhR, or both, function as repressor(s) of 1.6CAT in vSMCs, C/EBP-beta or AhR expression vectors were cotransfected with 1.6CAT. Overexpression of C/EBP-beta down-regulated the activity of 1.6CAT in response to BaP, but not Me2SO, whereas AhR up-regulated 1.6CAT in response to both BaP and Me2SO (Fig. 3A). Interestingly, overexpression of C/EBP-alpha also repressed 1.6CAT activity following BaP exposure (Fig. 3A). Paulson and co-workers (10) reported that AhR and C/EBP-alpha cooperate in the transactivation of rGST-Ya AhRE in HepG2 cells. To examine if AhR and C/EBPs cooperate in transcription of rGST-Ya, we found that both overexpression of C/EBP-beta and C/EBP-alpha decreases the positive effect of AhR on 1.6CAT in response to BaP in vSMCs (Fig. 3B).


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  		Fig. 3.   Repression of 1.6CAT activity by C/EBP-alpha or C/EBP-beta versus enhancement of 1.6CAT activity by AhR. A, cotransfection of 1.6CAT with C/EBP-alpha , C/EBP-beta , or AhR expression vectors. Fifteen µg of 1.6CAT was cotransfected with 5 µg of pcDNA3, C/EBP-alpha , C/EBP-beta , or AhR expression vectors into vSMCs by lipofection as described previously (13). Cells were treated with Me2SO (DMSO) or 3 µM BaP. A beta -galactosidase plasmid was cotransfected as an internal control. CAT and beta -galactosidase activities were determined as described previously (13). B, overexpression of C/EBP-alpha or C/EBP-beta decreases the stimulatory effect of AhR on 1.6CAT. Fifteen µg of 1.6CAT was cotransfected with pcDNA3, C/EBP-alpha , C/EBP-beta or AhR expression vectors as indicated (5 µg for each expression vector). Data are expressed as relative CAT activity. Values represent the mean ± S.E. from three separate experiments.

CBP Overrides Negative Regulation of Rat GST-Ya Gene-- CBP/p300 has been identified as a coactivator of various transcription factors (29). Activation of diverse signaling pathways may interfere with transcription factor function by competing for limiting intracellular levels of CBP/p300 (29). To test this hypothesis in vSMCs, increased amounts of CBP were cotransfected with 1.6CAT (Fig. 4A). The results showed that 10 µg of CBP significantly increased the activity of 1.6CAT in response to BaP as compared with Me2SO. To verify the function of CBP in this cell system, human adenovirus E1A was utilized to abrogate CBP/p300 function (33). Fig. 4B shows that E1A inhibited the function of CBP, whereas an E1A mutant (E1ADelta 2/36) did not. The E1A mutant utilized encodes for a protein without a complete CBP/p300 binding domain (41), confirming that limiting amounts of CBP are responsible for low induction of 1.6CAT in vSMCs.


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  		Fig. 4.   Limiting CBP mediates negative regulation of rGST-Ya gene in vSMCs. A, the effect of CBP overexpression on 1.6CAT. Fifteen µg 1.6CAT was cotransfected with different amounts of CBP in vSMCs as indicated (in µg). The total amount of DNA was kept constant by addition of CBP control vector (CBP-CV). B, inhibition by E1A of CBP cotransactivation of 1.6CAT. Cotransfection of 15 µg of 1.6CAT with 5 µg of E1A, E1ADelta 2/36, C/EBP-alpha , C/EBP-beta , or AhR vector in the presence of 10 µg of CBP vector in different combinations as indicated. The total amount of DNA was kept constant by addition of CBP-CV or pcDNA3 when appropriate. C, the effects of CBP, E1A, E1ADelta 2/36, C/EBP-alpha , C/EBP-beta or AhR overexpression on AhRECAT. Cotransfection of 15 µg fo AhRECAT with 2.5 µg of CBP, E1A, E1ADelta 2/36, C/EBP-alpha , C/EBP-beta , or AhR vector in different combinations as indicated. D, the effects of CBP, E1A, E1ADelta 2/36, C/EBP-alpha , C/EBP-beta , or AhR on ARE/EpRECAT. Cotransfection of 15 µg of ARE/EpRECAT with 2.5 µg of CBP, E1A, E1ADelta 2/36, C/EBP-alpha , C/EBP-beta , or AhR vector in different combinations as indicated. Data are expressed as relative CAT activity. Values represent the mean ± S.E. from three separate experiments. Me2SO (DMSO).

We also tested the effect of AhR or C/EBP-beta or C/EBP-alpha overexpression on 1.6CAT in the presence of CBP. The results showed that following overexpression of CBP, AhR dramatically increases 1.6CAT activity, whereas C/EBP-beta or C/EBP-alpha decreases 1.6CAT activity (Fig. 4B). These results are consistent with previous observations that C/EBP-beta or C/EBP-alpha , but not AhR, function as negative regulators of rGST-Ya gene expression (Fig. 3).

To verify further the major BaP-responsive element within 1.6CAT in vSMCs, we also examined the effects of these proteins on AhRE or ARE/EpRE individually. The various protein expression vectors in different combinations were cotransfected with AhRECAT or ARE/EpRECAT. Overexpression of CBP or AhR alone, or in combination, significantly increased transcription of AhRECAT by BaP (Fig. 4C). The promotive function of CBP or AhR alone, or in combination, was inhibited by cotransfection of C/EBP-beta or C/EBP-alpha expression vectors (Fig. 4C). The effects of E1A and E1ADelta 2/36 on AhRECAT suggest that limiting CBP levels also influence AhRE inducibility by BaP. Overexpression of AhR or C/EBP-alpha or C/EBP-beta , or AhR with C/EBP-alpha , or AhR with C/EBP-beta represses ARE/EpRECAT activity by BaP but not Me2SO (Fig. 4D). ARE/EpRE activation by BaP is also affected by limiting CBP amounts as shown by 1.6CAT or AhRECAT. These results suggest that BaP enhances cotransactivation of CBP on 1.6CAT by formation of BaP-AhR·DNA complexes through a specific region within rGST-Ya promoter and indicate that the AhRE, but not ARE/EpRE, is the major target sequence in the rGST-Ya promoter responsive to BaP in vSMCs.

The AhRE but Not ARE/EpRE Plays a Major Role in the Activation of 1.6CAT by BaP in vSMCs-- Our previous work showed that 1.6CAT and ARE/EpRECAT are not inducible by BaP, TCDD, or H2O2, whereas AhRECAT is induced by BaP or TCDD but not H2O2 (Fig. 5A), and that mutation or deletion of C/EBP site within the ARE/EpRE in 1.6CAT construct restores responsiveness to BaP and TCDD but not H2O2 (Fig. 5B) (13). To determine if the AhRE is the major element responsible for BaP inducibility in vSMCs, site-directed mutagenesis was performed to mutate or delete the AhRE core sequence (TTGCGTG) to TTGTATG or TTG_TG in the 1.6CAT-EpREm or 1.6CAT-EpREd construct, respectively. These double mutant constructs were referred to as 1.6CAT-AhREm&EpREm and 1.6CAT-AhREd&EpREd, respectively. The fold inductions of 1.6CAT-AhREm&EpREm by 3 µM BaP, 1 nM TCDD, and 400 µM H2O2 were 1.357 ± 0.072, 1.254 ± 0.211, and 1.147 ± 0.101, respectively, whereas those of 1.6CAT-AhREd&EpREd are 1.304 ± 0.11, 1.008 ± 0.11, and 1.245 ± 0.098, respectively (n = 3). Neither BaP, TCDD, nor H2O2 induced the double mutants (Fig. 5B), suggesting that in vSMCs the AhRE is the major sequence of rGST-Ya promoter that is responsive to BaP, whereas the remaining possible BaP target sequences, such as the proximal AP-1 like site within the ARE/EpRE of 1.6CAT, are not responsive to BaP or H2O2.


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  		Fig. 5.   The AhRE is the major BaP responsive element of 1.6CAT in vSMCs. A. The induction patterns of 1.6CAT, -164CAT, AhRECAT, and ARE/EpRECAT. Data were obtained from Ref. 13 and shown schematically for reference. B, the induction patterns of 1.6CAT, 1.6CAT-EpREm, .6CAT-EpREd, 1.6CAT-AhREm&EpREm, and 1.6CAT-AhREd&EpREd by BaP, TCDD, and H2O2 in vSMCs. Data except 1.6CAT-AhREm&EpREm and 1.6CAT-AhREd&EpREd were from Ref. 13. 1.6CAT-AhREm& EpREm and 1.6CAT-AhREd&EpREd were constructed by site-directed mutagenesis as described under "Experimental Procedures." Fifteen µg of 1.6CAT and mutant constructs were transfected into vSMCs. Cells were treated with 3 µM BaP, 1 nM TCDD, or 400 µM H2O2 for 24 h as described previously (13). Data are expressed as fold induction. +, induction; -, no induction.

The BaP inducibility of the proximal AP-1-like site within ARE/EpRE in 1.6CAT-AhREm&EpREm or 1.6CAT-AhREd&EpREd may be repressed by a yet to be identified mechanism. Therefore, we examined the function of the proximal AP-1-like site within ARE/EpRECAT using strictly the ARE/EpRE sequence. Since the C/EBP site within ARE/EpRE was identified as negative regulatory sequence (13), the C/EBP site of ARE/EpRECAT was mutated or deleted as described previously (13) to generate ARE/EpRECAT-EpREm and ARE/EpRECAT-EpREd, respectively. The fold inductions of ARE/EpRECAT-EpREm and ARE/EpRECAT-EpREd by 3 µM BaP were 1.02 ± 0.12 and 0.92 ± 0.083, respectively (n = 3). BaP did not induce these mutants, consistent with the observations in Fig. 5B that the proximal AP-1-like site in 1.6CAT double mutant constructs is not inducible by BaP in vSMCs. Taken together, these experiments indicate that the AhRE is the main BaP-responsive enhancer sequence within rGST-Ya promoter in vSMCs.

The Regulatory Mechanism of Mouse GST-Ya Gene Is Comparable with That of Rat GST-Ya ARE/EpRE-- Only two bases differ between mGST-Ya ARE/EpRE and rGST-Ya ARE/EpRE (39, 42) (Fig. 6A). Previous studies have shown that the ARE/EpRE is the major cis-acting element of mouse (m)GST-Ya responsive to chemical agents (43), whereas the rat promoter contains both AhRE and ARE/EpRE. The rat ARE/EpRE is not induced in vSMCs by BaP but exhibits significant activation in HepG2 cells (13). To determine if regulation of the mouse ARE/EpRE is comparable to that of rat ARE/EpRE, we introduced a mouse ARE/EpRE-luc reporter construct into vSMCs and HepG2 cells. Similar to the rat ARE/EpRE, mouse ARE/EpRE was not induced in response to BaP in vSMCs but was induced in HepG2 cells (Fig. 6B). EMSA experiments showed that comparable DNA-protein complex patterns are generated by mouse and rat ARE/EpRE probes using 0.3 µM BaP-treated vSMC nuclear extracts and that these two ARE/EpRE sequences exhibit similar protein binding specificities (Fig. 6C). These results suggest that mouse ARE/EpRE and rat ARE/EpRE exhibit similar regulatory functions in response to BaP in vSMCs.


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  		Fig. 6.   The regulatory mechanism of mouse GST-Ya gene is comparable to that of rat GST-Ya ARE/EpRE. A, comparison of mGST-Ya ARE/EpRE and rGST-Ya ARE/EpRE sequences. B, induction patterns of mouse ARE/EpRE luciferase (mouse ARE/EpRE-luc) construct in vSMCs or HepG2 cells. Fifteen µg of mouse ARE/EpRE-luc construct was introduced into vSMCs or HepG2 cells as described previously. Cells were treated with 0.3-30 µM BaP for 24 h. Luciferase activity was determined using a luciferase assay system following the manufacturer's instructions. Data are expressed as relative luciferase activity. Values represent the mean ± S.E. from three separate experiments. C, similar binding protein complex patterns between mouse ARE/EpRE and rat ARE/EpRE. Lane 1, reaction without nuclear extracts. Lane 2, cells treated with 0.3 µM BaP. Lanes 3-5, cells treated with 0.3 µM BaP and subsequently competed with 50× cold competitors as indicated. Lane 6, reaction without nuclear extracts. Lane 7, cells treated with 0.3 µM BaP. Lanes 8-10, cells treated with 0.3 µM BaP and subsequently competed with 50× cold competitors as indicated.

Competition of AhRE for the Same DNA-Protein Complexes with the ARE/EpRE and Induction Interference between AhRE and ARE/EpRE-- Since C/EBP-beta and AhR interact with the ARE/EpRE and AhRE via the sequence containing TTGC (Figs. 1B and 2C), we next examined if AhRE competes away the top ARE·EpRE-binding protein complex (Fig. 1B). Competition experiments were performed using the ARE/EpRE as probe, and AhRE and its mutants (m3, m4, and m6) as competitors. We found that the AhRE and m6 partially competed away the top ARE·EpRE-binding protein complex but m3 and m4 did not (Fig. 7A). These data indicate that the AhRE can compete with the ARE/EpRE via sequences containing TTGC, the AhR and C/EBP high affinity half-binding site (10, 44-45).


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  		Fig. 7.   Functional interference between AhRE and ARE/EpRE. A, competition of ARE/EpRE with AhRE or its mutants. Lane 1, reaction without nuclear extracts. Lane 2, cells treated with 0.3 µM BaP. Lanes 3-7, cells treated with 0.3 µM BaP and subsequently competed with 50× competitors as indicated (arrow). Shifted band competition. B, ARE/EpRE-luc construct decreased the activity of AhRECAT by BaP in vSMCs. C, AhRECAT decreased the activity of ARE/EpRE-luc construct by Me2SO or BaP in vSMCs. AhRECAT, -164CAT, mouse ARE/EpRE-luc construct, and pGL2-Basic luciferase construct (10 µg for each construct) were cotransfected into vSMCs as indicated. Cells were treated with 3 µM BaP for 24 h. Half of the cells were used for CAT assays, and the other half was used for luciferase assays as described previously. Data are expressed as relative CAT/luciferase activity. Values represent the mean ± S.E. from three separate experiments.

As shown in Fig. 6, A and B, mouse ARE/EpRE and rat ARE/EpRE have similar sequences and functions in vSMCs and HepG2 cells treated with BaP. In addition, both ARE/EpRE exhibited similar protein binding patterns and competed with each other in EMSA experiments (Fig. 6C). To examine if the presence of ARE/EpRE reduces the activation of AhRE and vice versa, AhRECAT and mouse ARE/EpRE luciferase reporter constructs were cotransfected into vSMCs followed by treatment with 3 µM BaP. Half the cells were used for CAT assays, and the other half was used for luciferase assays as described under "Experimental Procedures." Fig. 7, B and C, shows that mouse ARE/EpRE-luc construct, but not pGL2-Basic luciferase construct, hindered the inducibility of AhRECAT by BaP, whereas both luciferase constructs did not affect the activation of -164CAT. In contrast, AhRECAT, but not -164CAT, decreased the activity of mouse ARE/EpRE-luc construct by Me2SO or BaP. Neither CAT construct modulated the activity of the pGL2-Basic luciferase construct. These data suggest that in vSMCs the ARE/EpRE inhibits inducible activity of rGST-Ya via the AhRE by BaP and vice versa.

C/EBP-beta , C/EBP-alpha , mGST-Ya, and CBP Levels Are Not Affected by BaP-- Western blotting experiments were next conducted to determine if BaP affects the protein levels of C/EBP-beta , C/EBP-alpha , or CBP to regulate 1.6CAT function or influence mGST-Ya protein levels. Fig. 8 shows that BaP does not modulate C/EBP-beta , C/EBP-alpha , mGST-Ya, or CBP levels within 24 h of treatment as compared with controls. BaP did not change C/EBP-beta protein levels, and the two C/EBP-alpha isoforms (42 and 30 kDa, shown in the middle panels, respectively) were barely detectable in this cell system. The relative protein levels of C/EBP-beta and C/EBP-alpha in vSMCs are consistent with our previous results that C/EBP-beta , but not C/EBP-alpha , can be detected by supershift experiments using vSMC nuclear extracts (Figs. 1A and 2A). The inability of BaP to modulate mGST-Ya protein levels is consistent with the results of transfection experiments using the mouse ARE/EpRE luciferase construct (Fig. 6B) and collectively suggests that GST-Ya is refractory to BaP induction in vSMCs.


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  		Fig. 8.   Expression of C/EBP-beta , C/EBP-alpha , mGST-Ya, and CBP following BaP treatment in vSMCs. Fifty µg of total protein was obtained from vSMCs treated with 3 µM BaP for 1, 2, 4, 8, 16, and 24 h. Primary antibodies against C/EBP-beta , C/EBP-alpha , mGST-Ya, and CBP were applied at an appropriate dilution according to the manufacturer's instructions. Horseradish peroxidase-labeled secondary antibodies were applied at a dilution of 1:30,000. The membrane was detected by ECL Western blotting detection system according to the manufacturer's instructions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The rGST-Ya promoter contains AhRE and ARE/EpRE sequences located at -914 to -882 and -722 to -682, respectively (39). AhRE and ARE/EpRE are cis-acting regulatory elements involved in coordinate regulation of mammalian gene expression by BaP and related xenobiotics (46-48). The AhRE contains a 5'-TTGCGTG-3' consensus sequence that is recognized specifically by the AhR·ARNT protein complex. This element is activated following exposure to planar aromatic compounds such as BaP, TCDD, beta -naphthoflavone, and 3-methylcholanthrene (39, 46-49). The ARE/EpRE comprises two adjacent AP-1-like sites (5'-TGGCATTGC-3' and 5'-TGACAAAGC-3') that mediate inducibility by some planar aromatic compounds, phenolic antioxidants (e.g. tert-butyl hydroquinone), and pro-oxidants, such as hydrogen peroxide (H2O2) and menadione (46, 51-53). Transacting factors involved in ARE/EpRE signaling have yet to be identified definitively. AhRE and ARE/EpRE have been characterized as positive regulatory elements using CAT reporter constructs linked to the minimal promoter region of rGST-Ya in HepG2 cells (39). To date, little is known about functional interactions between these elements within the rGST-Ya gene. In the present study, we utilized the rGST-Ya promoter as a model system to determine molecular mechanisms of gene regulation by BaP mediated via these two elements in vSMCs.

On the basis of gene inducibility profiles and site-directed mutagenesis, we have shown the following. 1) In genomic context, ARE/EpRE does not function as a positive regulatory element in the rGST-Ya promoter. 2) Xenobiotic inducibility of rGST-Ya is lost when both AhRE and ARE/EpRE are present in genomic context within the full-length promoter (13). In the present study, we demonstrate for the first time that C/EBP-beta interacts with the C/EBP-like site (ATTGC) within rGST-Ya ARE/EpRE (Fig. 1, A and B) and that this interaction negatively regulates expression from the rGST-Ya promoter (Figs. 3 and 4).

In contrast to HepG2 cells where C/EBP-alpha , but not C/EBP-beta , is detectable (54-55), C/EBP-beta , but not C/EBP-alpha , is the primary C/EBP isoform present in vSMCs (Fig. 8). EMSAs established that C/EBP-beta , not C/EBP-alpha , interacts with the AhRE in vSMCs (Fig. 2A). Interestingly, C/EBP-beta or C/EBP-alpha significantly repressed the activity of AhRECAT and reversed the stimulatory effect of AhR overexpression on AhRECAT in vSMCs (Fig. 3B). Similar competitive events occurred between AhR and the upstream stimulatory factor 1 that share TCGCGTGACT binding sequence in rabbit kidney RK13 cells (56-57). Conversely, it has been suggested that C/EBP-alpha cooperates with AhR in the regulation of a test plasmid with three rGST-Ya AhRE sequences in HepG2 cells (10). However, C/EBP-alpha also plays a negative regulatory role on beta 2-adrenergic receptor gene in DDT1 MF-2 hamster smooth muscle cell line and rat hepatocytes (20). C/EBP-beta is also known to exert a strong negative effect on albumin promoter in HeLa or L cells, whereas C/EBP-beta or C/EBP-alpha stimulate transcription of the same promoter in HepG2 cells (17, 54). Collectively, these observations suggest that the expression and/or activities of C/EBP-beta and C/EBP-alpha are cell-specific. The C/EBP-beta gene contains two repression domains (RD1 and RD2). RD1 regulates the activation domain of the protein and does not affect cell type specificity of the protein, whereas RD2 controls cell type-specific transactivation (23). Furthermore, the attenuator domain of C/EBP-alpha diminishes the transactivation of other activation domains of C/EBP-alpha (16, 18). Taken together, it seems plausible that the RD domains of C/EBP-beta and attenuator domain of C/EBP-alpha contribute to repression of rGST-Ya gene in vSMCs. This hypothesis needs further investigation.

The negative effect of limiting intracellular CBP on the transcription of rGST-Ya gene may be mediated in at least two ways. Given that a variety of transcription factors can directly interact with CBP (29), our findings (Fig. 4) suggest that competition for limiting intracellular CBP levels by nuclear factors interferes with rGST-Ya gene transcription. Similar events have been reported for transcriptional interference between CREB and c-Jun and between liganded nuclear hormone receptor and AP-1 (31-32). It is also possible that interaction of CBP with multiple nuclear factors on the same promoter simultaneously may decrease its coactivational ability. This suggestion is consistent with a previous study showing that p300, a functional homologue of CBP, with different domains bind v-Myb and C/EBP-beta simultaneously to interact with Myb and C/EBP-binding sites, respectively, on the chicken mim-1 promoter (58). Thus, evidence from the literature suggests that the amino terminus of C/EBP-beta is responsible for interactions with the domain from amino acids residues 1752-1859 of p300 that overlap with the E1A-binding region (58-59). In support of this interpretation, others (60) have shown that the activation domain of Arnt, but not AhR, interacts with the CREB-binding domain of CBP/p300. Our supershift experiments showed that both AhR and C/EBP-beta are part of the AhRE or ARE·EpRE protein complex in BaP-treated nuclear extracts (Figs. 1 and 2). Physical interactions between these proteins may in fact be facilitated by the short distance between AhRE and ARE/EpRE of less than 170 bases (39). Because mouse CBP has a molecular mass of 265 kDa (37) relative to ~95-kDa AhR (61), ~85.4-kDa Arnt (34), and ~34- or 38-kDa C/EBP-beta (62), the size of CBP may span the distance between AhRE and ARE/EpRE.

On the basis of these findings, we hypothesize that interactions of CBP with C/EBP-beta and Arnt on AhRE, and with C/EBP-beta and other possible nuclear factors on ARE/EpRE at the same time, may restrict the ability of CBP to form stable complexes with the components of the basal transcription complex machinery (e.g. TBP, TBIIF and RNA polymerase II) (29). Thus, competition by activators (e.g. AhR·Arnt in our case) and repressors (e.g. C/EBP-beta ) for CBP on the same promoter may lead to repression of gene expression. As such, the relative levels of activators to repressors may contribute to positive or negative transcription of xenobiotic-regulated genes (Figs. 3 and 4). Collectively, our results suggest that CBP coordinates negative and positive signals transmitted to rGST-Ya promoter in vSMCs by BaP.

Interestingly, overexpression of AhR decreased ARE/EpRECAT activity by BaP in the presence of CBP (Fig. 4D). In addition, overexpression of AhR with C/EBP-alpha or C/EBP-beta gave similar results. Although we have shown that AhR is part of the ARE·EpRE-binding protein complex (Fig. 1C), the mechanism by which AhR interacts with C/EBP-beta or other proteins on the ARE/EpRE requires further investigation. The roles of the AhRE and ARE/EpRE in genomic context were examined using site-directed mutagenesis to construct mutant 1.6CATs with altered AhRE or ARE/EpRE sequence or mutant ARE/EpRECATs with changed sequence (Fig. 5) (13). The induction patterns of these mutants show that the AhRE is the critical enhancer inducible by BaP in the 1.6CAT construct in vSMCs.

The pattern of mGST-Ya gene induction by BaP in vSMCs and HepG2 cells was consistent with that for 1.6CAT, showing that negative regulation of GST-Ya is not restricted to heterologous systems. The mouse ARE/EpRE is the only cis-regulatory element of mGST-Ya promoter (47) and is required for induction of tert-butyl hydroquinone, beta -naphthoflavone, diphenols, H2O2, dimethylfumarate, 3-methylcholanthrene, phorbol ester, and phenobarbital in HepG2 cells (47, 63, 64). As shown in Fig. 6, A and B, mouse ARE/EpRE and rat ARE/EpRE have similar sequences and functions in vSMCs and HepG2 cells treated with BaP. In addition, both ARE/EpREs exhibit similar protein binding patterns and compete with each other using BaP-treated vSMC nuclear extracts in EMSA experiments (Fig. 6C). Several components of rGST-Ya model apply to mGST-Ya gene regulation in vSMCs since 1) C/EBP-beta , as a repressor, interacts with the ARE/EpRE of mGST-Ya gene; 2) limiting intracellular CBP levels compromise induction of mGST-Ya gene; and 3) liganded AhR alone, or in combination with C/EBP-beta , also functions as a repressor of the ARE/EpRE. Although GST-Ya protein was constitutively expressed in murine vSMCs, the gene was refractory to BaP induction (Fig. 8). These findings are consistent with the results of transfection experiments using the mouse GST-Ya ARE/EpRE luciferase construct and collectively suggest that BaP negatively regulates GST-Ya transcription in vSMCs through multiple pathways involving the ARE/EpRE. Although functional interactions between AhRE and ARE/EpRE are unique to the rGST-Ya gene, the C/EBP site within the ARE/EpRE core sequence functions as a repressor of both rat and mouse GST-Ya regulation. Since BaP can be metabolized to cytotoxic and genotoxic intermediates in vSMCs (65), and GSTs participate in the detoxification of reactive BaP metabolites, the biological consequences of GST-Ya repression in vSMCs need to be explored further.

Taken together, the results presented in this paper suggest multiple mechanisms for negative regulation of GST-Ya gene expression in vSMCs by BaP (Fig. 9). These mechanisms may interact with each other leading to negative regulation of GST-Ya gene in vSMCs by BaP. First, in the case of rGST-Ya, the C/EBP site within the ARE/EpRE may inhibit chemical inducibility of the AhRE by forming an AhRE·C/EBP-beta ·ARE·EpRE complex that prevents function of the basal transcription machinery (Fig. 9, I). Conversely, the AhRE may also reduce the inducible activity of the ARE·EpRE by BaP. Second, C/EBP-beta appears to function as a repressor by competing for binding to the C/EBP site within the AhRE with liganded AhR·Arnt complex or by competing with ARE/EpRE-binding proteins for the ARE/EpRE (Fig. 9, II). Third, limiting intracellular CBP protein levels diminish the inducibility of GST-Ya gene by BaP (Fig. 9, III). Finally, liganded AhR alone, or along with C/EBP-beta , may function as a repressor of the ARE/EpRE, whereas liganded AhR·Arnt functions as an activator of the AhRE (Fig. 9, IV). Clearly, the expression of ARE/EpRE-regulated genes, especially from promoters also containing other xenobiotic-responsive elements, such as the AhRE, is complicated and should be better understood and the proteins involved in transcription control identified.


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  		Fig. 9.   Working model for the regulation of GST-Ya gene in vSMCs by BaP. BaP is metabolized by cytochrome P450 enzymes present in vSMCs to quinones (BaP-Qs) that redox cycle and generate reactive-oxygen species that activate ARE·EpRE-binding proteins (ARE·EpRE-BPs) to stimulate gene transcription (65). BaP is also a ligand for and an activator of AhR-mediated signal transduction in these cells (66). BaP elicits transcription of rGST-Ya gene through an AhR-mediated pathway involving the AhRE, the major BaP-responsive sequence in vSMCs (Fig. 5). However, in the case of rGST-Ya, a C/EBP site within ARE·EpRE inhibits inducibility of the AhRE within this promoter (I). C/EBP-beta , as a repressor, interacts with the AhRE or ARE/EpRE (II). The promotive effect of liganded AhR with Arnt on AhRE is decreased by protein-protein interactions with C/EBP-beta , whereas liganded AhR is associated with repressive functions on ARE/EpRE (IV). Limiting intracellular CBP protein levels also contribute to low induction potential of this gene (III). In addition, competition for the same activator or coactivator proteins between AhRE and ARE/EpRE may act to diminish induction of this gene.


    ACKNOWLEDGEMENTS

We thank Dr. C. B. Pickett for kindly providing rat GST-Ya CAT constructs; Dr. J. P. Whitlock, Jr. for mouse AhR and Arnt expression vectors; Dr. D. W. Hum for E1A and mutant (E1ADelta 2/36) expression vectors; Dr. R. H. Goodman for mouse CBP expression vector; Dr. O. A. MacDougald for mouse C/EBP-alpha and C/EBP-beta expression vectors; and Dr. J. A. Johnson for mGSTA1-ARE/EpRE luciferase reporter construct.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant ES 04849 and ES 09106 (to K. S. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843-4466. Tel.: 979-845-5993; Fax: 979-882-4929; E-mail: kramos@cvm.tamu.edu.

Published, JBC Papers in Press, May 18, 2000, DOI 10.1074/jbc.M000405200

    ABBREVIATIONS

The abbreviations used are: GST, glutathione S-transferase; C/EBP, CCAAT/enhancer-binding protein; CBP, CREB-binding protein; BaP, benzo(a)pyrene; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; vSMCs, vascular smooth muscle cells; AhRE, aryl hydrocarbon response element; ARE/EpRE, antioxidant/electrophile response element; HepG2, human hepatoma cells; AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; r, rat; m, mouse.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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