Introduction

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The cytosolic glutathione S-transferases are a superfamily of dimeric enzymes of subunit molecular mass of 20-30 kD (Rushmore and Pickett, 1993; Hayes and Pulford, 1995). These proteins are classified as families based on their degree of sequence identity and on their ability to form heterodimeric proteins whose catalytic functions are intermediate relative to their respective homodimers (Mannervik et al., 1985). These proteins play a major role in the detoxification of xenobiotic chemicals by catalyzing the conjugation of glutathione with their electrophilic centers, thus preventing protein or nucleic acid alkylation reactions. In addition, some glutathione S-transferases seem to serve as intracellular binding proteins for nonsubstrate ligands (Listowsky, 1993).

The molecular events involved with the transcriptional activation of rat gstA2 subunit gene have been well characterized by Pickett and coworkers (Paulson et al., 1990; Rushmore et al., 1991). The gstA2 subunit named using the nomenclature proposed by J. D. Hayes (Hayes and Pulford, 1995) is the rat gene whose cDNA was isolated as clone pGTB45-15 (Telekowski-Hopkins et al., 1988). Single copies of two cis-acting responsive elements, the AHRRE and ARE (Rushmore et al., 1990), have been characterized for their role in the induction of this protein subunit by xenobiotic compounds. The AHRRE core sequence (5'-TNGCGTG-3') also is found in multiple copies in the 5'-flanking region of the CYP1A1 gene, for which we have characterized the potentiation of the PAH-dependent induction by GC (Mathis et al., 1989; Xiao et al., 1995; Prough et al., 1996). This responsive element is activated on binding the heterodimeric complex of the AHR and ARNT protein (AHR nuclear translocater). The ligands for the cytosolic AHR are planar chlorinated compounds such as 2,3,7,8-tetrachlorodibenzo-p-dioxin or PAHs such as BA and -naphthoflavone. The ARE core sequence (GTGACNNNGC) is required for induction of gstA2 message and protein by metabolites of aromatic compounds, phenolic antioxidants, phorbol esters, and hydrogen peroxide, suggesting a mechanism for activation involving reactive oxygen species (Rushmore et al., 1991; Nguyen et al., 1994). Similar ARE regulatory sequences are found in the human (Jaiswal et al., 1988) and rat (Favreau and Pickett, 1993) NAD(P)H:quinone acceptor oxidoreductase genes.

Several other responsive elements have been implicated in the constitutive expression of this protein (Paulson et al., 1990; Mendel and Crabtree, 1991; Pimental et al., 1993), namely for the hepatic nuclear factors HNF-1, HNF-4, and C/EBP. Genes regulated by these families of transcription factors use characteristic consensus sequences and may display developmental control. For example, genes regulated by HNF-1 in rats (Mendel and Crabtree, 1991) are expressed during neonatal life, whereas those regulated by C/EBP may not be fully expressed until adolescence (Lee et al., 1994).

We have shown that GCs modulate the PAH-dependent induction of the gstA2 subunit protein (Sherratt et al., 1990; Linder and Prough, 1993; Xiao et al., 1995; Prough et al., 1996). In neonatal rats, GCs potentiate the PAH-dependent induction of the gene, whereas in adolescent rats, which display higher basal levels of expression, GCs suppress PAH induction (Linder and Prough, 1993). In both cases, these effects were shown to be regulated at the transcriptional level. Cell culture experiments conducted with both fetal (Sherratt et al., 1990) and adult (Xiao et al., 1995; Prough et al., 1996) rat hepatocytes have suggested two possible mechanisms of GC regulation. The first occurs at concentrations of the synthetic GC, DEX, of <1 × 10⁷ M (i.e., in the concentration range expected for agonist/GC receptor binding) that potentiate the PAH-dependent induction of the subunit in fetal cells but suppress the effect in adult hepatocytes. A second mechanism is observed at higher concentrations of GC (1 × 10⁶ to 1 × 10⁴ M), resulting in increased expression of this protein. This second mechanism has been observed in both fetal (Sherratt et al., 1990) and adult (Xiao et al., 1995; Prough et al., 1996) rat hepatocytes.

In examining the 5'-flanking region of the gstA2 subunit gene, we discovered several sequences between 1.65 and 1.15 kb that have homology to the canonical consensus hexanucleotides [5'-TGT(T/C)CT-3'] for the GC receptor. This report explores the hypothesis that the gstA2 subunit gene is under regulatory control of GC due to binding of the ligand-activated GC receptor to its functional canonical consensus response element in the 5'-flanking region of the gene.

	Experimental Procedures

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Materials. Restriction endonucleases, pGL2-basic and T4 ligase, were purchased from Promega (Madison, WI) or New England Biolabs (Beverly, MA). pCR II cloning kits were obtained from InVitrogen (San Diego, CA). The 5'-flanking constructs of gstA2 gene, p4.0YaCAT, p1.6YaCAT, p1.15YaCAT, and p0.164YaCAT, have been described previously (Telakowski-Hopkins et al., 1988; Paulson et al., 1990). pCMV- was obtained from Clonetech (Palo Alto, CA). The reporter construct p2XDEX-LUC and pRSVGR, the expression vector for the human GC receptor, were a kind gift from Michael Mathis (LSU Medical Center, Shreveport, LA). pGR-PPAR has been described previously (Boie et al., 1993). pcDNA3 was purchased from InVitrogen.

Cells and culture conditions. E. coli DH5 and HB 101 cells were transformed routinely with plasmids of interest. pCRII-derived plasmids were grown in E. coli of the ONE-SHOT strain (InVitrogen). The human hepatoblastoma cell line HepG2 (HB8065, American Type Culture Collection, Rockville, MD) was maintained in Dulbecco's modified Eagle's medium supplemented with 250 µg/ml Fungizone, 10 units/ml penicillin, 10 units/ml streptomycin, and 10% fetal bovine serum. The hepatoma cells were incubated at 37° in a 5% carbon dioxide atmosphere and were subcultured every 2-3 days.

PCR products. A 1651-bp PCR product used to construct p1.62YaLUC was synthesized using an upstream primer that contains bp complementary to position 1620 to 1578 of p1.6YaCAT and a 5' NdeI site with extra bp (YaGRETOP, 5'-GGAATTCCAT ATGTGGGAGC ATTCCAGAACA AGCTGTACCA CCAAGGGTCA CT-3') and a 33-mer downstream primer with a 5' HindIII extension and extra bp (BMYaUNI, 5'-AGACTAAGCT TGGGTTGTAA AAGAGAGTAC TGA-3'). A 371-bp PCR product used to construct 0.164YaLUC was synthesized from 0.164YaCAT using the upstream primer PRIMNDE1 (5'-GTGAGCGAGGAAGCGGAAGA-3'), which is complementary to bp 2523-2503C of pRSVoCAT and BMYaUNI. PCR was performed in a Thermolyne Amplitron II thermal cycler (Barnstead/Thermolyne, Dubuque, IA) with 2 mM Mg²⁺. The products were generated through 20 cycles of the following steps: denaturing temperature at 94° for 0.5 min, annealing temperature 50° for 1 min, and elongation temperature 72° for 1 min. A 604-bp product was generated from p1.6YaCAT using YaGRETOP and a downstream primer complementary to bp 1052 to 1032 of the 5'-flanking region of the gstA2 gene and a restriction site for NdeI with extra bp (YaHALFBOT, 5'-GGAATTCCAT ATGGCCATTT GCCTGTGGTC ACG-3'). These were designed for cloning DNA products into the unique NdeI restriction site of p0.164YaLUC vector.

Plasmid constructs. pCMV-GR was produced by subcloning the XhoI/KpnI fragment from pRSVGR containing the coding region for the human GC receptor into the unique XhoI/KpnI sites of pcDNA3. p1.52YaCAT was synthesized by digesting p1.6YaCAT with XbaI and NdeI, followed by treatment with Klenow fragment and religation of the resultant fragment. pCR-GREMUT was generated by subcloning the mutated 1060-bp PCR product described previously into a pCRII vector. pKCF28, a construct nearly identical to p1.6YaCAT but containing a mutated pGRE at bp 1609 to 1594, was made by digesting both p1.6YaCAT and pCR-GREMUT with XbaI and NdeI and by religating the 90-bp fragment containing the mutated GRE into the XbaI/NdeI-restricted p1.6YaCAT parent vector.

Transfection of hepatoma cells. HepG2 cells were transfected at 40% confluence, treated with various agents, and harvested after 24 hr using methods described previously (Rushmore et al., 1990). All cells were cotransfected with pCMV- as a transfection control and pRSVGR, the expression plasmid for human GR. Routinely, 2 µg of plasmids with viral promoters (pCMV- or pRSVGR) or 4 µg of the respective gstA2 promoter CAT or luciferase construct was added per flask. The inducing agents, BA, DEX and nafenopin, were added as 500× concentrated stocks in dimethylsulfoxide; controls received dimethylsulfoxide alone. Cells transfected with luciferase reporter plasmids were harvested with 0.5 ml of cell lysis buffer (Promega, Madison, WI) according to manufacturer's instructions.

Assays of CAT, -galactosidase, and luciferase activity. The CAT assay used in this study was a variation of the method of Gorman et al. (1982), which includes xylene extraction of the products and liquid scintillation quantification. Reactions were performed in 100-µl reactions of cell extract (120 µg of protein) in 0.25 M Tris·HCl, pH 7.5, containing 3.7 mM chloramphenicol (25 nCi) and 5 µg of n-butyryl CoA for 1 hr at 37°. Samples initially were extracted with 300 µl of xylene, and after reextraction of 250 µl of the xylene phase with 100 µl of reaction buffer, the enzyme activity was calculated as the volume-adjusted ratio of radioactivity in 200 µl of organic and 50 µl of aqueous phase. This method gave identical results (not shown) to the thin layer chromatography method described by Gorman et al. (1982). Luciferase activity was determined using the luciferase assay system from Promega. Luciferase activity was measured with 20 µl of cell extract over a 10-sec time period in a Berthold model LB9501 Lumat luminometer (Wallac, Gaithersburg, MD). For the -galactosidase assays, cell extracts (30 µg of protein) were incubated with chlorophenol red -galactopyranoside at 37° for 1 hr. Activity was determined spectrophotometrically at 595 nm on a Titretek Uniskan II plate reader (Flow Laboratories, McLean, VA).

Electrophoretic mobility shift assays. Nuclear extracts were prepared from rat liver as described previously (Gorski et al., 1986), placed into aliquots, and stored at 70°. Polyclonal anti-human GC receptor antibodies (PA1-511) were obtained from Affinity Bioreagents (Golden, CO). Nuclear extracts were incubated with radiolabeled probe at 30° for 30 min before resolution on a polyacrylamide gels using low ionic strength buffers (Chodosh, 1995). The gels were dried and analyzed by exposure to a Molecular Dynamics Phosphor Screen in a Molecular Dynamics PhosphorImager (Sunnyvale, CA). HepG2-GR4 cells were produced by selection of cells transfected with pCMV-GR, which formed clonal colonies in the presence of 1.8 mg/ml (~2 weeks). Individual colonies thereafter were maintained on 0.9 mg/ml Geneticin. Both concentrations of geneticin were toxic to untransfected cells. The colony HepG2-GR4 was selected for its GC responsiveness in transfection assays with p2XDEX-LUC, a reporter containing two copies of the MMTV-GRE, relative to HepG2 cells cotransfected with pRSV-GR.

Statistical analysis. Student's t tests were used to discriminate significance between groups. Fold induction and the degree of repression were analyzed by fitting to theoretical equations with the least-squares regression program Kineti77 (Clark and Carrol, 1986).

	Results

Top Summary Introduction Procedures Results Discussion References

Deletion and mutational analysis. In preliminary experiments, we tested the ability of DEX to effect expression of CAT constructs containing various segments of the 5'-flanking region of the rat gstA2 gene. Although DEX did down-regulate gstA2 gene expression slightly in HepG2 cells, the results were not consistent when the expression vector for the GC receptor was omitted. In our hands, consistent repression of 1.6YaCAT expression occurred only when the GC receptor expression plasmid was cotransfected with the CAT construct (data not shown). We determined that 2 µg of pRSVGR, the expression plasmid for the human GC receptor, gave consistent responsiveness to DEX, suggesting that HepG2 cells express the GC receptor (Lui et al., 1993) at levels much lower than those found in hepatic tissues in vivo.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Induction of CAT activity in HepG2 cells transiently transfected with 5'-deletion constructs of gstA2. A, Diagrammatic representation of the 5'-constructs used. pGRE, palindromic GRE. B, Activity of various deletion CAT constructs of gstA2. CAT and -galactosidase assays were performed on lysed HepG2 cell transiently transfected with YaCAT constructs and the expression plasmids for pCMV and pRSVGR and subsequently treated with either 50 µM benzanthracene, 1 µM DEX ,or a combination for 24 hr as described in Experimental Procedures. CAT activity is expressed as the percent conversion of chloramphenicol to its acetyl derivative relative to -galactosidase activity and is the mean ± standard deviation of three flasks. Activities were normalized for BA-induced transcription rate. *, Statistically different from PAH-induced CAT activity (p < 0.05).

Effects of RU38486. To document the involvement of the GC receptor in this regulation, we used the GC antagonist RU38486 to inhibit receptor function (Fig. 2). Administration of 10 µM RU38486 alone had little or no significant effect on either basal or BA-induced expression of p1.6YaCAT. RU38486 antagonized the DEX-dependent repression of PAH induction because expression levels of p1.6YaCAT were identical in samples treated with either BA or BA plus DEX when RU38486 was present. Because RU38486 is a type II antagonist of the GC receptor (i.e., it is translocated to the nucleus but does not form a transcriptionally-active DNA/protein complex), our results support the involvement of both the GC receptor and DNA binding of the receptor in the repressive effect of GCs on gstA2 gene expression.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Effects of RU38486 on CAT activity of HepG2 cells transiently transfected with p1.6YaCAT and treated with BA, DEX, or a combination. CAT and -galactosidase assays were performed on lysates from HepG2 cells that had been transiently transfected with p1.6YaCAT and the expression plasmids for pCMV and pRSVGR and then treated with either 50 µM benzanthracene, 1 µM DEX, 10 µM RU38486, or combinations of these compounds for 24 hr as described in Experimental Procedures. CON, control. The normalized CAT activity is the percent conversion of chloramphenicol to its acetyl derivative relative to -galactosidase activity and is the mean ± standard deviation of three flasks. *, Statistically different from CAT activity of BA-treated cells (p < 0.01).

Effects of BA and DEX on luciferase expression of p1.62YaLUC. Because DEX had no apparent effect on the very low basal level expression of gstA2 CAT constructs, we constructed a plasmid similar to p1.6YaCAT that contains the luciferase structural gene. In comparison to CAT constructs, luciferase reporter gene systems have low backgrounds and high assay sensitivity (Alam and Cook, 1990). The effects of BA and DEX on expression of p1.62YaLUC are presented in Fig. 3. As anticipated, BA caused a 15-fold induction, whereas t-butylhydroquinone caused a significant induction (~5-fold) in reporter expression (results not shown). Administration of DEX caused a 75 ± 7% and 82 ± 6% (average ± standard deviation) suppression in both basal and PAH-induced expression of this reporter system, respectively. The levels of suppression are similar to those observed in animals (Linder and Prough, 1993) or isolated primary rat hepatocytes (Xiao et al., 1995) and clearly demonstrates that GCs inhibit basal and PAH-induced gene expression. Basal expression of this gene is regulated transcriptionally by HNF1 and ARE responsive elements (Paulson et al., 1990; Rushmore et al., 1990). Suppression of both basal and induced expression is consistent with DEX negative regulation being independent of the action of the AHR (Xiao et al., 1995).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effects of DEX on the basal and PAH-induced luciferase activity in HepG2 cells transiently transfected with the plasmid p1.62YaLUC. Luciferase and -galactosidase assays were performed on lysed HepG2 cells that had been transiently transfected with p1.62YaLUC and the expression plasmids for pCMV and pRSVGR subsequently treated with either 50 µM benzanthracene, 1 µM DEX, or in combination for 24 hr as described in Experimental Procedures. CON, control. The normalized luciferase activity is expressed as relative light units divided by -galactosidase activity and is the mean ± standard deviation of three flasks. *, Statistically different from control cells (p < 0.05). **, Statistically different from cells treated with BA alone (p < 0.05).

Concentration-dependent repression of basal expression of p1.62YaLUC by DEX. To determine whether the response may be due to interaction with the GC receptor or to "nonclassic" mechanisms, the concentration dependence of the effects of DEX on the basal expression of p1.62YaLUC was tested (Fig. 4). DEX also suppressed the PAH-dependent induction at all concentrations tested except at 1 × 10¹¹ M (data not shown). The repression was greatest at GC concentrations of 1 × 10⁷ M, with significant reductions being observed with doses as low as 1 × 10⁹ M. The concentration dependence of DEX suppression was the same as that observed with BA-induced CAT activity from p1.6YaCAT (results not shown). This concentration-dependent, monotonic decline is consistent with the process being mediated by the GC receptor. The concentration-dependence curve was extended to 1 × 10⁵ M to examine whether any nonclassic mechanism of GC induction occurred as observed in both the fetal (Sherratt et al., 1990) and adult (Xiao et al., 1995) hepatocytes. No evidence of a biphasic concentration-response relationship was observed in the expression of p1.62YaLUC or p1.6YaCAT in HepG2 cells. Therefore, nonclassic induction mechanisms apparently do not influence this transient transfection system or affect the results with the concentration of DEX (1 × 10⁶ M) routinely used in this study. Our work is similar to that observed by others in which nonclassic mechanism of GC action could be observed only in whole animals or primary cultures of hepatocytes (Schuetz et al., 1984). The concentration dependence is similar to that observed in adult hepatocytes at low DEX concentrations (Prough et al., 1996).

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of DEX repression of luciferase activity in HepG2 cells transiently transfected with the plasmid p1.62YaLUC. Luciferase and -galactosidase assays were performed on lysed HepG2 cells that had been transiently transfected with p1.62YaLUC and the expression plasmids for pCMV and pRSVGR and subsequently treated with varying doses of DEX for 24 hr as described in Experimental Procedures. The normalized luciferase activity is expressed as the relative light units divided by -galactosidase activity and is the mean ± standard deviation of three flasks. *, Statistically different from untreated cells (p < 0.05).

Electrophoretic mobility shift assays. To test whether the palindromic GRE is capable of binding the GC receptor, we performed electrophoretic mobility shift experiments using the ³²P-labeled double-stranded oligonucleotides (Fig. 5A) whose sequence is identical to the pGRE of gstA2 and nuclear extracts from rat liver, HepG2 cells, or HepG2-GR4 cells. HepG2-GR4 cells are stably transfected with a expression vector for the human GC receptor as described in Experimental Procedures. As can be seen in Fig. 5B, a specific DNA/protein complex was observed when the pGRE oligonucleotide of gstA2 was mixed with rat nuclear extract and resolved by gel electrophoresis. The DNA/protein complex formed could be competed for effectively by double-stranded oligonucleotides whose sequence was identical to either the pGRE from the MMTV long terminal repeat or the pGRE from gstA2 itself but not by an unrelated oligonucleotide, such as an oligonucleotide identical to the AHRRE from CYP1A1. When the MMTV pGRE was used as radiolabeled probe, 100- and 200-fold molar ratios of gstA2 pGRE to MMTV pGRE double-stranded-oligonucleotide blunted DNA/protein complex formation by >80%; cold MMTV pGRE double-stranded oligonucleotide completely reversed complex formation, demonstrating that both pGREs compete for GR binding but that MMTV pGRE has a slightly higher affinity for the receptor than gstA2 pGRE (data not shown). Using the same mutation strategy used in the transient transfection experiments, competition for DNA/protein complex formation by an oligonucleotide containing a mutated pGRE from gstA2 was diminished significantly. A reduction in the ability of the mutated pGRE oligonucleotide to compete for complex formation is consistent with a loss of binding affinity for the GC receptor. Thus, complex formation could be prevented by inclusion of unlabeled oligonucleotides with sequence identity to the GC hexanucleotide consensus sequence (Fig. 5A), and mutation of the gstA2 pGRE core sequence greatly reduced the ability of the oligonucleotide to bind protein.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Interaction between the pGRE located between bp 1611 and 1594 in the 5'-flanking region of gstA2 and rat nuclear extract using electrophoretic mobility shift assay. A, Double-stranded oligonucleotides used for the 32P-labeled probe and for competition are shown (coding strand only). Boldface, consensus nucleotides to the GRE palindrome (Schule et al., 1988). Smiley faces, aligned mutated sequences. B, Nuclear extract from livers of untreated male rat was incubated with the radiolabeled probe and resolved using a low ionic strength polyacrylamide gel electrophoresis. Lanes 1-10, probe and nuclear extract. Lanes 2 and 7, 50- and 100-fold excess of MMTV GRE, respectively. Lanes 3 and 8, 50- and 100-fold excess of gstA2 pGRE, respectively. Lanes 4 and 9, 50- and 100-fold excess of mutant pGRE, respectively. Lanes 5 and 10, 50- and 100-fold excess of the unrelated oligonucleotide AHRRE, respectively. C, Nuclear extract from either HepG2 or HepG2-GR4 cells was incubated with probe in the absence or presence of a polyclonal antibody elicited against human GR. Lanes 1, probe alone. Lanes 2, probe and HepG2 nuclear extract. Lane 3, probe and HepG2 nuclear extract plus antibody. Lanes 4, probe and HepG2-GR4 nuclear extract. Lanes 5, probe and HepG2-GR4 nuclear extract plus antibody. The DNA/protein responses were measured using a PhosphorImager.

Palindromic GRE CAT constructs. Because the pGRE of the gstA2 gene apparently binds the GC receptor, we sought to establish whether introduction of these sequences into reporter constructs possibly accounts for the negative regulation of this gene by GC. To facilitate this, we synthesized oligonucleotides containing the pGRE, either 25 or 47 mers, and ligated them as double-stranded oligonucleotides into the NdeI site of p0.164Ya CAT. Of the plasmids tested (Fig. 6), only one was GC responsive; this plasmid pGRE5CAT contained three copies of the pGRE. All other plasmids tested contained either one or two copies of the GRE in several orientations and displayed levels of basal expression similar to the minimal promoter construct p0.164YaCAT; none were GC responsive. What is striking is the fact that the CAT activity of this plasmid is induced 15-fold by GC, whereas the PAH-induced CAT activity of the reporter gene containing the 1.6-kb 5'-flanking region of the native gene is repressed >60% by GCs. The 47-mer constructs were made to establish whether spacing between pGREs or the immediate flanking sequences were critical, as suggested by Schule et al. (1988); at least two palindromic sequences or a palindrome and several half-sites apparently are required for GC responsiveness, and adequate spacing must exist between the palindromes, half-sites, or both to ensure the optimal geometry for cooperativity of receptor binding and function. Our results (Fig. 6) suggest that unlike other strong pGREs (Lanz et al., 1994) that confer GC responsiveness when present in two copies, at least three copies of the gstA2 palindromic GRE are required for CAT-reporter constructs to be GC responsive. This response may be related in part to the strength of binding of GR to this pGRE. Schule et al. (1988) have shown that binding to weak GREs have greater synergistic effects than those to strong GREs, such as those found in MMTV, which forms a hormone-responsive element when present in only two copies. Because the magnitude of induction of pGRE5CAT is much smaller (15-fold) than that we have observed with plasmids containing two copies of the MMTV-GRE (40-100-fold; results not shown), the functional interaction of the GR with this specific sequence seems weaker than that seen with the MMTV-GRE, and therefore greater synergistic effects might be expected (Lanz et al., 1994).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Induction of CAT activity by DEX in HepG2 cells transiently transfected with CAT constructs containing the palindromic GRE minimal sequences of gstA2. CAT and -galactosidase assays were performed on lysates of HepG2 cells that had been transiently transfected with GRE-containing CAT constructs containing either one or more copies of a 25- or 47-mer oligonucleotide spanning the palindromic GRE and the expression plasmids for pCMV and pRSVGR. Arrows, orientation and number of oligonucleotide insert. The HepG2 cells were treated with 1 µM DEX for 24 hr as described in Experimental Procedures. The normalized CAT activity is the percent conversion of chloramphenicol to its acetyl derivative relative to -galactosidase activity and is the mean ± standard deviation of three flasks. *, Statistically different from untreated cells (p < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Induction of luciferase activity by DEX in HepG2 cell transiently transfected with homologous promotor constructs containing bp 1620 to 1032 of gstA2. Luciferase and -galactosidase assays were performed on lysed HepG2 cells that had been transiently transfected with various LUC constructs that contained a 588-bp PCR product cloned into p0.164YaLUC and the expression plasmids pCMV and pRSVGR. Arrows, orientation of the insert. The HepG2 cells were treated with 1 µM DEX for 24 hr as described in Experimental Procedures. The normalized luciferase activity is expressed as relative light units divided by -galactosidase activity and is the mean ± standard deviation of three flasks. *, Statistical difference from cells treated with BA alone (p < 0.5).

Chimeric PPAR-GR receptor specificity. To test further the hypothesis that the GC receptor acts differently in regulating the expression of p1.6YaCAT construct than for a CAT construct containing three copies of the pGRE (pGRE5CAT), we determined the ability of a chimeric receptor GR-PPAR to activate either pGRE5CAT or p1.6YaCAT. This receptor contains the GC receptor DNA binding domain fused to the PPAR ligand binding domain (Fig. 8A). Previous work (Boie et al., 1993) has shown that this chimeric PPAR receptor is capable of activating genes with functional positively acting GREs and is inducible by peroxisome proliferators. When pGRE5 was cotransfected with GR-PPAR (Fig. 8B), a significant (2.0-fold) increase in basal level transcription was observed, and this activity was induced 17-fold on the addition of 50 µM nafenopin, a potent peroxisome proliferator. Nafenopin had no effect on these reporter genes when administered in the absence of the chimeric receptor or after cotransfection of the GC receptor (results not shown). With pGRE5CAT, similar effects were observed when other peroxisome proliferators, ciprofibrate and clofibrate, were administered (results not shown). Interestingly, cotransfection of GR-PPAR in either the presence or absence of nafenopin had no significant effect on the fold induction of CAT activity of p1.6YaCAT by PAH (Fig. 8C). There was a modest increase in basal expression, suggesting that although the chimeric receptor does contain the domains required for trans-activation of some genes regulated by the GC receptor, it does not have the domains required to mediate the negative regulation of the native rat gstA2 gene.

View larger version (37K): [in this window] [in a new window]   Fig. 8.   The effects of nafenopin and BA on CAT expression in HepG2 cells transiently transfected with GSTYaCAT constructs and a chimeric receptor, GR-PPAR. A, Diagrammatic representation of the chimeric construct between the GC and peroxisome proliferator activated receptor. Transfected pGRE5CAT (B) and transfected p1.6YaCAT; CAT, and -galactosidase assays (C) were performed on lysates from HepG2 cells that had been transiently transfected with the expression vector pCMV and either pRSVGR (GC receptor) or pGR-PPAR (chimeric GC receptor). pGRE5CAT or p1.6YaCAT also were cotransfected, and the HepG2 cells were subsequently treated with 50 µM benzanthracene, 1 µM DEX, 50 µM nafenopin, or in combination for 24 hr as described in Experimental Procedures. The normalized CAT activity is expressed as the percent conversion of chloramphenicol to its acetyl derivative relative to -galactosidase activity and is the mean ± standard deviation of three flasks. *, Statistically different from control cells (p < 0.05). **, Statistically different from cell treated with BA (p < 0.05).

	Discussion

Top Summary Introduction Procedures Results Discussion References

Our results demonstrate that the negative regulation of the gstA2 gene by GCs occurs via a GC receptor-dependent process and is similar to the responses observed in intact animal models (Linder and Prough, 1993) and in adult hepatocytes (Prough et al., 1996). The expression of the rat gstA2 gene is known to be under multiple regulatory processes and differs during the various stages of development from the fetal to adult state (Abramovitz et al., 1989; Sherratt et al., 1990; Linder and Prough, 1993; Xiao et al., 1995; Prough et al., 1996). In addition, Paulson et al. (1990) and Rushmore et al. (1990) have demonstrated the presence of two functional xenobiotic responsive elements that allow regulation by the AHR and a novel responsive element that allows transcriptional regulation by a variety of antioxidants/electrophilic chemicals termed the ARE. These two elements, controlled by exogenous chemicals, seem to function independently of each other. Other liver-specific transcription factors also may regulate expression of this gene, as was shown for C/EBP by Pimental et al. (1993). Because GCs apparently play a role in regulating the expression of gstA2 in adrenalectomized animals or animals deficient in normal circulating levels of this steroid hormone (Linder and Prough, 1993), we identified putative GREs in the 5'-flanking region of the gstA2 subunit gene: one palindromic consensus GRE (1609 to 1694 bp) and four consensus GRE half-sites (1637, 1361, 1063, and 646 bp). Therefore, we sought to demonstrate that these were functional GREs, accounting for some of the changes in expression shown in intact animals.

In both primary hepatocytes and immortalized cells, significant changes are observed in the levels of expression of constitutive transcription factors, such as HNF-1. These changes are thought to be important in the basal expression of the gstA2 gene. In cultured primary hepatocytes, expression of the gstA2 gene falls during the first 24 hr of culture (results not shown). In human renal tumors, there is a strong correlation between the expression of HNF-1 and the levels of GST expression (Clairmont et al., 1994). Consequently, the basal expression of CAT constructs in HepG2 cells is likely to be low. In our studies, we measured CAT activities 24 hr after treatment. The inability to observe maximal negative regulation (only 40-60%) of the basal expression of gstA2 promoter-driven CAT plasmids simply may be due to the relatively short dosing period in cultured HepG2 cells and the inherent stability of the CAT protein. However, the basal expression of our luciferase reporter constructs was more clearly repressed by GCs. Because the fold suppression of the basal expression of the LUC construct is similar to that observed with PAH-dependent induction and the basal rate is affected by the presence of the ARE, HNF-1, and a moderately strong promoter (Rushmore et al., 1990), the DEX-dependent repression phenomenon seems to be independent of gene activation by the AHR (Prough et al., 1996). However, our results do show that the structural reporter gene used in transient transfection assays may affect the magnitude of both the suppressive GC effect and the PAH-dependent induction that were observed. With the luciferase reporter construct, the degree of suppression was similar or slightly greater than that observed for the native gene either in vivo or in primary hepatocyte models over the same time period (Linder and Prough, 1993; Prough et al., 1996).

Previously, we have shown that GC negatively regulates inducible activities of the gstA2, NAD(P)H:quinone oxidoreductase, and aldehyde dehydrogenase 3 but potentiated the AHR-dependent activation of CYP1A1 and UDP-glucuronosyl transferase 1A6 proteins in cultured adult hepatocytes (Xiao et al., 1995; Prough et al., 1996). In concert, these results suggest it is unlikely that the effects of GC on these genes involve the direct interaction of the liganded GC receptor with the AHR. Other interactions, possibly with constitutive transcription factor elements, also may be involved.

Several mechanisms of regulation have been described involving the GC receptor (Starr et al., 1996). Negative mechanisms of regulation include removal of essential factors from the nucleus before receptor binding. This mode of inhibition (squelching) is seen with genes such as nuclear factor-B (Mukaida et al., 1994) and is distinguished from the mode of inhibition displayed in our current work in that the presence of a GRE is not required and RU38486 serves as an effective agonist of gene expression rather than an antagonist. Other mechanisms of negative regulation involve overlapping composite response elements, as seen in the proliferin gene (Miner and Yamamoto, 1992), or competition for transcription factor binding to the promoter element for the TATA box, as seen in the osteocalcin gene (Stromstedt et al., 1991). The GREs found in gstA2 are located upstream from all other known cis-acting elements, in an area that does not seem to significantly affect basal activity and acts as a positive hormone-responsive element when placed with the minimal promoter. These results collectively suggest that negative interaction is not simply due to competition of transcription factor binding to a composite pGRE response element. In the pro-opiomelanocortin gene (Drouin et al., 1993), a negative regulatory sequence has been described that binds three GC receptor molecules. In this gene, the response element has a 2-bp difference compared with the GRE consensus palindrome found in the MMTV GRE. The negative GRE of the pro-opiomelanocortin gene is characterized by not forming GC-sensitive plasmid constructs when the pGRE is present in three copies. Because the palindromic sequence found in gstA2 has identity with the consensus palindromic GRE described by Beato (1989) and is positively GC responsive when present in three copies or when a portion of the 5'-flanking region (1032 to 164 bp) is omitted, its regulation clearly is different from that of the proopiomelanocortin gene.

Our results demonstrate that although the response does involve receptor binding to its canonical response element, the response is complex. Clearly, the normal function of the pGRE is not a classic response element because it is negatively regulated by GC. Furthermore, chimeric receptor studies suggest that the domains involved in the repressive effect are different from those involved with positive trans-activation. In concert, these results suggest that the GC receptor interacts with other elements of the 5'-regulatory region of gstA2 gene (between bp 1032 and 164) through protein/protein interactions, which may involve DNA looping. Identification of these elements will be a focus for further study.