Dioxin induces transcription of fos and jun genes by ah receptor-dependent and -independent pathways

TOXICOLOGYANDAPPLIEDPHARMACOLOGY141, 238-247 (1996) ARTICLENO. 0280

Dioxin Induces Transcription of fos and jun Genes by Ah ReceptorDependent and -Independent Pathways A M Y HOFFER, CHING-YI CHANG, AND A L V A R O P U G A 1

Center for Environmental Genetics and Department of Environmental Health, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati, Ohio 45267-0056 Received April 26, 1996; accepted July 8, 1996

Dioxin Induces Transcription offos andjun Genes by Ah Receptor-Dependent and -Independent Pathways. HOFFER, A., CHANG, C.-Y., AND PUGA, A. (1996). ToxicoL Appl. Pharmacol. 141, 238247. Halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), and polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are environmental contaminants that cause many apparently unrelated toxic effects. In a previous study, we have shown that treatment of mouse hepatoma cells with TCDD or B(a)P results in an increase in mRNA levels of the immediate-early protooncogenes c-fos, c-jun, junB, and junD, and the concomitant increase of the DNA-binding activity of the transcription factor AP-1, a dimer of FOS and JUN proteins. To analyze the mechanism of fos/jun activation by TCDD we have used electrophoretic mobility shift and transient expression assays of reporter gene constructs containing response elements for 12-O-tetradecanoylphorbol-13-acetate (TRE), serum (SRE), cAMP (CRE), and aromatic hydrocarbons (AhRE) from thefos and jun genes fused to the firefly luciferase gene under the control of the SV40 minimal promoter. In mouse hepatoma Hepa-1 cells, which have Ah receptor (AHR) and Ah receptor nuclear translocator (ARNT) proteins, inclusion of TRE, SRE, and the AhRE motifs from cjun and junD, but not CRE or the AhREs from c-fos, fosB, and junB, causes a large TCDD-dependent increase in luciferase expression. In agreement with these results, c-jun and junD, but not c-fos, fosB, andjunB AhREs, competed with a canonical CyplAl AhRE for binding to the AHR-ARNT heterodimeric complex. In African Green Monkey CV-1 cells, which lack AHR, expression plasmids with AhRE motifs require coexpression of AHR and ARNT for TCDD to stimulate luciferase expression. In contrast, SRE-containing expression plasmids respond equally well to TCDD whether or not AHR and ARNT are coexpressed. These results suggest that TCDD induces expression of the immediate-early response genes los and jun by activation of possibly three separate signal transduction pathways, at least one of which does not require a functional Ah receptor complex. © 1996 AcademicPress, Inc.

1 To whom correspondence should be addressed. Fax: (513) 558-0925. 0041-008X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is the prototype congener of a large group of halogenated polycyclic hydrocarbons that cause a profusion of apparently unrelated toxic effects. In rodents, TCDD is one of the strongest tumor promoters ever tested in animal model systems; it causes an elevated incidence of hepatic carcinoma and pulmonary and skin tumors (Kociba et al., 1978; Schtiller, 1991; Flodstr6m et al., 1991), and promotes tumor formation at one 100th the dose of the classical tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in the skin of hairless mice (Poland and Knutson, 1982; Poland et aL, 1982; Knutson and Poland, 1982). Because TCDD is not a mutagen, it has been proposed that it promotes neoplastic transformation in cells that have already been initiated (Wassom et al., 1977; Poland and Glover, 1979; Geiger and Neal, 1981; Neal et al., 1982), but the molecular basis of this activity has yet to be resolved. In humans, exposure to dioxin and to various other polychlorinated phenolic agents causes chloracne, a long-lasting skin disease characterized by the hyperkeratinization of follicular sebocytes (Suskind, 1985; Zugerman, 1990). In addition, recent long-term epidemiological studies have established a link between exposure to high doses of TCDD and certain types of cancers and cardiovascular disease (Manz et al., 1991; Fingerhut et aL, 1991; Flesch-Janys et al., 1995). TCDD is a ligand for the cytosolic aromatic hydrocarbon (Ah) receptor (AHR) and is one of the most potent inducers of the mammalian cytochrome P450 CyplA1 gene. The AHR is a ligand-activated basic r e g i o n - h e l i x - l o o p - h e l i x (bHLH) transcription factor (Burbach et al., 1992; Ema et al., 1992) that forms a heterodimeric complex with a second b H L H transcription factor, the Ah receptor nuclear translocator (ARNT) (Swanson and Bradfield, 1993; Hankinson, 1993); this complex binds to aromatic hydrocarbon-responsive elements (AhREs, also termed XREs and DREs) in the 5' flanking region of the CYPIA1 gene and of other genes in the Ah-responsive gene battery, and acts as a transcriptional activator. Exogenous ligands for this receptor include halogenated as well as polycyclic aromatic hydrocarbons. Much

238

DIOXIN INDUCES los AND jun GENE EXPRESSION intensive research in this and in m a n y other laboratories has elucidated the major aspects of r e c e p t o r - l i g a n d interactions, nuclear translocation, and transcriptional activation (Nebert, 1989; Landers and Bunce, 1991; Nebert et al., 1993; Swanson and Bradfield, 1993; H a n k i n s o n , 1993; Whitlock, 1993). It is generally accepted, based largely on genetic evidence in mice, that T C D D b i n d i n g to the receptor is an essential step required for its toxicity (reviewed in Nebert, 1989; Landers and Bunce, 1991). Some k n o w n effects of T C D D , however, have not b e e n shown to involve the Ah receptor, including induction of protein kinases ( B o m b i c k et al., 1985) and phospholipase C (Beebe et al., 1990), and effects on plasma m e m b r a n e s and low-density lipoprotein receptor ( B o m b i c k et al., 1984; M a t s u m u r a et al., 1984). In a previous study, we have shown that treatment of mouse hepatoma cells with polycyclic or halogenated aromatic hydrocarbons such as T C D D and b e n z o [ a ] p y r e n e (B[alP) causes an increase in the steady state m R N A levels of the immediateearly protooncogenes c-fos, c-jun, j u n B , and junD, and a c o n c o m i t a n t increase in the D N A - b i n d i n g activity of the transcription factor AP-1 (Puga et al., 1992). Interestingly, induction of c-fos and j u n B appears to be i n d e p e n d e n t of A H R and A R N T , since it also takes place in variant hepatoma cell lines with highly reduced levels of A H R or lacking A R N T (Puga et aI., 1992). I n d u c t i o n of immediate-early response protooncogenes by T C D D is likely to play a major role in early molecular events surrounding T C D D - i n d u c e d t u m o r promotion, and therefore, delineation of molecular events i n d e p e n d e n t of the formation of A H R " A R N T complexes is critical for an understanding of the m e c h a n i s m s of T C D D toxicity. In this article we analyze the role of various gene regulatory sequences in protooncogene activation by T C D D and test the hypothesis that the A H R - A R N T c o m p l e x m a y not be required for this effect of T C D D . W e find that this is indeed the case for the activation of c-fos expression. METHODS

239

from trypsinization,the cells were treated for 16-24 hr with the compounds indicated or with an equivalent volume of DMSO vehicle (for TCDD), acetone (for TPA), or medium (for cAMP). Hereafter, cells treated with vehicle will be referred to as untreated. Alternatively,cells were transfected directly in the wells of a 12- or 24-well plate using 0.3 #g or reporter plasmid and 0.1 >g of pCMV/3gal. The latter method was employed routinely in experiments designed to test the role of serum in reporter expression, in which case cells were placed in medium containing0.1 or 5% serum prior to transfection. Cell extracts were prepared and luciferase and jS-galactosidase activities were determined with the Promega Luciferase Assay System, following the manufacturer's specifications.Luciferase activities were measured in a Turner 20e luminometer. Data were normalized for differences in transfection efficiency by determination of the relative amount of arbitrary light units per unit of/3-galactosidaseactivity. All determinationswere done in duplicate or triplicate plates, and experimentswere repeated at least three times; the values shown are the means _+ SEM. Plasmids for transfeetion. Reporter plasmids were constructedby ligation of the sequences shown in Table 1 upstream of the minimal SV40 promoter in the pGL2 vector of Promega (Madison, Wl), Oligonucleotides were prepared in an Applied Biosystems 380B DNA Synthesizer. Number of copies and identity of all the clones constructed were confirmed by sequencing. In addition, two other expression vectors were used in some experiments. One of these was the plasmid pCMVB6AhR, constructed in our laboratory, which contains the cDNA coding for the high-affinityAh receptor from C57B1/6J mice and expresses this protein under the control of the CMV promoter; the other is plasmid pcDNAINeo/mARNT, which expresses the murine ARNT protein (a generous gift of Dr. Oliver Hankinson, UCLA). Electrophoretic mobility shift assays. Hepa-1cells were grown to 80% confluence ill a-MEM containing 5% fetal bovine serum. One-half of each culture was treated with l0 nM TCDD for 6 hr at 37°C, and the other half was mock-treated with vehicle (0.01% DMSO). Nuclear extracts were prepared as described (Dignam et al., 1983; Carder et al., 1992) and used for binding reactions that were carried out for 20 min at 4°C in 20 >1 of a buffer containing 20 mM Hepes, pH 7.8, 1 n'~MEDTA, 1 mM DTT, 120 mM KC1, 1 #g of poly(dl.dC)-poly(dkdC), 10% glycerol, 10/zg of nuclear protein extract, and 0.1 ng of a 20-mer AhRE-3 probe (sp act 1 × l0 s dpm/ #g), labeled with T4 polynucleotide kinase in the presence of [y-32p]ATP as previously described (Cartier et al., 1994). For competition reactions, prior to addition of the probe, extracts were preincubated for 5 rain with an excess of the oligonucleotides containing AhRE sequences shown in Table 1. After incubation, samples were analyzed in nondenaturing 4% polyacrylamide gels in 0.5× TBE running at 200 V. After electrophoresis, the gels were dehydrated, dried under vacuum, and exposed to X-ray film. Enzymatic assays. CyplA1 activity in crude cell extracts was measured spectrophotofluorometrically,as previously described (Nebert and Gelboin, 1968; Carrier et al., 1992) by deterrrdnation of the rate of hydroxylated benzo[a]pyrene formation.

Cell lines, transfections, and growth conditions. MouseHepa-1 hepatoma cells (Bernard et al., 1973) and African Green Monkey adult kidney epithelium CV-1 cells (Jensen et al., 1964) were maintained routinely as RESULTS monolayer cultures in a-MEM (Gibco/BRL) supplemented with 5% fetal bovine serum. For transient transfection experiments, constructs carrying the fireflyluciferase gene were used as reporters (see below). Approximately Regulatory sequences of fos and j u n genes contain A h R E 5 #g of the appropriatereporter plasmid was transfectedby standardcalcium motifs. Computer analyses of genes in the murine and rat phosphate techniques (Graham and Van der Erb, 1973; Puga et al., 1990) f o s and j u n families revealed that all the family members into semiconfluent cells grown in 25-cm2 tissue culture flasks. To control for variations due to differences in transfection efficiency, all cultures were contain the A h R E core sequence 5 ' - C A C G C - 3 ' in their cotransfected with 1 #g of plasmid pCMV/3gal (CloneTech), which ex- proximal promoter region. Table 1 shows a s u m m a r y of presses the bacterial/3-galactosidasegene under the control of the cytomeg- A h R E sequences (only one strand shown) found in the mualovirus immedi~Ie-earlyenhancer and promoter. Expression of/3-galactosi- fine genes with their coordinates in the appropriate G e n B a n k dase under regulation of this promoter is independent of treatment to the cells. Twelve to 16 hr after transfection, the cells were washed with fresh loci. c-los and f o s B each contain one A h R E copy, c-jun medium and 1-2 hr later were trypsinized and seeded into 12- or 24-well contains two t a n d e m copies, sharing one base, and j u n B and tissue culture plates. Five to 6 hr later, when the cultures had recovered j u n D each have two copies of the A h R E motif. The presence

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DIOXIN INDUCES fos AND jun GENE EXPRESSION of these motifs raised the possibility that induction of fos and jun expression by TCDD might result from the same molecular mechanisms responsible for induction of the CyplA1 gene, namely, binding of a liganded Ah receptor. A R N T complex to regulatory AhRE domains. If this were the case, fos andjun AhREs might be expected to be as efficient as canonical CyplA1 AhREs in TCDD-dependent transcriptional activation. To test this possibility, we synthesized the AhRE oligonucleotides shown in Table 1, and their complementary strands, and cloned them upstream of the SV40 minimal promoter in the luciferase reporter plasmid pGL2. We also prepared positive control clones containing a canonical AhRE from the mouse CyplA1 gene and, as a negative control, a corresponding plasmid containing a mutated AhRE. To determine whether the effect of TCDD could be mediated by other known response elements in the promoters of the immediate-early protooncogenes, we also synthesized and cloned into pGL2 the sequence motifs shown at the bottom of Table., 1. These included a consensus TPA response element (TRE) and TREs from c-fos and c-jun, a consensus cAMP response element, and a c-fos serum response element (SRE) that forms a ternary complex with the serum response factor and Elk-1. (For a recent description of these binding sites and their properties see Karin (1995) and references therein).

c-jun andjunD AhREs compete with a canonical CyplA1 AhRE for binding to the AHR" ARNT complex. Binding of the AHR" ARNT heterodimeric complex to AhRE motifs in their regulatory domains is a required step for transactivation of Ah battery genes (reviewed in Hankinson, 1995). If AhRE motifs in fos and jun genes were responsible for activation of these genes by TCDD, they might also be expected to bind to A H R - A R N T complexes. To test this hypothesis, the double-stranded AhRE oligonucleotides shown in Table 1 were used in gel retardation experiments as binding competitors for a 32p-labeled, canonical CyplA1 AhRE probe. The c-jun AhRE site and the junD site at position 528 competed the binding of the probe as efficiently as a positive control with unlabeled CyplA1 AhRE; the junD site at 698 was a slightly less efficient competitor, and the sites in c-fos, fosB, and junB, as well as the negative control with a mutated AhRE, did not compete for binding of the probe (Fig. 1). These results indicate that c-jun and junD AhREs are recognized by the ,~dtR. ARNT complex. Since the other sites tested did not bind to the complex, it follows that not all the AhREs are synonymous, and that the sequence context surrounding the AhRE motif itself must provide important recognition elements for the DNA/protein interaction. This is in agreement with numerous other studies that have shown that more than the AhRE core sequence is necessary for AHR" ARNT binding (reviewed in Whitlock, 1993).

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FIG. 1. Competition electrophoretic mobility shift analysis of AhRE motifs in fos and jun family members. Nuclear protein extracts (10 #g) from Hepa-1 cells treated with 10 nMTCDD were incubated with increasing amounts (50- and 200-fold excess) of the competitoroligonucleotides indicated above each lane and described in Table 1 prior to incubation with a 32p-labeled AhRE probe. U, nuclear extract from untreated cells; N, nuclear extract from TCDD-treated cells, showing a band corresponding to the probe/AHR. ARNT complex, in the absence of added competitoroligonucleotide.

c-jun and junD AhREs function as TCDD-dependent enhancers. The results shown above imply that those AhREs that do not form complexes with A H R - A R N T are unlikely to function as cis-acting transcriptional enhancers; conversely, those AhREs that do form complexes with A H R . ARNT have at least a potential for being biologically functional. To test their biological activity as cis-acdng enhancers, the AhRE-containing luciferase reporters shown in Table 1 were used in transient transfection assays in TCDDtreated and untreated Hepa-1 cells. TCDD treatment had a significant effect on luciferase expression directed by plasraids containing c-jun and junD AhREs, whereas it had no effect on expression directed by plasmids containing c-fos, fosB, and junB AhREs (Fig. 2). In the case of the c-jun sequences, inclusion of one copy of the tandem repeat in plasmid pcjAh resulted in a 2-fold stimulation of expression in the presence of TCDD, while inclusion of two copies in plasmid p2cjAh gave a 3.5-fold stimulation. The junD AhRE at position 528 in pjD1Ah responded significantly more efficiently to TCDD than the motif at position 698, and pjD12Ah, the construct containing both motifs, did not shown synergy or even additivity of the effects of the two motifs. The positive control plasmid p2AhRE, containing two copies of the canonical CyptA1 AhRE-3, showed a 4fold stimulation of luciferase after TCDD treatment, whereas p2mAhRE and pGL2 plasmids, containing mutated or no AhRE motifs, respectively, did not respond to TCDD (Fig. 2). These results are in good agreement with the findings of gel retardation experiments, and indicate that activation of at least two of the immediate-early genes under study, namely c-jun and junD, could result from TCDD-dependent

242

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FIG. 2. Effect of TCDD on the transient expression of luciferase reporter plasmids containing AhRE motifs fromfosand jun family members. The values in the ordinate represent luciferase activity normalized for relative transfection efficiency to/%galactosidase activity directed by the control plasmid, pCMV/3gal. The plasmids used are described in Table 1. PGL2 is the empty vector containing the luciferase gene fused to the SV40 minimal promoter. Results were compared using two-sample t tests; the one-tailed p values for the paired comparisons of TCDD-treated and untreated samples are: pcjAh, p = 0.003; p2cjAh, p = 0.0002; pjB12Ah, p = 0.034; pjD1Ah, p = 0.0007; pjD2Ah, p = 0.077; pjD12Ah, p = 0.003; p2AhRE, p = 0.0007. All other paired comparisons were not statistically significant.

activation of the AHR and subsequent formation of an AHR• A R N T transactivation complex.

TRE and SRE, but not CRE, mediate TCDD activation of c-fos in quiescent cells. The results described above, however, could not explain activation of c-fos, since the potential AhRE motifs from this gene did not bind A H R . A R N T complexes and did not enhance luciferase expression in TCDD-treated cells. To identify the regulatory sequences responsible for c-fos activation by TCDD, we constructed luciferase reporter plasmids containing the TPA and serum response elements (TRE, SRE) from the mouse c-fos gene, as well as plasmids with the consensus TRE motif, the TRE motif from c-jun, and the consensus cAMP response element (CRE). The enhancer sequences included in these plasmids are also shown in Table 1. The effect of TCDD on luciferase expression directed by these plasmids was determined by transient transfection assays in TCDD-treated and untreated Hepa-1 cells. As positive controls for transfections with TRE- and CRE-containing plasmids, we included TPA and cAMP treatments, respectively. Assays were conducted in exponentially growing cells as well as in cells made quiescent by incubation in medium containing 0.1% serum. In low serum medium, one copy of the c-fos TRE showed significant enhancer activity in the presence of TCDD as well as TPA; basal, TPA-, and TCDD-induced activities were markedly increased in the reporter containing two cop-

ies of this TRE sequence (Fig. 3A). In comparison, two copies of the c-jun TRE had a much reduced effect on basal expression levels, which were also stimulated by TCDD but to a much lower extent (approximately 7 times less) than those directed by two copies of the c-fos TRE. In 5% serum medium, basal levels were much higher for all the plasmids tested, and TCDD or TPA treatments did not cause significant increases in luciferase activity (Fig. 3A). The CRE motif did not mediate a response to TCDD treatment in either low serum or high serum medium, although it directed a 6- to 7-fold stimulation in the presence of cAMP in cells maintained in high serum medium (Fig. 3B). In contrast, two or four copies of the c-fos SRE motif mediated the highest TCDD-dependent effects. In cells maintained in 0.1% serum, basal levels of expression were nearly negligible, and TCDD enhanced luciferase expression by 5- to 10-fold; in the presence of serum, the basal expression level was so elevated that it masked any possible effect of TCDD (Fig. 3C). In low serum medium, the TCDD effect mediated by the c-fos SRE was dose dependent and closely parallelled the dose-dependent effects of a plasmid containing two copies of the CyplA1 AhRE (Fig. 4). These data implicate both c-fos TRE and SRE on the activation of cfos by TCDD.

TCDD activation of the SRE does not require the Ah receptor. To determine whether activation of c-fos TRE and SRE required a functional Ah receptor, we used for transfection experiments the CV-1 cell line, which has no detectable Ah receptor by Western blot but has normal levels of ARNT (Matsushita et al., 1993; Fukunaga et al., 1995; Fukunaga and Hankinson, 1996). The lack of a functional Ah receptor complex was confirmed in assays of cell extracts from TCDD-treated and untreated ceils for B(a)P (CYP1A1) hydroxylase activity. Control TCDD-treated Hepa-1 cells showed a 12-fold stimulation of enzymatic activity over the level detected in untreated cells, whereas CV-1 cells, whether treated with TCDD or untreated, had undetectable levels of B[a]P hydroxylase (Table 2). In pilot experiments to test expression of AhRE reporter plasmids (not shown) we noticed that, even though CV-1 cells express immunoreactive ARNT, cotransfection of an ARNT expression vector caused a large increase in the level of reporter gene expression. Consequently, we included an ARNT expression plasmid in all our cotransfection studies. Transient transfection assays in CV-1 cells included plasmids, such as pjD12Ah and p2AhRE, that contained AhRE motifs, and therefore were expected to require a functional A H R . ARNT complex for expression, a negative control plasmid (p2mAhRE) containing a mutated AhRE site, and the two plasmids (p2cfTRE and p4cfSRE) to be tested. To test the role of the A H R . ARNT complex one set of transfection dishes received no additional plasmids, and another set was cotransfected with the two expression plasmids

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pCVMB6AhR and pcDNAINeo/mARNT, which express the high-affinity C57BL/6J mouse Ah receptor and ARNT, respectively. Luciferase activity directed by AhRE-containing plasmids was not affected by TCDD in the absence of AHR• ARNT expression, but it was stimulated by two- to fourfold when AHR. ARNT expression plasmids were also included in the transfection. The negative control reporter, p2mAhRE, showed no TCDD- or AHR" ARNT-dependent effects (Fig. 5). In contrast, p4cfSRE, carrying four copies of the c-fos SRE, responded to TCDD equally well in the presence or the absence of coexpressed AHR. ARNT, whereas p2cfTRE did not respond to TCDD in either case (Fig. 5). These results demonstrate that AhRE-mediated activation of the jun genes by TCDD is, like the activation of the CyplAl gene itself, Ah receptor dependent, whereas activation of cfos by TCDD, when mediated by the SRE, is independent of the Ah receptor. ARNT complex.

DISCUSSION In this article we present experiments designed to analyze the molecular mechanisms whereby TCDD induces the expression of immediate early protooncogenes. Our results strongly suggest that these mechanisms are diverse and include Ah receptor-dependent as well as -independent pathways. Stimulation of c-jun andjunD appears to be mediated mainly by AhRE motifs in the regulatory region of these genes; these AhRE motifs behave in a similar fashion to the canonical AhREs in the CyplA1 gene, i.e., they bind to the AHR. ARNT complex and mediate TCDD-dependent stimulation of expression. Therefore, we conclude that induction of c-jun and junD expression by TCDD requires the Ah receptor. ARNT complex. On the contrary, stimulation of c-fos is mediated by TRE and SRE motifs. Stimulation through the SRE occurs equally well in Ah receptor-positive

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Hepa-1 cells as in Ah receptor-negative CV-1 cells and, in the latter cell line, coexpression of AHR. ARNT causes no change in reporter expression levels. Consequently, we conclude that induction of c-fos expression by TCDD takes place by two pathways, one of which does not require a functional AHR. ARNT complex. These results are in good agreement with our earlier observations based on TCDDinduced mRNA levels in variant Hepa-1 cells (Puga et al., 1992). Potential AhRE motifs from fosB and junB do not recognize AHR. ARNT, nor do they mediate TCDD-dependent luciferase expression in our reporter plasmids. Of these

TABLE 2 Benzo[a]pyrene Hydroxylase (CYPIA1) Activity in the Cells Used in These Studies Nanomoles of phenolic B[a]P formed per minute per milligram of protein Cell line

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p4cfSRE

FIG. 5. Effect of TCDD on transient expression of AhRE-, TRE-, and SRE-containing plasmids in CV-1 ceils cotransfected with plasmids expressing A H R and ARNT. The four combinations of transfection and treatment are shown: untreated and TCDD-treated ceils, cotransfected or not in each case with the two plasmids p C M V B 6 A h R and pCDNAINeo/mARNT, expressing A H R and ARNT, respectively. Note the change of ordinate scale for the three sets of transfecdons with p2mAHRE, p2cfrRE, and p4cfSRE. Statistical analyses were by two-sample t tests. The one-tailed p values for the statistically significant differences were: pjD12Ah (in the presence of AHR/ARNT), p = 0.003; p2AHRE, p = 0.0006; p4cfSRE ( - A H R / A R N T ) , p = 0.003; p4cfSRE (+AHR/ARNT), p = 0.017. All other paired comparisons were not statistically significant.

two protooncogenes onlyjunB has been shown to be induced by TCDD; it is likely that its induction results from Ah receptor-independent mechanisms similar to those responsible for c-fos activation. The c-jun TRE differs from other TREs by a one-basepair insertion which makes it more easily recognized by cJUN. ATF-2 heterodimers than by conventional AP-1 complexes (van Dam et al., 1993, 1995). We find that both cjun and consensus TRE motifs confer very low levels of expression and low TCDD inducibility to their respective reporter plasmids, whereas a nonconsensus c-fos TPA response element (Gutman et al., 1991) gives much higher basal as well as TCDD-induced reporter expression in Hepa1 cells (Fig. 3A) but not in CV-1 cells (Fig. 5). These results suggest that the response to TCDD mediated by this TRE motif is not the result of the activation of c-JUN. ATF-2 heterodimers and that it involves at least one member of the AP-1 complex that may not be expressed in CV-1 cells. Complete characterization of the immediate-early genes expressed in CV-1 cells will be required to confirm this conclusion. The SRE mediates the response of c-fos to TCDD in a serum- (Fig. 3C) and dose-dependent manner (Fig. 4) that

DIOXLN INDUCES fos AND jun GENE EXPRESSION is i n d e p e n d e n t of the A h receptor (Fig. 5). The S R E also mediates c-fos induction b y growth factors, cytokines, U V irradiation, oxidants, and other stimuli that activate mitogenactivated protein kinases ( M A P K s ) ( A r o n h e i m et al., 1994; S a c h s e n m a i e r et al., 1994; r e v i e w e d in Karin, 1995). Phosp h o r y l a t i o n o f the ternary c o m p l e x factors (TCFs) such as E L K - 1 or SAP-1 b y M A P K s results in the rapid formation o f a transcriptionally active ternary c o m p l e x c o m p o s e d o f a p h o s p h o r y l a t e d T C F , the serum response factor, and the S R E m o t i f itself (Karin, 1995). M i t o g e n i c signals that activate M A P K s originate at the cell surface and include activation of e p i d e r m a l growth factor receptors, and a cascade o f cytop l a s m i c signals i n v o l v i n g R A S , R A F - 1 , and S R C activation ( S m e a l et al., 1991; Binetruy et al., 1991; van den Berg et al., 1991; D e v a r y et al., 1992; R a d l e r - P o h l et al., 1993; W e s t w i c k et aL, 1994; A r o n h e i m et al., 1994; E n g e l b e r g el al., 1994; D e n g and Karin, 1994; Derijard et al., 1994; S a c h s e n m a i e r et al., 1994). T C D D has been shown to affect m a n y o f the c o m p o n e n t s o f this signal transduction cascade, including activation o f receptor tyrosine kinases (Beebe et aI., 1990) and other unidentified tyrosine kinases ( B o m b i c k et aI., 1985; B o m b i c k et al., 1988; M a et al., 1992; M a and Babish, 1993; D e V i t o et al., 1994; L e e et al., 1996), as well as R A S activation ( B o m b i c k et al., 1984; M a d h u k a r et al., 1984; Tullis et aL, 1992; E b n e r et aI., 1993; Enan and Matsumura, 1994, 1995). Therefore, it is likely that the activation o f the S R E ternary c o m p l e x b y T C D D m a y start b y a signal triggered at the cell surface that precedes binding of T C D D to the cytosolic A h receptor. In this context, it is worth noting that T C D D has b e e n shown to disrupt p l a s m a m e m b r a n e s , p o s s i b l y b y activation of PLA2 and formation of lysophospholipids ( B o m b i c k et al., 1984; M a t s u m u r a et al., 1984) and to inhibit gap j u n c t i o n intercellular communication, although in a m a n n e r not c o m p l e t e l y i n d e p e n d e n t o f the A h receptor (De H a a n et al., 1994; B a k e r et al., 1995). E x p r e s s i o n o f several genes, in addition to the i m m e d i a t e early p r o t o o n c o g e n e s and the phase I and phase II m e m b e r s o f the A h battery, is altered b y T C D D exposure. E x p r e s s i o n o f p l a s m i n o g e n activator inhibitor-2 and interleukin-1/3 (Sutter et al., 1991), transforming growth factors-ce and -/32 (Gaido et al., 1992; L e e et al., 1996), and c y c l i n - d e p e n d e n t kinases ( M a and Babish, 1993) are up-regulated, whereas e p i d e r m a l growth factor receptors ( K a r e n l a m p i et aI., 1983; M a d h u k a r et al., 1984; K a w a m o t o et al., 1989; L i n e t al., 1991) and l o w - d e n s i t y lipoprotein receptors ( B o m b i c k et al., 1984) are d o w n - r e g u l a t e d b y T C D D . It is p o s s i b l e that s o m e o f the effects o b s e r v e d in the regulation o f these genes are d o w n s t r e a m effects resulting from the activation o f i m m e d i ate-early response genes. ACKNOWLEDGMENTS We thank Oliver Hankinson for a gift of plasmid pcDNAINeo/mARNT and Cindy Bachurski and Daniel W. Nebert for a critical reading of the

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