Transcriptional Activation of c-fos Protooncogene by 17ß-Estradiol: Mechanism of Aryl Hydrocarbon Receptor-Mediated Inhibition

Renqin Duan, Weston Porter, Ismael Samudio, Carrie Vyhlidal, Michael Kladde and Stephen Safe

Department of Veterinary Physiology and Pharmacology (R.D., W.P., I.S., S.S.) and Department of Biochemistry and Biophysics (C.V., M.K.) Texas A&M University College Station, Texas 77843


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
17ß-Estradiol (E2) induced c-fos protooncogene mRNA levels in MCF-7 human breast cancer cells, and maximal induction was observed within 1 h after treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibited the E2-induced response within 2 h. The molecular mechanism of this response was further investigated using pFC2-CAT, a construct containing a -1400 to +41 sequence from the human c-fos protooncogene linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In MCF-7 cells transiently transfected with pFC2-CAT, 10 nM E2 induced an 8.5-fold increase of CAT activity, and cotreatment with 10 nM TCDD decreased this response by more than 45%. {alpha}-Naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, blocked the inhibitory effects of TCDD; moreover, the inhibitory response was not observed in variant Ah-nonresponsive MCF-7 cells, suggesting that the AhR complex was required for estrogen receptor cross-talk. The E2-responsive sequence (-1220 to -1155) in the c-fos gene promoter contains two putative core pentanucleotide dioxin-responsive elements (DREs) at -1206 to -1202 and -1163 to -1159. In transient transfection assays using wild-type and core DRE mutant constructs, the downstream core DRE (at -1163 to -1159) was identified as a functional inhibitory DRE. The results of photo-induced cross-linking, gel mobility shift, and in vitro DNA footprinting assays showed that the AhR complex interacted with the core DRE that also overlapped the E2-responsive GC-rich site (-1168 to -1161), suggesting that the mechanism for AhR-mediated inhibitory effects may be due to quenching or masking at the Sp1-binding site.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
c-fos Protooncogene is widely expressed in mammalian tissues and plays an important role in both normal and transformed cells (1, 2, 3, 4). c-Fos protein forms a heterodimer with c-jun to give the activating protein-1 (AP-1) transcription factor complex that modulates expression of multiple genes through interactions with AP-1 cis-elements in their corresponding promoters (1, 2, 3, 4, 5, 6). c-fos protooncogene expression is modulated by multiple endogenous and exogenous factors including hormones, growth factors and related mitogens, cytokines, and protein kinase inducers/inhibitors (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). c-fos Transactivation by these factors is highly cell-specific and dependent on interactions of nuclear proteins with multiple cis-elements in the c-fos protooncogene promoter. For example, GH induces c-fos gene expression in 3T3-F442A cells by activating the MAPK pathway, resulting in phosphorylation of serum response factor and associated proteins that bind the serum response element (SRE) at -322 to -314 and phosphorylation of Stat 1 and Stat 3 that bind the sis-inducible element (SIE) (18). Other modulators of fos gene expression also act through the SRE or SIE motifs (20, 25, 26, 27, 28). Vitamin D also stimulates c-fos expression via interaction of the vitamin D and retinoid X receptor with a CCAAT-binding factor at a composite cis element between -178 and -144 in the fos gene promoter (14).

Recent studies in this laboratory have shown that induction of c-fos expression in MCF-7 human breast cancer cells by 17ß-estradiol (E2) involves interaction of an estrogen receptor (ER)/Sp1 complex with a distal GC-rich promoter element at -1168 to -1161 (11). This novel pathway for ER action involves binding of the ER to Sp1 protein and not DNA (29), and other E2-responsive GC-rich motifs have been identified in the retinoic acid receptor {alpha}-1, cathepsin D, and adenosine deaminase gene promoters (30, 31, 32). This pathway for DNA binding-independent ER action is similar to results reported for ER/AP-1 interactions with promoters containing AP-1 sites (33, 34, 35). Studies in this laboratory and others have focused on the indirect antiestrogenic activity of aryl hydrocarbon receptor (AhR) agonists and the mechanisms associated with AhR-ER cross-talk (36, 37, 38, 39). Results obtained for at least two E2-responsive genes, namely cathepsin D and pS2, have identified GCGTG pentanucleotide sequences that are required for AhR-mediated inhibition of ER action (38, 39). This motif corresponds to the core-binding nucleotides required for a dioxin-responsive element (DRE) (40) and have been designated as inhibitory DREs (iDREs) (38, 39). This study demonstrates that in MCF-7 cells, the potent AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), inhibits E2-induced c-fos protooncogene expression and reporter gene activity with constructs containing c-fos gene promoter inserts. Deletion analysis of this promoter has identified two GCGTG (core DRE) motifs in the -1220 to -1155 region of the promoter, and only the downstream sequence at -1163 to -1159 is a functional iDRE.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inhibition of E2-induced Transactivation of c-fos by TCDD and ICI 182,780
E2 rapidly induces c-fos mRNA levels in MCF-7 cells, and a 8.7-fold increase was observed after treatment for 1 h (Fig. 1AGo). In cells cotreated with E2 (1 h) and 10 nM TCDD for 1, 2, 4, 12, or 24 h, the hormone-induced mRNA levels were significantly decreased within 2 h, and inhibition was observed for up to 24 h. pFC2 contains the -1400 to +41 region from the c-fos gene promoter linked to a chloramphenicol acetyl transferase (CAT) reporter gene in pBLCAT2. Ten nanomolar E2 caused a more than 8-fold increase in CAT activity in MCF-7 cells transiently transfected with pFC2, and in cells cotreated with E2 and 10 nM TCDD, the hormone-induced response was significantly decreased (Fig. 1BGo). The inhibitory responses mediated by TCDD in MCF-7 cells were similar to those observed for the antiestrogen ICI 182,780; however, the latter compound was a more potent inhibitor (Fig. 1CGo).



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Figure 1. Inhibition of E2-Induced Transactivation of c-fos by TCDD and Antiestrogens

A, mRNA levels. Cells were treated with DMSO (lane 1), 10 nM E2 alone for 1 h (lane 2), or cotreated with 10 nM E2 (1 h) and 10 nM TCDD for 1, 2, 4, 12, or 24 h (lanes 3–7, respectively), and c-fos mRNA levels (relative to ß-tubulin mRNA) were determined as described in Materials and Methods. Fos mRNA levels relative to DMSO (arbitrarily set at 100 ± 75) were 870 ± 163, 1080 ± 370, 410 ± 137, 167 ± 45, 209 ± 45, and 165 ± 60 for cells treated with E2 alone and E2 plus TCDD for 1, 2, 4, 12, and 24 h, respectively. Induction of c-fos mRNA by E2 (8.7-fold) was significantly (P < 0.05) decreased by TCDD at all time points >=2 h. B, Inhibition of E2-induced CAT activity by TCDD. MCF-7 cells were transiently transfected with pFC2-CAT (-1400 to +14) and treated with various hormones/chemicals, and CAT activity was determined as described in Materials and Methods. Significant (P < 0.05) induction was observed for E2 alone ({dagger}), and this response was inhibited by TCDD (ET) ({ddagger}); however, the inhibitory effect was reversed by {alpha}NF ({alpha}NF/ET). C, Antiestrogenic activity of ICI 182,780. Using the same protocol described in panel B, the induction response by E2 was inhibited by ICI 182,780 (IE) ({ddagger}), but this inhibition was not reversed by {alpha}NF ({alpha}NF/IE). D, Inhibitory effects of TCDD in MCF-7BaP cells. Using the same protocol described in panel B, the induction of CAT activity by E2 (*) was not significantly inhibited by TCDD (ET) in Ah-nonresponsive MCF-7BaPr cells (42 ). All results were determined in triplicate (three separate experiments) and expressed as means ± SD (B–D).

 
Inhibition of E2-Induced Responses by TCDD: Requirement for the AhR
Previous studies in MCF-7 cells have demonstrated that the AhR antagonist {alpha}-naphthoflavone ({alpha}NF) inhibits formation of the liganded nuclear AhR complex and thereby blocks inhibitory effects of TCDD on E2-regulated responses (41). The results illustrated in Fig. 1BGo show that {alpha}NF alone did not significantly affect CAT activity in MCF-7 cells transfected with pFC2; however, in combination with E2 plus TCDD, {alpha}NF significantly blocked the inhibitory effects of TCDD. In contrast, {alpha}NF did not affect the antiestrogenic action of ICI 182,780 and the inhibitory effects of ICI 182,780 were greater than those observed for TCDD (Fig. 1CGo). These data illustrate the specificity of {alpha}NF, and the results are consistent with a role for the AhR in this inhibitory process. Benzo[a]pyrene (BaP)-resistant MCF-7 cells are E2 responsive and Ah-nonresponsive due to a defective AhR-Arnt heterodimer that does not bind DREs (42). E2 induced CAT activity (3.6-fold) in BaP-resistant MCF-7 cells transfected with pFC2, and TCDD alone did not affect CAT activity compared with control cells (Fig. 1DGo). In cells cotreated with E2 plus TCDD, the hormone-induced response was not decreased, confirming that the inhibitory activity of TCDD requires an intact nuclear AhR complex that binds DNA.

Characterization of a Functional iDRE in the c-fos Gene Promoter
Previous studies have demonstrated that the GC-rich motif at -1168 to -1161 is required for E2 responsiveness in MCF-7 cells, and pF1 contains a -1220 to -1155 fos gene promoter insert with downstream GC-rich elements and an upstream imperfect palindromic estrogen response element (ERE) that is required for ER action in HeLa cells (43). E2 induced CAT in MCF-7 cells transfected with pF1 (Fig. 2Go, A and B), and this was similar to results of previous studies (11). The -1120 to -1155 region of the fos promoter contains two pentanucleotide GCGTG motifs that may function as iDREs, which have been identified in the cathepsin D and pS2 gene promoters (38, 39). pF1.d1m and pF1.d2m contain mutations in core DRE1 (-1206 to -1202) and 2 (-1163 to -1159), respectively, and in transient transfection assays in MCF-7 cells, both constructs were E2 responsive. In contrast, TCDD significantly inhibited hormone-induced activity in cells transfected with pF1.d1m but not pF1.d2m, indicating that the downstream GCGTG sequence that overlaps the GC-rich motif is a functional iDRE. In a separate experiment (Fig. 2BGo), ICI 182,780 inhibited E2-induced transactivation using the same wild-type and mutant constructs (note: E2-responsiveness of the constructs was somewhat variable between experiments).



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Figure 2. Comparative Inhibitory Activities of TCDD and ICI 182,780 Using Wild-Type and Core DRE Mutant pF1 Constructs

A, Effects of TCDD. MCF-7 cells were transfected with various constructs and treated with E2, TCDD, or TCDD plus E2 (ET), and CAT activity was determined as described in Materials and Methods. E2 significantly (P < 0.05) induced activity in cells transfected with all three constructs ({dagger}), and TCDD significantly inhibited E2-induced activity only with pF1 and pF1d1m ({ddagger}). B, Antiestrogenic activities of ICI 182,780. Experiments were carried out as described in Fig. 1AGo, and ICI 182,780 inhibited E2-induced activity (EI) using all three constructs ({ddagger}). Results are expressed as means ± SD for three separate experiments for each treatment group.

 
Gel Mobility Shift Assays and Photoinduced Cross-Linking of the AhR Complex with iDRE2
Previous studies (11) show that the GC-rich site (-1168 to -1161) is required for Sp1 and ER/Sp1 binding in gel mobility shift assays, and Sp1 protein binds [32P]fos4 (-1175 to -1153) (Fig. 3AGo, lane 2); the intensity of this band is enhanced by coincubation with AhR/Arnt (lane 5). Excess unlabeled wild-type but not mutant Sp1 oligonucleotides decreased binding (lanes 6 and 7), and cold DRE also slightly decreased binding. This pattern of AhR/Arnt interaction with Sp1 and ER/Sp1 binding was previously observed using a 32P-labeled Sp1(N)4DRE(core) motif from the cathepsin D gene promoter (44, 45) where the core DRE motif was required for enhanced Sp1-DNA binding and transactivation. Core DRE2 (-1163 to -1159) in the fos gene overlaps the GC-rich sequences (-1168 to -1161). A T-G (at -1160) mutation to give fos4.m1 results in a key mutation of the core DRE but [32P]fos4.m still binds Sp1 protein in a gel mobility shift assay (Fig. 3BGo, lane 2), and this mutation did not affect E2 responsiveness in transactivation (Fig. 2Go). ER but not AhR/Arnt enhanced Sp1-DNA binding (lanes 3 and 4), and in unlabeled oligonucleotide competition studies (lanes 6–8), Sp1 but not mutant Sp1 or wild-type DRE competitively decreased Sp1-DNA binding. These results demonstrate that the core DRE motif that overlaps the GC-rich Sp1-binding site in [32P]fos4 is required for modulation of Sp1-DNA binding, and these results are similar to those previously reported for the [32P]Sp1(N)4DRE(core) oligonucleotide from the cathepsin D gene promoter (44, 45).



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Figure 3. Gel Mobility Shift Assay and Role of Core DRE2

A, Wild-type [32P]fos4. Proteins, unlabeled oligonucleotides, and [32P]fos4 were incubated and separated by gel mobility shift assays as described in Materials and Methods. The assay was carried out in triplicate, and relative band intensities compared with control (lane 2, arbitrarily set at 100) were (lanes 3–8) 283 ± 51, 188 ± 7, 368 ± 43, 24 ± 4, 333 ± 48, and 242 ± 11, respectively (means ± SE). Incubation with unprogrammed lysate in vitro-translated AhR or Arnt proteins alone did not give a retarded band (data not shown and Ref. 11). B, Mutant [32P]fos4.m. The assay was determined in duplicate as described above, and although Sp1 protein bound DNA (lane 2) and ER enhanced binding (lane 3, 3-fold), the AhR complex did not significantly modulate Sp1-DNA binding (lanes 3 and 5), and competition with unlabeled excess DRE did not decrease intensity of the Sp1-DNA complex (lane 8). These results were similar in both replicate experiments. Experiments with ER and AhR/Arnt were determined in the presence of E2 and TCDD; however, these interactions with Sp1 can be observed in the presence or absence of ligand (11 29 44 ).

 
Gel mobility shift assays with the [32P]bromodeoxyuridine (BrdU)-F2 oligonucleotide from the c-fos promoter (-1130 to -1182) and nuclear extracts from MCF-7 cells did not give a specifically bound AhR-complex (data not shown); this was consistent with previous results (38, 39, 40) indicating that the core pentanucleotide sequence was not sufficient for detecting binding of the AhR complex using this assay. In contrast, photocross-linking of nuclear extracts from MCF-7 cells with bromodeoxyuridine-substituted c-fos iDRE2 resulted in formation of a specifically bound 200-kDa cross-linked band (Fig. 4Go, lane 1). Intensity of the cross-linked band was decreased after coincubation with 100-fold excess wild-type consensus DRE (lane 2) or AhR antibody (lane 4) but unaffected by 100-fold excess mutant DRE (lane 3) or nonspecific IgG (lane 5). The competition with wild-type DRE and AhR antibodies suggest that the 200-kDa cross-linked band contained AhR/Arnt. However, effects of Arnt antibodies were not determined. These results parallel previous photoinduced cross-linking studies with functional iDREs in the cathepsin D and pS2 gene promoters (38, 39).



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Figure 4. Cross-Linking the AhR Complex to Core DRE2 (fos Gene)

[32P]BrdU-F2 (-1182 to -1131) was incubated with nuclear extracts from MCF-7 cells treated with TCDD and other oligonucleotides or antibodies, and photoinduced cross-linking was carried out as described in Materials and Methods. Formation of the 210-kDa cross-linked band (lane 1) was inhibited by coincubation with unlabeled DRE oligonucleotide (lane 2) and AhR antibodies (lane 4) but not by mutant DRE (lane 3) or nonspecific IgG (lane 5). Only AhR antibodies were used in this experiment; however, both Arnt and AhR antibodies immunoprecipitate the heterodimeric AhR complex.

 
In Vitro SssI Footprinting
Figure 5Go summarizes in vitro SssI DNA methylation footprinting of the c-fos gene promoter in the region of the distal GC-rich (-1168 to -1161) sites and the core pentanucleotide DRE motifs. SssI methylates only the 5'-cytosine of a CG pair and therefore is useful for footprinting promoters with GC-rich motifs. Incubation of pFC2-CAT with Sp1 protein alone (lane 1) did not protect DRE1 or DRE2. Moreover, coincubation with ER plus Sp1 proteins (lane 3) did not protect either site. In contrast, incubation of pFC2-CAT with ER protein alone protected DRE1 and DRE2, and protection of the former site is consistent with an imperfect palindromic ERE that overlaps DRE1 (43). In addition, an ERE half-site (-1178 to -1182) is adjacent to DRE2, and we hypothesize that the protection of DRE2 by ER alone (lane 2) may be associated with binding to this site. The failure to observe protection after coincubation of ER with Sp1 is consistent with Sp1-ER (protein-protein) binding (29) that competes with ER-DNA interactions. The transformed AhR complex alone very weakly footprinted DRE2 (lane 4), whereas DRE1 was not protected (lane 4); however, after coincubation with ER/Sp1 proteins, a greater than 2-fold increase in binding to core DRE2 was observed (lanes 5 and 6), as evidenced by a dose-dependent decrease in methylation with increasing amounts of AhR/Arnt (3U and 6U, lanes 5 and 6, respectively). This enhanced binding (decreased methylation) at core DRE2, but not DRE1, was observed in replicate experiments and demonstrates direct binding of the heterodimeric AhR complex to core DRE2.



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Figure 5. Footprinting the Proximal Region of the fos Gene Promoter by SssI DNA Methyltransferase-Mediated Methylation (60 )

A restriction fragment of pFC2-CAT was incubated with various proteins, and DNA methylation patterns in the region of DRE2 and the GC-rich site (-1168 to -1161) were determined as described in Materials and Methods. Incubation of Sp1, ER, Sp1/ER, or transformed AhR complex alone (lanes 1–4, respectively) did not decrease DNA methylation in the -1168 to -1161 region of the c-fos gene promoter. However, transformed AhR/Arnt coincubated with ER/Sp1 significantly decreased DNA methylation (lane 6) in the region of core DRE2 (~2-fold decrease), and this was observed in replicate experiments. One microliter of in vitro translated AhR and Arnt proteins was arbitrarily defined as 1 U. AhR/Arnt was transformed with TCDD before incubation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
c-fos Protooncogene expression is modulated by different endogenous and exogenous agents that act through multiple pathways. Some of these responses involve alterations of nuclear transcription factors and their interactions with proximal SRE, SIE (20, 25, 26, 27, 28), and vitamin D response elements (14) and an upstream imperfect palindromic ERE (-1212 to -1200) (43) and GC-rich motif (-1168 to -1661) (11). Interestingly, elements associated with E2 responsiveness were cell specific since the distal imperfect palindromic ERE (-1212 to -1200) was functional in HeLa cells (43), whereas ER/Sp1 interaction with the GC-rich Sp1-binding site is required for ER action in MCF-7 cells (11).

Previous studies have demonstrated that AhR agonists, typified by TCDD, inhibit E2-induced responses in the rodent uterus and mammary and in human breast cancer cells (reviewed in Refs. 36, 37). For example, TCDD inhibited spontaneous and 7,12-di-methylbenzanthracene-induced mammary tumor development and growth, and similar results were obtained in a mouse xenograft model (46, 47, 48, 49). These studies have also led to development of relatively nontoxic AhR-based compounds that have potential utility for treatment of breast cancer (50, 51). This study utilizes c-fos protooncogene as a model for understanding the molecular mechanism of ER-AhR cross-talk in breast cancer. TCDD rapidly inhibits E2-induced c-fos mRNA levels (Fig. 1AGo) and CAT activity in MCF-7 cells transiently transfected with pFC2 containing the -1400 to +41 region of the promoter (Fig. 1BGo), and results with TCDD were comparable to those observed for the direct-acting antiestrogen ICI 182,780 (Fig. 1CGo). Although TCDD totally inhibited E2-induced c-fos gene expression, the inhibitory response was lower in transient transfection experiments where ICI 182,780 was the more potent inhibitor.

The role of the AhR in mediating the inhibitory effects of TCDD on ER action was confirmed in studies using {alpha}NF, an AhR antagonist. Previous studies have demonstrated that 1 µM {alpha}NF blocks formation of the nuclear AhR complex and some of the suppressive effects of TCDD including inhibition of E2-induced cell proliferation (41). Similar results were observed in the present study where {alpha}NF blocks the effects of TCDD on E2-induced transactivation using pFC2 (Fig. 1BGo), whereas {alpha}NF does not affect the antiestrogenic activity of ICI 182,780 (Fig. 1CGo). Further confirmation for the role of a functional AhR in mediating the inhibitory effects of TCDD was obtained using BaP-resistant MCF-7 cells that express a defective nuclear AhR complex that does not bind DNA (42). E2 induces CAT activity in BaP-resistant MCF-7 cells, whereas TCDD does not inhibit this response, and this is consistent with results of previous studies showing that TCDD does not inhibit other E2-induced responses in this cell line (42).

The rapid inhibition of E2-induced fos gene expression in MCF-7 cells (Fig. 1AGo) suggests that this may be related to direct genomic effects of the AhR complex as previously reported for inhibition of E2-induced cathepsin D and pS2 expression (38, 39). In both of these studies, core iDREs (GCGTG) were identified as functional inhibitory cis-elements required for AhR-mediated activity, and this involved weak interactions with the AhR complex that could be detected only by photoinduced cross-linking studies. Two pentanucleotide GCGTG sequences at -1206 to -1202 and -1163 to -1159 were identified in the -1220 to -1155 region of the c-fos gene promoter that also contains the GC-rich region (-1168 to -1161) required for E2 responsiveness. The results illustrated in Fig. 2Go compare the antiestrogenic action of TCDD and ICI 182,780 in MCF-7 cells transfected with pF1 or constructs mutated in the upstream or downstream core DREs (pF1.d1m and pF1.d2m). All three constructs were E2 inducible, and ICI 182,70 inhibited more than 90% of the hormone-induced response. In contrast, the inhibitory effects of TCDD were observed only for wild-type pF1 and pF1.d1m, whereas no significant inhibition was observed in cells transfected with pF1.d2m. These results clearly distinguish between the direct antiestrogenic effects of ICI 182,780 and the indirect inhibitory effects of TCDD that were dependent on an intact core DRE2 that overlaps the GC-rich Sp1-binding site at -1168 to -1161.

Previous studies on the cathepsin D gene promoter have demonstrated that Sp1 and the AhR complex physically interact, and estrogen responsiveness of the -145 to -119 promoter region was dependent on an Sp1(N)4DRE (core pentanucleotide) motif (44). The GC-rich site was necessary but not sufficient for ER/Sp1 action, and hormone-induced transactivation was dependent on cooperative interactions between proteins bound to the GC-rich and core DRE sites. Subsequent studies showed that the nuclear AhR complex (in the absence of endogenous ligand) cooperatively interacted with ER/Sp1 complex or Sp1 protein to modulate hormone-induced or basal gene expression, respectively (44, 45). The cooperative ER/Sp1-AhR/Arnt interactions also decreased with increasing distance between the GC-rich and core DRE-binding sites (45). The downstream core DRE motif in the c-fos gene promoter overlaps the GC-rich sequence at -1168 to -1161 and therefore differs from the Sp1(N)4DRE(core) sequence in the cathepsin D gene promoter. Mutation of core DRE2 (to give pF1.d2m) did not significantly affect basal or E2-inducibile responses compared with those observed for pF1 (Fig. 2Go), and therefore the overlapping core DRE2 does not play a critical role in ER/Sp1 action at the GC-rich motif (-1168/-1161). In contrast, both basal and inducible responses were decreased by >85% by mutation of the core DRE in the cathepsin D-derived construct (44), suggesting that the overlap between the GC-rich and core DRE elements may have functional significance (45).

Evidence for interaction of the ligand-transformed AhR complex with the core DRE2 site was further investigated by gel mobility shift, photoinduced cross-linking, and SssI DNA methyltransferase footprinting assays. Direct binding of the AhR complex to [32P]fos4 was not observed in gel mobility shift assays (data not shown), and these results were consistent with previous studies showing that AhR/Arnt binding in this assay requires not only the core DRE but additional flanking sequence (38, 39, 40). Interactions of the 200-kDa AhR complex with core DRE2 were confirmed by cross-linking studies (Fig. 4Go) to give a specifically bound cross-linked AhR complex, and these results were comparable to cross-linking studies using core DREs from the cathepsin D and pS2 gene promoters (38, 39). In vitro SssI DNA footprinting showed consistent interactions of the transformed nuclear AhR complex with core DRE2 (but not DRE1) only in the presence of ER and Sp1 proteins (Fig. 5Go). These results are consistent with the inhibition of E2-induced responses by the liganded AhR/Arnt complex, whereas TCDD alone did not significantly affect basal responses (e.g. Fig. 1Go, B–D, and Fig. 2Go). Ongoing studies will further extend applications of the SssI DNA footprinting assay for determining site- specific binding of specific nuclear proteins and nuclear extracts from breast cancer cells to hormone-regulated gene promoters.

Previous gel mobility shift studies using 32P-labeled Sp1(N)4DRE(core) (cathepsin D gene promoter) demonstrated that although the nuclear AhR complex did not directly bind the oligonucleotide, the on-rate and Bmax value for Sp1-DNA complex formation were significantly increased after coincubation with the AhR complex (44, 45). The results in Fig. 3Go, A and B, utilizing [32P]fos4 and core DRE mutant ([32P]fos4.m) show that the transformed AhR complex also enhanced Sp1-DNA complex formation (>2-fold) using the wild-type oligonucleotide without forming a ternary supershifted complex. Enhanced Sp1 binding to [32P]fos4 by ER and AhR/Arnt was determined in the presence of E2 and TCDD, respectively; however, similar effects can be observed using other GC-rich oligonucleotides in the presence or absence of ligand (11, 29, 30, 32, 44, 45). The failure of the AhR complex to induce formation of a supershifted ternary AhR/Sp1-DNA complex is not unprecedented since it has also been reported that other nuclear proteins, including human T-cell leukemia virus, Type-I Tax, sterol-regulatory element-binding protein, and cyclin D1 enhanced binding of bZIP, Sp1, and ER to their cognate enhancer elements (52, 53, 54). Sp1-DNA binding was not enhanced by the transformed AhR complex using [32P]fos4.m (mutant DRE2), showing that enhanced binding was dependent on both the AhR complex and an intact core DRE. These results are consistent with repressors and activators that can co-occupy overlapping DNA sequences and suggests that AhR-mediated inhibition of ER/Sp1 action in the fos gene promoter may be due to quenching or masking at the distal GC-rich site (55).

Interestingly, the inhibitory core iDREs identified in the c-fos (present study), cathepsin D (38), and pS2 (39) gene promoters modulate E2-responsiveness via different pathways. The iDRE in the cathepsin D gene promoter is located between the Sp1(N)23ERE (half-site), and results of in vitro studies suggest that the nuclear AhR complex-iDRE interaction prevents formation of the functional ER/Sp1 complex. This may be due to steric interactions since the iDRE is located within the Sp1(N)23ERE (half-site) motif required for ER/Sp1-DNA complex formation. The functional iDRE in the pS2 gene promoter is required for AhR-AP-1 protein interactions that modulate ER action at a downstream imperfect palindromic ERE, and the mechanisms of these interactions are unknown (39). Thus, inhibition of E2-induced transactivation by the transformed AhR/Arnt heterodimer is complex and gene promoter specific. Moreover, recent studies in this laboratory have identified other E2-responsive genes that are also inhibited by AhR agonists via core iDRE-independent pathways (our unpublished results) and these are currently being investigated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chemicals, Cell Lines, and Oligonucleotides
MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The BaP-resistant MCF-7 (MCF-7BaPr) cell line was developed in this laboratory (42). Cells were maintained in MEM with phenol red and supplemented with FBS (Intergen, Purchase, NY)), plus 10 ml antibiotic/antimycotic solution (Sigma Chemical Co., St. Louis, MO) in an air/carbon dioxide (95:5) atmosphere at 37 C. For Northern blot analysis, cells were seeded in DMEM/F12 medium without phenol red and 5% FBS treated with dextran-coated charcoal and grown for 2–3 days. The same media without FBS were then used for at least 48 h before addition of E2, TCDD, and other chemicals. For transient transfection studies, cells were grown for 1 day in DMEM/F12 medium without phenol red and 5% FBS treated with dextran-coated charcoal. The human ER (hER) was provided by Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). pFC2-CAT plasmid, which contains the -1400 to +41 5'-flanking sequence from human c-fos gene linked to a bacterial CAT reporter gene, was kindly provided by Dr. Alessandro Weisz (Universita di Napoli, Naples, Italy) (43). TCDD was synthesized in this laboratory to >98% purity as determined by gas chromatographic analysis. E2 was purchased from Sigma Chemical Co.. [32P]dCTP (300 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA). Dimethyl sulfoxide (DMSO) was used as solvent for E2 and the antiestrogens. 4'-Hydroxytamoxifen was purchased from Sigma Chemical Co., and ICI 182,780 was provided by Alan Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK). All other chemicals and biochemicals were the highest quality available from commercial sources.

Oligonucleotides derived from the c-fos protooncogene promoter and other oligonucleotides were synthesized by the Gene Technologies Laboratory, Texas A&M University (College Station, TX). The complementary strands were annealed, and the 5'-overhangs were used for cloning. The structures of these oligonucleotides are summarized below, and the GC-rich Sp1 site is underlined. Pentanucleotide core DREs are indicated in bold letters. Mutations incorporated into mutant oligonucleotides are denoted by an asterisk. fos1 (-1220/-1155) 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG GGG CGT GGC ACG-3' fos2.d1m (-1220/-1155) 5'-AGC TTG GCT GAG CCG GCA T*A*T* TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG GGG CGT GGC ACG-3' fos1.d2m (-1220/-1155) 5'-AGC TTG GCT GAG CCG GCA GCG TGA CCC CGG CTG TCC TAC GCA GCA GGG CAG GAG ATT GGG GGG CGG* GGC ACG-3' BrdU-F2 (-1131/-1182) (antisense) 5'-TGG GGA GGC AAG GTG CTC CAG AGT GTG CCA CGC CCC CCA ATC TCC TGC CCT GCT-3' fos4 5'-AGC TTG GAG ATT GGG GGG CGT GGC ACA CG-3' fos4.m 5'-AGC TTC GAG ATT GGG GGG CGG* GGC ACA CG-3' Consensus Sp1 oligonucleotide 5'-AGC TTA TTC GAT CGG GGC GGG GCG AGC G-3' Mutant Sp1 oligonucleotide 5'-AGC TTA TTC GAT CGA* A*GC GGG GCG AGC G-3' Wild-type DRE (antisense) 5'-GAT CTC CGG TCC TTC TCA CGC AAC GCC TGG GG-3' Mutant DRE (antisense) 5'-GAT CTC CGG TCC TTC TA*C* A*T*C AAC GCC TGG GG-3' Primer sequence used for BrdU-F2 5'-AGC AGG GCA GGA GAT-3'

Cloning
The pBLTATA-CAT plasmid was made by digesting the pBLCAT2 vector with BamHI and XhoI to remove the thymidine kinase promoter; the double-stranded E1B oligonucleotide (29) containing complementary 5'-overhangs was then inserted into the corresponding sites. The fos1, fos1.d1m, and fos1.d2m oligonucleotides were cloned into the pBLTATA-CAT vector at the HindIII and BamHI sites to give the pF1, pF1.d1m, and pF1.d2m constructs, respectively, as previously described (29).

Northern Blot Analysis
Plasmids containing c-fos and ß-tubulin genes were purchased from ATCC. RNA was extracted from MCF-7 cells treated with DMSO (control), E2, and/or TCDD using the acidic guanidinium thiocyanate extraction procedure followed by separation on a 1.2% agarose gel electrophoresis and transfer to a nylon membrane. The membrane was then exposed to UV light for 5 min to cross-link RNA to the membrane and baked at 80 C for 2 h. The membrane was prehybridized in a solution containing 0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrollidone, 10% dextran sulfate, 1% SDS, and 5x SSPE (0.75 M NaCl, 50 mM NaH2PO4, 5 mM EDTA) for 18–24 h at 65 C and hybridized in the same buffer for 24 h with the 32P-labeled DNA probe (106 cpm/ml). DNA probes were labeled with [{alpha}-32P]dCTP using the random-primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). The resulting blots were quantitated using a Betagen Betascope 603 blot analyzer (Intelligenetics, Inc., Mountain View, CA) and visualized by autoradiography. c-fos mRNA levels were standardized against ß-tubulin mRNA.

Transient Transfection and CAT Assay
Cultured MCF-7 and MCF-7BaPr cells were transiently transfected utilizing the calcium phosphate method with 10 µg of the pFC2-CAT plasmid and 10 µg of wild-type hER expression plasmid. Overexpression of E2-responsive constructs containing promoter inserts from the progesterone receptor, cathepsin D, pS2, retinoic acid receptor {alpha}1, and heat shock protein 27 genes requires cotransfection with hER (11, 29, 30, 31, 32, 38, 39, 56, 57, 58, 59). After 18 h, the media were changed, and cells were treated with DMSO (0.2% total volume), 10 nM E2, 10 nM TCDD, or their combinations in DMSO for 44 h. Cells were washed with PBS and scraped from the plates. Cell lysates were prepared in 0.15 ml of 0.25 M Tris-HCl (pH 7.5) by three freeze-thaw-sonication cycles (3 min each). Protein concentrations were determined using BSA as a standard, and analysis for CAT activity in cell lysates used a constant amount of protein from each treatment group. Lysates were incubated at 56 C for 7 min to remove endogenous deacetylase activity. CAT activity was determined by incubating aliquots of the cell lysates with 0.2 mCi d-threo-[dichloroacetyl-1-14C]chloramphenicol and 4 mM acetyl-CoA. Acetylation was allowed to proceed to less than 20–25% completion (linear range), and acetylated metabolites were analyzed by TLC. After TLC, acetylated products were visualized and quantitated using a Betagen Betascope 603 blot analyzer. CAT activity was calculated as fraction of that observed in cells treated with DMSO alone (arbitrarily set at 100), and results are expressed as means ± SD. The experiments were carried out at least in triplicate. The TLC plates were subjected to autoradiography using X-Omat film ( Eastman Kodak Co., Rochester, NY).

Electrophorectic Mobility Shift Assays
Pure Sp1 protein was purchased from Promega Corp. (Madison, WI). Expression plasmids for hER, AhR, and Arnt were used to in vitro translate proteins in 1x binding buffer (20 mM HEPES, 5% glycerol, 100 mM potassium chloride, 5 mM magnesium chloride, 0.5 mM dithiothreitol, and 1 mM EDTA in a final volume of 25 µl). Equal volumes (1 µl) of lysate containing the AhR and Arnt complex were transformed with 20 nM TCDD for 2 h at 25 C. The hER (1 µl) was transformed with 20 nM E2 for 15 min on ice. Sp1 (2.5 ng) and 32P-labeled oligonucleotides (60,000 cpm) were then added to the reaction mixtures in the presence of 1 µg poly [d(I-C)] and incubated for 15 min at 25 C. In competition experiments, different amounts of unlabeled oligonucleotides were also incubated in the incubation mixtures. Aliquots of these mixtures were loaded onto a 4% polyacrylamide gel (acrylamide/bisacrylamide ratio, 30:0.8) and run at 110 V in 0.09 M Tris/0.09 M borate/2 mM EDTA (pH 8.0). [32P]DNA and DNA-protein bands were visualized by autoradiography and quantitated by densitometry using the Zero-D software package (Molecular Dynamics, Inc., Sunnyvale, CA) and a JX-330 scanner (Sharp Electronics Corp., Mahwah, NJ).

UV-DNA Cross-Linking
For cross-linking studies, 10 pmol of the synthetic oligonucleotide (c-fos-DRE) were annealed to 10 pmol of a cross-linked primer sequence. The annealed template was end-filled with the Klenow fragment of DNA polymerase in the presence of 0.1 µM dGTP, dATP, and BrdU and 1 mM [32P]dCTP as previously described (38, 39) and was designated the BrdU-substituted DRE oligonucleotide. Nuclear extracts (10 µg) from MCF-7 cells treated with appropriate chemicals were incubated with the BrdU-DRE for 15 min at 20 C after a 15-min incubation at 20 C with 400 ng of poly(dI-dC) in HEGD buffer (2.5 mM HEPES, 1.5 mM EDTA, 10% glycerol, 1.0 mM dithiothreitol, pH 7.6) for 10 min followed by a 5-min incubation at 20 C with unlabeled excess competitor. Incubation mixtures were irradiated using a UV transilluminator (Fotodyne, Inc., New Berlin, WI) at more than 205 nm for 30 min at 20 C. Samples were then mixed with 10 µl of an SDS loading buffer, heated to 95 C for 5 min, and then subjected to electrophoresis on SDS-polyacrylamide gels. Molecular weights of UV cross-linked nuclear ligand-AhR complexes were calculated from 14C-methylated standards obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). Immunodepletion of the AhR was carried out by incubating 10 µg of nuclear extract with 1 µg of either AhR antibody or nonspecific mouse IgG for 1 h at 25 C. The immunodepleted extract was used in the UV cross-linking studies as described above.

In Vitro SssI Footprinting
Fifty micrograms of plasmid pFC2-CAT (which contains the region of the human c-fos promoter from -1400 to +41) were restricted with XbaI and diluted to a concentration of 10 ng/µl. One microliter of the diluted plasmid was incubated with human recombinant Sp1 (Promega Corp.), ER proteins (PanVera Corp., Madison, WI), and in vitro translated Ah and Arnt, and transformed with TCDD, both Sp1 and ER proteins, and varying concentrations of in vitro translated and TCDD-transformed Ah and Arnt in the presence of both ER and Sp1 proteins. Binding reactions were carried out in 1x TNS binding buffer [0.02 M HEPES, 0.1 M KCl, 0.005 M MgCl2, 0.004 mM EDTA, 5% glycerol, 4% TNT lysate (Promega Corp.), 50 mM SAM] in a volume of 25 µl. The binding reactions were incubated on ice for 5 min and then equilibrated to room temperature for 20 min. Two microliters of 1:4 dilution of purified SssI (New England Biolabs, Inc., Beverly, MA) were added to the equilibrated reactions, which were then incubated at 30 C for 15 min. After 15 min at 75 C, 10 µl of freshly made deamination denaturation buffer (0.9 N NaOH, 25 mM EDTA, 0.2 mg/ml of sheared salmon sperm DNA) were added. After 5 min at 98 C, 200 µl of a saturated solution of sodium metabisulfite were added, and the samples were processed as described by Kladde and co-workers (60). The primers used to amplify from the purified deaminated plasmid DNA were cfosb1 (5'-AAACCCAAAAAATAAAAAAAAAA-AAAC-3') and cfosb2 (5'-GTTTTAGGGGTAGGGAGTGTGAG-3'). PCR products were purified and cleaned using the Wizard PCR prep kit from Promega Corp. Purified PCR products were sequenced with radiolabeled cfosb1 primer in the presence of a 5 µM solution of dATP, dCTP, and dTTP using 50 µM cddGTP as the stop nucleotide. Sequitherm 10X buffer and Sequitherm Thermostable SNA Polymerase (Epicentre Technologies, Madison, WI) were used for the sequencing reactions. Sequencing reactions were run on 5% PAGE-urea sequencing gels. The dried gels were exposed to a phosphor screen for 12 h and analyzed on a Storm 860 (Molecular Dynamics, Inc.).


    FOOTNOTES
 
Address requests for reprints to: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466.

This work was supported by the NIH Grants ES-04176 and ES-09106, the Welch Foundation, and the Texas Agricultural Experiment St ation. S. Safe is a Sid Kyle Professor of Toxicology.

Received for publication February 11, 1999. Revision received May 4, 1999. Accepted for publication May 25, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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