Molecular Mechanism of Inhibitory Aryl Hydrocarbon Receptor ...

0888-8809/06/$15.00/0 Printed in U.S.A.

Molecular Endocrinology 20(9):2199–2214 Copyright © 2006 by The Endocrine Society doi: 10.1210/me.2006-0100

Molecular Mechanism of Inhibitory Aryl Hydrocarbon Receptor—Estrogen Receptor/Sp1 Cross Talk in Breast Cancer Cells Shaheen Khan, Rola Barhoumi, Robert Burghardt, Shengxi Liu, Kyounghyun Kim, and Stephen Safe Department of Veterinary Physiology and Pharmacology (S.K., S.S.), Department of Veterinary Integrated Biology (R.B., R.B.), Texas A&M University, College Station, Texas 77843; and Institute of Biosciences and Technology (S.L., S.S.), Texas A&M University System Health Science Center, Houston, Texas 77030 The trifunctional carbamoylphosphate synthetase/ aspartate transcarbamyltransferase/dihydroorotase (CAD) gene is hormone responsive in MCF-7 and ZR-75 breast cancer cells, and this response is inhibited by the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Estrogen-dependent induction of CAD mRNA and reporter gene activity in cells transfected with constructs (pCAD) containing hormone-responsive GC-rich CAD promoter inserts involves estrogen receptor ␣ (ER␣)/Sp1 interactions with these proximal GC-rich motifs. TCDD also inhibits hormone-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD constructs. The mechanism of inhibitory AhR-ER␣/Sp1 cross talk was further investigated by chromatin immunoprecipitation (ChIP), and the results show that ER␣/Sp1 and the AhR are constitutively bound

to the CAD gene promoter and only minor changes are observed after treatment with 17␤-estradiol, TCDD, or their combination. However, examination of interactions of these transcription factors by fluorescence resonance energy transfer shows that E2 enhances ER␣-Sp1 interactions, whereas cotreatment with TCDD significantly decreases interaction of these proteins. These results suggest that inhibitory AhR-ER␣/Sp1 cross talk is due, in part, to enhanced association of AhR and ER␣ (also determined by fluorescence resonance energy transfer), which coordinately dissociates ER and Sp1 and decreases ER␣/Sp1-mediated transactivation, whereas remaining associated with the CAD promoter. This represents a novel interaction between two ligand activated receptors where one receptor inhibits activation of the second receptor. (Molecular Endocrinology 20: 2199–2214, 2006)

STROGEN RECEPTOR ␣ (ER␣) AND ER␤ are members of the nuclear receptor superfamily of transcription factors, and studies in ER knockout mice and humans show the important role for this receptor in reproductive tract development, neuronal and vascular function, and bone growth (1–6). ER expression and activation by estrogens also play pivotal roles in mammary tumor development and growth (7, 8), and early stage ER-positive breast cancer has been successfully treated with antiestrogens such as tamoxifen and other selective ER modulators (9–11). Although

tamoxifen has been extensively used in clinical applications, there is evidence that prolonged use may lead to an increased risk for endometrial cancer or development of tumors resistant to endocrine therapy (12). An alternative approach for inhibiting estrogen-dependent mammary tumor growth using ligands for the aryl hydrocarbon receptor (AhR) has been investigated in this laboratory (13, 14). For example, the AhR agonist 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF) activates inhibitory AhR-ER␣ cross talk in breast and endometrial cancer cells, the rodent uterus, and rodent mammary tumors in vivo (13, 14) and 6-MCDF significantly inhibited 7,12-dimethylbenz[a]anthracene-induced mammary tumor growth in female Sprague Dawley rats (Harlan, Houston, TX) at doses as low as 50 ␮g/kg䡠d (15). Moreover, in combination with tamoxifen, 6-MCDF synergistically inhibited mammary tumor growth in the rat model and protected against tamoxifen-induced estrogenic responses in the uterus but did not affect bone lengthening induced by tamoxifen (15). Hormone-mediated mammary tumor growth is dependent on modulation of gene expression and in breast cancer cells, AhR agonists, such as 6-MCDF or the high-affinity AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), inhibit 17␤-estradiol (E2)-

E

First Published Online May 4, 2006 Abbreviations: Ahr, Aryl hydrocarbon receptor; CAD, trifunctional carbamoylphosphate synthetase/aspartate transcarbamyltransferase/dihydroorotase; ChIP, chromatin immunoprecipitation; CYP, cyan fluorescent protein; E2, 17␤estradiol; ER, estrogen receptor; FRET, fluorescence resonance energy transfer; GAPDH, human glyceraldehyde3-phosphate dehydrogenase; iDRE, inhibitory dioxin-responsive element; 6-MCDF, 6-methyl-1,3,8-trichlorodibenzofuran; pCAD, CAD promoter plasmid; SDS, sodium dodecyl sulfate; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; YFP, yellow fluorescent protein. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

2199

2200

Mol Endocrinol, September 2006, 20(9):2199–2214

induced progesterone receptor, prolactin receptor, cathepsin D, heat shock protein 27, c-fos, pS2, and cyclin D1 mRNA and/or protein expression (16–23). Based on results of promoter analysis, one inhibitory mechanism involves direct interaction of the AhR complex with inhibitory dioxin-responsive elements (iDREs) in E2-responsive gene promoters (16, 20, 21, 23). Both E2 and TCDD induce proteasome-dependent degradation of ER␣ (24, 25) and in breast cancer cells cotreated with E2 plus TCDD, the resulting low levels of ER␣ may become limiting and thereby decrease expression of some hormone-dependent genes. In this study, we have investigated the mechanism of inhibitory AhR-ER␣ cross talk using the hormone-responsive trifunctional carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase (CAD) gene as a model (26). Hormonal activation of CAD and a number of other E2-responsive genes involved in nucleotide biosynthesis and cell cycle progression is dependent on ER␣/Sp1 interactions with GC-rich promoter sequences (27, 28). In this study, we show that TCDD inhibits hormone-induced activation of CAD mRNA levels and reporter gene activity in MCF-7 and ZR-75 breast cancer cells transfected with constructs (pCAD) containing E2-responsive regions of the proximal region of the CAD gene promoter. Using fluorescence resonance energy transfer (FRET), it was shown that both E2 and TCDD enhanced AhR-ER␣ interactions. E2 also induced interactions between ER␣ and Sp1; however, cotreatment with TCDD abrogated this effect. Results of ChIP assays of the CAD gene promoter coupled with the transactivation and FRET data demonstrate a unique model of AhR-ER␣ cross talk where the liganded AhR inhibits ER␣-Sp1 interactions and also recruits ER␣ to Ah-responsive gene promoters (e.g. CYP1A1).

RESULTS TCDD Inhibits Hormone-Induced Activation of CAD Gene Expression and pCAD Reporter Gene Activity E2 induces CAD gene expression in ZR-75 and MCF-7 breast cancer cells, and this response is mediated through interaction of ER␣/Sp1 with proximal GC-rich motifs (⫺90 to ⫹25) (26). This study uses the CAD gene as a model for investigating the mechanisms of inhibitory AhR-ER␣ cross talk in which ER␣/Sp1 is the hormone-activated transcription factor complex. Results in Fig. 1, A and B, show that E2 induced CAD mRNA levels in ZR-75 and MCF-7 cells, respectively, as previously reported (26) and hormone-induced CAD mRNA levels were inhibited in cells cotreated with E2 plus TCDD. Similar results were observed after cotreatment with the antiestrogen ICI 182,780 (data not shown). These data demonstrate inhibitory AhR-ER␣ cross talk associated with hormonal regulation of CAD

Khan et al. • Inhibitory AhR-ER␣ Cross Talk Mechanism

Fig. 1. Regulation of CAD mRNA Levels in ZR-75 (panel A) and MCF-7 (panel B) Cells ZR-75 cells (panel A) were treated with Me2SO (D), 10 nM E2 (E), or 10 nM TCDD (T), or in combination with E2 for 12 h. CAD mRNA levels were determined by Northern blot analysis as described in Materials and Methods. Using a similar approach, CAD mRNA levels were also determined in MCF-7 cells (panel B) treated with Me2SO, 10 nM E2, 10 nM TCDD, or E2 plus TCDD.

gene expression in ER-positive ZR-75 and MCF-7 cells. The inhibitory effects of TCDD on E2-induced transactivation were also investigated in ZR-75 cells transfected with constructs containing CAD gene promoter inserts. E2 induced transactivation in cells transfected with pCAD1 and pCAD2, which contains the E2responsive ⫺90 to ⫹115 and ⫺90 to ⫹25 promoter inserts. After cotreatment with E2 plus TCDD, the induced luciferase activity was significantly decreased (Fig. 2, A and B). In a similar set of experiments, ZR-75 cells were transfected with pCAD1 or pCAD2 and treated with Me2SO, 10 nM E2, E2 plus ICI 182,780, or 1 ␮M ICI 182,780 alone (Fig. 2, C and D). The results show that, like TCDD, ICI 182,780 also inhibited E2induced transactivation and the classical antiestrogen was a more effective inhibitor in the transient transfection studies. The results also show that the proximal E-box motifs are not required for E2 responsiveness or the inhibitory effects of TCDD or ICI 182,780. Moreover, these elements in the CAD promoter repress luciferase activity as previously reported (26). We also carried out a comparable set of experiments in MCF-7 cells transfected with pCAD1 or pCAD2 and treated with E2 alone or in the presence of 10 nM TCDD (Fig. 3, A and B) or ICI 182,780 (Fig. 3, C and D). The results were comparable to those observed in ZR-75 cells and both TCDD and ICI 182,780 inhibited E2-induced transactivation. Inhibitory AhR-ER␣ cross talk was also investigated in ZR-75 and MCF-7 cells transfected with pCAD1 and constructs containing mutants in critical GC-rich sites


Mol Endocrinol, September 2006, 20(9):2199–2214 2201

Fig. 2. Regulation of CAD Constructs in ZR-75 Cells ZR-75 cells were transfected with pCAD1 (panels A and C) or pCAD2 (panels B and D), treated with Me2SO, 10 nM E2, 10 nM TCDD, or E2 plus TCDD (panels A and B) or Me2SO, 10 nM E2, 1 ␮M ICI 182,780, or E2 plus ICI 182,780 (panels C and D) and luciferase activity determined as described in Materials and Methods. Results are expressed as means ⫾ SE for three replicate determinations for each treatment group and significant (P ⬍ 0.05) induction by E2 (*) or inhibition of this response by TCDD or ICI 182,780 (**) are indicated.

and a potential iDRE motif (Fig. 4A). The upstream GC-rich site nos. 1 and 2 were previously identified as the major E2-responsive motifs in the CAD gene promoter (26), and the results in Fig. 4B demonstrate that induction by E2 was enhanced in ZR-75 cells transfected with pCAD1m1 (mutated site no. 2), whereas in cells transfected with pCAD1m2, hormone inducibility was not observed, indicating that GC-rich site no. 2 was sufficient for hormone inducibility. TCDD inhibited E2-induced transactivation in cells transfected with pCAD1m1. Inhibitory AhR-ER␣ cross talk for cathepsin D, heat shock protein 27, and c-fos has been linked to direct interactions of the AhR complex with iDREs containing the core CACGC motif that binds the AhR complex (16, 20, 21, 23). The CAD promoter also contains a CACGC motif at ⫺45; however, E2 induced luciferase in ZR-75 cells transfected with pCAD1m3

(mutated DRE), and in cells cotreated with E2 plus TCDD, the induced response was significantly inhibited (Fig. 4C). Transfection of plasmids that contain the mutant iDRE and also mutations of GC-rich site no. 1 (pCAD1m4) or nos. 1 and 2 (pCAD1m5) resulted in loss of E2 responsiveness, and TCDD had no effect on this activity. These results suggest that the antiestrogenic activity of TCDD in ZR-75 cells was iDRE independent; however, this would not preclude interaction of the AhR complex with the GC-rich CAD promoter because previous studies show that the AhR interacts with both ER␣ and Sp1 (29, 30). A parallel set of experiments were carried out in MCF-7 cells transfected with pCAD1, pCAD1m1, and pCAD1m2 (Fig. 4D), pCAD1, pCAD1m3, pCAD1m4, or pCAD1m5 (Fig. 4E). Although pCAD1m1 was hormone inducible and this response was inhibited by cotreatment with TCDD

2202

Mol Endocrinol, September 2006, 20(9):2199–2214


Fig. 3. Regulation of CAD Constructs in MCF-7 Cells Cells were transfected with pCAD1 (panels A and C) or pCAD2 (panels B and D), treated with Me2SO, 10 nM E2, 10 nM TCDD, or E2 plus TCDD (panels A and B) or Me2SO, 10 nM E2, 1 ␮M ICI 182,780, or E2 plus ICI 182,780 (panels C and D). Luciferase activity was determined as described in Materials and Methods. Results are expressed as means ⫾ SE of three replicate determinations for each treatment group, and significant (P ⬍ 0.05) induction by E2 (*) or inhibition after cotreatment with TCDD or ICI 182,780 (**) are indicated.

(Fig. 4D), the magnitude of the induction response by E2 was significantly decreased, suggesting a more important role for GC-rich site no. 2 in mediating activation by E2 in MCF-7 cells. Compared with results in ZR-75 cells (Fig. 4C), hormone-induced transactivation was decreased in MCF-7 cells transfected with pCAD1m3 (Fig. 4D), suggesting that in MCF-7 cells, the CACGC sequence may also influence hormoneinduced transactivation through the GC-rich site no. 2. This observation was not unprecedented because a previous study in MCF-7 cells showed that hormoneinduced transactivation of a GC-rich motif in the cathepsin D promoter was also dependent on a proximal CACGC site (31). The pCAD constructs are clearly activated by E2 in ZR-75 and MCF-7 cells and ER␣/ Sp1 activation is a critical component of this process. However, results with the mutant constructs demonstrate that cell context also plays a role in differential activation of specific promoter elements. Despite

these differences in hormone-induced transactivation of wild-type and mutant CAD promoter constructs in ZR-75 and MCF-7 cells, inhibitory AhR-ER␣ cross talk was observed for both gene and reporter gene expression in both cell lines. Ligand-Dependent AhR Activation Inhibits ER␣Sp1 Interactions as Determined by FRET FRET has been used to study interactions of nuclear receptors and coregulator proteins or peptides in living cells (32–35), and using yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) chimeras of ER␣ and Sp1, we observed ligand-induced interactions of ER␣ with Sp1 in MCF-7 cells (36). Figure 5A illustrates the YFP/CFP chimeras used in this study in MCF-7 cells. ZR-75 cells exhibit lower transfection efficiencies and were not used for the FRET studies. In a previous report (36), it was shown that the YFP/CFP-



Fig. 4. Inhibitory AhR-ER␣ Cross Talk in Cells Treated with Wild-Type or Mutant CAD Constructs Cells Panel A, Summary of CAD constructs and their cis elements. Transfection of wild-type and mutant CAD constructs in ZR-75 (panels B and C) and MCF-7 (panels D and E) cells. Cells were transfected with the constructs, treated with Me2SO, 10 nM E2, 10 nM TCDD, or E2 plus TCDD, and luciferase activity determined as described in Materials and Methods. Results are expressed as means ⫾ SE for three replicate determinations for each treatment group and significant (P ⬍ 0.05) induction by E2 (*) or inhibition by E2 plus TCDD (**) are indicated.

ER␣ and Sp1 chimeras were functional in transactivation assays. Figure 5B summarizes the effects of Me2SO, E2, and E2 plus TCDD on distribution of transfected YFP-AhR in MCF-7 and COS-1 cells. In MCF-7

cells treated with Me2SO and E2, the AhR was detected in the cytosolic and nuclear fractions, whereas after treatment with E2 plus TCDD or TCDD alone, the receptor was localized exclusively in the nucleus and

2204 Mol Endocrinol, September 2006, 20(9):2199–2214


Fig. 5. Chimeric Expression Plasmids and Activity of YFP-AhR Cells A, Summary of chimeric constructs used for FRET studies. B, Ligand-dependent subcellular trafficking of YFP-AhR. MCF-7 or COS-1 cells were transfected with YFP-AhR, treated with Me2SO, 10 nM E2, 10 nM TCDD, or E2 plus TCDD, and localization of YFP-AhR was determined as described in Materials and Methods. C, Ah responsiveness of COS-1 cells transfected with YFP-AhR. COS-1 cells were transfected with pDRE3 and different amounts of YFP-AhR expression plasmid, treated with Me2SO or 10 nM TCDD, and luciferase activity determine as described in Materials and Methods. Results are expressed as means ⫾ SE for three replicate determinations for each treatment group and significant (P ⬍ 0.05) induction by TCDD is indicated (*). Similar results were obtained in COS-1 cells transfected with an AhR expression plasmid in pcDNA3 (data not shown). DMSO, Dimethylsulfoxide.

exhibited a punctate staining pattern. In a parallel experiment with COS-1 cells that do not expressed ER or AhR, the transfected YFP-AhR was both cytosolic/ perinuclear and nuclear in cells treated with Me2SO or E2, whereas E2 plus TCDD or TCDD alone induced formation of a nuclear AhR complex as observed in

MCF-7 cells. The functionality of the YFP-AhR chimera was also investigated in COS-1 cells treated with Me2SO or 10 nM TCDD and transfected with pDRE3 which contains three tandem consensus DREs linked to firefly luciferase (Fig. 5C). In the absence of YFPAhR, TCDD did not induce luciferase activity; however,


induction by TCDD was observed after cotransfection of YFP-AhR indicating that the chimeric YFP-AhR protein was functional. Ligand activation of the AhR complex inhibits induction of ER␣/Sp1-dependent activation of CAD gene/ gene promoter expression in MCF-7 and ZR-75 cells (Figs. 1–4), and the effects of AhR and ER␣ ligands on interactions of CFP-ER␣ and YFP-AhR in living cells were determined by FRET in MCF-7 and COS-1 cells (Fig. 6). In solvent (Me2SO)-treated MCF-7 cells transfected with YFP-AhR and CFP-ER, both receptors were primarily localized in the nucleus and this was also observed in cells treated with 10 nM E2, 10 nM TCDD, or E2 plus TCDD (Fig. 6A). Cells were pretreated with TCDD for 10 min and then treated with E2 (alone or in combination) for an additional 8 min. A

A


punctate nuclear pattern was observed in all the ligand-treated groups and was most pronounced in cells treated with E2 plus TCDD. Excitation of CFP-ER at 410 nm and emission at 488 illustrates the blue fluorescent emission of the nuclear CFP-ER. The yellow fluorescence was detected in the FRET channel at 525 nm, and this represents the CFP-YFP interaction and energy transfer. The emission intensities in the FRET channel were enhanced in the treated cells. The results in Fig. 6A also quantitate the FRET efficiencies in the various treatment groups, and there was a significant increase in FRET efficiencies in cells treated with E2, TCDD, and E2 plus TCDD. The overlay of the CFP and FRET signals shown in Fig. 6A confirm the enhanced emission observed in the FRET channel for the treatment groups. A parallel set of experiments

MCF-7 DMSO

E2

TCDD

E2+TCDD

CFP channel

*

FRET Efficiency

60

FRET channel

40

* 20 0

overlay

B

D

E

*

T

E+T

COS-1

CFP channel

FRET channel

overlay

E2

TCDD

E2+TCDD

*

100 FRET Efficiency

DMSO

*

80 60 40

*

20 0

D

E

E+T

T

Fig. 6. Ligand-Dependent Interactions of YFP-AhR and CFP-ER␣ Cells MCF-7 (A) and COS-1 (B) cells were transfected with YFP-AhR and CFP-ER␣, treated with Me2SO, 10 nM E2, 10 nM TCDD, and TCDD plus E2 where TCDD was added 10 min before treatment with E2. Representative FRET images in each treatment group were acquired after 8 min as described in Materials and Methods. FRET efficiencies in MCF-7 and COS-1 cells transfected with YFP-AhR and CFP-ER␣ were acquired from images taken from 8–18 min after treatment. For each treatment group, 10–15 images were acquired and each image contained one to five cells that were subsequently analyzed. Background signals from the images were subtracted as described in Materials and Methods, and significant (P ⬍ 0.05) induction of FRET efficiency in the various treatment groups is indicated by an asterisk. DMSO, Dimethylsulfoxide.


was also carried out in COS-1 cells that do not express endogenous AhR or ER␣. In COS-1 cells transfected with CFP-ER and YFP-AhR (Fig. 6B), the results of excitation/emission studies were similar to those observed in MCF-7 cells and FRET efficiencies were significantly increased in COS-1 cells treated with E2, TCDD, and E2 plus TCDD (Fig. 6B). Direct interactions of chimeric Sp1 and AhR proteins were not observed in the FRET assay (data not shown), and this is not unexpected due to the high molecular weights of these proteins which preclude adequate distance (1–10 nm) between the fluorophores to observe energy transfer. We therefore investigated the effects of the liganded AhR complex on hormone-dependent activation of ER␣/Sp1 in MCF-7 cells that express endogenous AhR. Cells were transfected with CFP-Sp1 and YFP-ER␣ and treated with solvent (Me2SO) control, E2, TCDD or E2 plus TCDD. Cells were pretreated with TCDD for 10 min before


addition of E2 for 8 min (Fig. 7A). Cells treated with Me2SO or TCDD exhibit low FRET efficiencies, whereas after treatment with E2, there was a significant increase in the FRET signal. This ligand-dependent increase was consistent with our recent FRET study showing ER␣-Sp1 interactions in breast cancer cells (36). However, the intensity of the E2-induced FRET emission is significantly decreased in cells treated with E2 plus TCDD, and quantitation of the FRET efficiencies summarized in Fig. 7B confirms this observation. These data indicate that the liganded AhR complex induces a rapid change in ER␣/Sp1 interactions, which correlates with the observed inhibitory AhR-ER␣ cross talk on the CAD gene/gene promoter (Figs. 1–4). We further investigated ER-AhR interactions in MCF-7 cells treated with Me2SO, E2, TCDD, and E2 plus TCDD and transfected with FLAGAhR. Cell lysates were immunoprecipitated with nonspecific IgG or FLAG antibodies and analyzed for ER␣

Fig. 7. Ligand-Dependent Interactions between YFP-ER and CFP-Sp1, Treated with Me2SO, 10 nM E2, 10 nM TCDD, or TCDD Plus E2 where TCDD Was Added 10 min before Treatment with E2 Cells Representative images in each treatment group were acquired after 8 min as described in Materials and Methods. FRET efficiencies in the various groups were determined 8–18 min after treatment. For each treatment group, 10–15 images were acquired and each image contained one to five cells that were analyzed for FRET efficiency by subtracting background signals as described in Materials and Methods. Significant (P ⬍ 0.05) induction of FRET efficiency by E2 (*) and inhibition of this response by cotreatment with TCDD (**) are indicated. DMSO, Dimethylsulfoxide.


by Western blot analysis (Fig. 8A). ER␣ was detected in IgG precipitates, and the lower levels were observed in the E2 plus TCDD treatment group. Interactions of ER␣ with the AhR were determined in the FLAG antibody immunoprecipitates in which higher levels of ER␣ were observed in the TCDD and E2 plus TCDD treatment groups. These results were consistent with the enhanced AhR-ER␣ interactions observed by FRET in MCF-7 and COS-1 cells treated with TCDD and E2 plus TCDD (Fig. 6). Levels of Sp1 protein did not show any consistent treatment-related effects in the immunoprecipitation in COS-1 cells transfected with pDRE3 and FLAG-AhR studies (data not shown). Results in Fig. 8B show that TCDD induced transactivation (compared with Me2SO), thus confirming that the FLAG-AhR chimera was functional.

Fig. 8. FLAG-AhR Interaction with ER␣ A, Coimmunoprecipitation experiments. MCF-7 cells were transfected with FLAG-AhR expression plasmid, and cells were treated with Me2SO, 10 nM TCDD, 10 nM E2, or TCDD plus E2 for 30 min, and whole cell lysates were immunoprecipitated (IP) with IgG or FLAG antibodies and then analyzed by Western blot assays for ER␣ as outlined in Materials and Methods. B, Ah responsiveness of FLAG-AhR. COS-1 cells were transfected with pDRE3 and FLAG-AhR or AhR expression (in pcDNA3) plasmids, cells were then treated with Me2SO or TCDD luciferase activity determined as described in Materials and Methods. Results are means ⫾ SE for three replicate determinations for each treatment group and significant (P ⬍ 0.05) induction by TCDD is indicated by an asterisk.


Analysis of ER␣, AhR and Other Transcription Factor Interactions with the CAD and CYP1A1 Gene Promoters in MCF-7 and ZR-75 Cells Results of the FRET experiments suggest that the AhR complex either forms a transcriptionally inactive AhR: ER/Sp1 complex where the AhR suppresses ER/Sp1 action or the AhR competitively dissociates ER␣ from interactions with Sp1 or both pathways are operative. AhR-dependent dissociation of ER␣ from Sp1 is supported, in part, by recent studies showing that treatment of cells with TCDD alone or in combination with E2 recruits the ER/AhR complex to promoters of Ahresponsive genes such as CYP1A1 (37, 38). We therefore investigated simultaneous interactions of AhR/ Arnt and ER␣ on the endogenous CAD and CYP1A1 gene promoters (Fig. 9A) using a ChIP assay. Initial studies examined interactions of ER␣, Sp1, AhR, and Arnt with the CAD gene promoter in ZR-75 cells after treatment with Me2SO, 10 nM E2, 10 nM TCDD, and E2 plus TCDD for 1 h (Fig. 9B). There was evidence that all of these transcription factors were associated with the E2-responsive (GC-rich) region of the CAD promoter in the solvent (Me2SO)-treated group and similar results were obtained in MCF-7 cells (data not shown). Arnt and Sp1 levels exhibited minimal changes in band intensities in the various treatment groups. The ER␣ band increased and decreased in cells treated with TCDD and E2 plus TCDD, respectively, and in cells treated with TCDD, there was a decrease in AhR interaction with the CAD promoter. These results show some treatment-related differences at one specific time point (1 h) and, to more accurately define AhR/ ER␣ interaction with the CAD promoter during conditions of inhibitory AhR-ER␣/Sp1 cross talk (i.e. E2 plus TCDD), we determined the time-dependent interactions of transcription factors with the CAD promoter in ZR-75 (Fig. 9C) and MCF-7 (Fig. 9D) cells cotreated with E2 plus TCDD. In ZR-75 cells, band intensities associated with Arnt and Sp1 were similar at all time points (0, 15, 60, or 120 min), whereas after 60 or 120 min, there was increase in bands associated with the AhR and a decrease in the ER␣ band. These results are similar to the 60-min ChIP assay results in the E2 plus TCDD treatment group (Fig. 9B). As a positive control for this experiment in ZR-75 cells, we also showed that treatment with TCDD plus E2 recruited AhR, Arnt, and ER␣ to the Ah-responsive region of the CYP1A1 promoter (Fig. 9C). The time-dependent recruitment of AhR, ER␣, Arnt and Sp1 to the CAD and CYP1A1 promoters were also determined in MCF-7 cells treated with E2 ⫹ TCDD (Fig. 9D). In MCF-7 cells, only minimal changes in ER␣ and AhR band intensities were observed after 120 min; however, recruitment of AhR, Arnt and ER␣ to the CYP1A1 promoter was comparable in both cell lines (Fig. 9, C and D). As a positive control for these interactions, we also compared hormone-induced changes in the interaction of ER␣ with the CAD promoter and the region of the pS2 gene promoter containing a functional ERE (20, 39). The



Fig. 9. ChIP Assay A, Promoter and primer cells. The E2-responsive GC-rich and ERE sites on the CAD and pS2 promoters and Ah-responsive sites (DREs) on the human CYP1A1 promoter and primer locations are indicated. ChIP assay on the CAD (B) and CAD/CYP1A1 (C) in ZR-75 cells and CAD/CYP1A1 (D) in MCF-7 cells. Cells were treated with various reagents for 1 h (B) or 15 min, 1 or 2 h, and analysis of proteins interacting with promoters of the CAD and CYP1A1 genes were determined as described in Materials and Methods. E, Interactions of ER␣ with the CAD and pS2 gene promoters. MCF-7 or ZR-75 cells were treated with E2 for 30 or 60 min, and analysis of proteins interacting with the E2-responsive regions of both promoters was determined by ChIP as described in Materials and Methods. F, Control binding of TFIIB. The control ChIP assay illustrates binding of TFIIB to the GAPDH promoter but not exon 1 of the CNAP1 gene (negative control). DMSO, Dimethylsulfoxide.

results (Fig. 9E) were obtained using two ER␣ antibodies and show that treatment with E2 strongly enhanced ER␣ interactions with the pS2 promoter as

previously described (40–44). In contrast, ER␣ (and Sp1) are constitutively bound to the CAD gene promoter in MCF-7 and ZR-75 cells and treatment with E2


does not markedly affect ER␣ binding to the promoter. We have observed constitutive binding of ER␣ and Sp1 to GC-rich promoters of other E2-responsive genes (data not shown), suggesting that our previous report of E2-dependent recruitment of ER␣ to the CAD promoter may be an experimental artifact (26). Figure 9F is a control experiment showing that the transcription factor TFIIB binds the human glyceraldehyde-3phosphate dehydrogenase (GAPDH) promoter but not exon 1 of the CNAP gene as previously reported. Results of the ChIP assay suggest that AhR-ER␣/Sp1 cross talk in cells cotreated with E2 plus TCDD involves ligand-induced disruption of ER␣/Sp1 by the AhR, and this is accompanied by slightly decreased ER␣ interactions with the CAD gene promoter only in ZR-75 cells, whereas ER␣ is recruited to the CYP1A1 promoter in both cell lines. It was also apparent from replicate experiments that full dissociation of ER␣ from the CAD gene promoter in ZR-75 cells was not observed, suggesting that AhR may act by both suppressing ER␣/Sp1 action and sequestering ER␣.

DISCUSSION The classical mechanism of E2-dependent induction of gene expression involves initial formation of a liganded nuclear ER homodimer that binds to consensus or nonconsensus EREs in hormone-responsive gene promoters (45, 46). The discovery of ER␤ as a second ER subtype suggest that in some cell contexts ER␤/ ER␣ heterodimers may also be functional because both proteins can interact in in vitro assays (47, 48). There is also evidence that one or more ERE half-sites alone or in cooperation with other transcription factors such as Sp1 are functional hormone-responsive motifs, and E2 also activates gene expression through interaction of ER with other DNA-bound transcription factors such as AP-1 and Sp1 (28, 49). Research in this laboratory has identified several genes involved in nucleotide synthesis and proliferation of breast cancer cells that are activated through ER␣/Sp1-mediated pathways (28). Hormone-induced transactivation can be inhibited by antiestrogens and also through cross talk with other ligand-activated receptors including the retinoic acid, vitamin D and Ah receptors (50–52). Research in this laboratory has identified selective aryl hydrocarbon receptor modulators such as 6-MCDF that are highly effective as inhibitors of rodent mammary tumor growth (13–15), and we have also focused on determining the mechanisms of inhibitory AhR-ER␣ cross talk (50). Functional iDREs have been identified in promoters of at least four E2-responsive genes (heat shock protein 27, cathepsin D, c-fos, and pS2) (16, 20, 21, 23). There is also evidence that after cotreatment of breast cancer cells with E2 plus TCDD, ER␣ levels may be limiting and thereby decrease expression of some E2-responsive genes (25).


The CAD gene is one of several hormone-responsive genes in breast cancer cells regulated by ER␣/ Sp1 (28), and this gene was used as a model to investigate inhibitory AhR-ER␣/Sp1 interactions in ZR-75 and MCF-7 cells. TCDD inhibited E2-induced CAD mRNA levels (Fig. 1) and reporter gene activity in cells transfected with pCAD constructs (Figs. 2–4). Inhibitory AhR-ER␣/Sp1 cross talk was independent of an iDRE at ⫺45 in the CAD gene promoter. It was not possible to determine whether the low levels of ER␣ in cells cotreated with E2 plus TCDD were limiting because the proteasome inhibitors, such as MG132, which block degradation of ER␣ (25) also decreased CAD mRNA levels in the presence or absence of hormone (data not shown). Thus, it is possible that decreased ER␣ levels in MCF-7 and ZR-75 cells cotreated with E2 plus TCDD may contribute to the antiestrogenic effects. Studies in this laboratory recently reported liganddependent interactions of ER␣ and Sp1 proteins in living MCF-7 and COS-1 cells using FRET (36), and these same constructs and YFP-AhR (Fig. 5) were used to investigate AhR-ER␣/Sp1 interactions. Interactions of AhR and Sp1 using the FRET assay were not observed due to the high molecular weights of both proteins (data not shown). However, E2, TCDD, and E2 plus TCDD increased FRET efficiency in MCF-7 and COS-1 cells transfected with CFP-ER and YFP-AhR (Fig. 6) demonstrating that both E2 and TCDD induce ER␣ and AhR interactions in living cells, and this complements results of other studies showing interactions of the two receptors (25, 29, 53). The molecular mechanisms of inhibitory AhR-ER␣/ Sp1 on a GC-rich E2-responsive promoter such as CAD could involve several pathways that may be solely responsible or contribute to this response. The AhR interacts not only with ER␣ but also Sp1 (30) proteins. Therefore, AhR could repress ER␣/Sp1 through formation of a transcriptionally inactive AhR/ ER␣/Sp1 complex or squelch ER␣/Sp1-mediated transactivation through competitively displacing ER␣ from binding Sp1 and forming and AhR/ER␣ complex. This latter possibility is supported by recent studies showing that in cells cotreated with E2 plus TCDD, there is an increased formation of AhR/ER␣ bound to promoter regions of Ah-responsive genes such as CYP1A1 (37, 38). Because direct interaction of chimeric Sp1 and AhR could not be directly detected by FRET due to the high molecular weights of both proteins, we investigated the effects of TCDD on ER␣Sp1 interactions in living cells by FRET (Fig. 7). Compared with cells treated with Me2SO or TCDD alone, treatment with E2 alone significantly increased FRET efficiency and this enhanced interaction of ER␣ and Sp1 was totally blocked in cells cotreated with E2 plus TCDD (Fig. 7). Disruption of hormone-activated ER␣/ Sp1 by the liganded AhR complex in living cells (Fig. 7) correlated with inhibition of ER␣/Sp1-mediated activation of CAD gene/reporter gene expression (Figs. 1–4). Thus, in cells cotreated with E2 plus TCDD, there


was increased association of AhR with ER␣ (Fig. 8), and this was coupled with decreased association of ER␣/Sp1 based on FRET efficiencies. The ligand-dependent retention and/or loss of these transcription factors on the CAD gene promoter was further investigated in a ChIP assay that compared protein-DNA interactions on the CAD and CYP1A1 gene promoters after treatment with E2 plus TCDD (Fig. 9). The time-dependent effects of cotreatment of MCF-7 and ZR-75 cells with TCDD plus E2 showed that ER␣ and AhR/Arnt were also recruited to the CYP1A1 promoter, and similar results were observed after treatment with TCDD alone (data not shown) as previously reported (37, 38). Because this was also accompanied by a slight time-dependent decrease in the interactions of ER␣ with the CAD gene promoter in ZR-75 cells, it is possible that sequestering of ER␣ by the AhR complex may contribute to inhibitory AhRER␣/Sp1 cross talk. Interactions of Arnt with the CAD promoter were similar in the presence or absence of hormone; however, it was evident that although both AhR and ER␣ binding to the CAD gene promoter was consistently increased and decreased, respectively, in ZR-75 (but not MCF-7) cells cotreated with E2 plus TCDD, these changes were small compared with E2dependent recruitment of ER␣ to the region containing the functional ERE in the pS2 gene promoter and recruitment of ER␣ to the CYP1A1 promoter in cells treated with E2 plus TCDD. Results of FRET experiments show that E2, TCDD, or their combination induced AhR-ER␣ interactions (Fig. 6), whereas treatment with E2 plus TCDD decreased ER␣-Sp1 interactions (Fig. 7). Because ER␣ and the AhR remain associated with the CAD gene promoter in ZR-75 and MCF-7 cells after treatment with E2 plus TCDD (Fig. 9), this suggests that the AhR complex represses ER␣/ Sp1-mediated transactivation by preventing formation of a transcriptionally active ER␣/Sp1 complex which remains, in part, bound to with the CAD promoter. Thus, the liganded AhR complex acts like a corepressor of hormone-activated ER␣/Sp1 due to its interaction with both proteins. Multiple mechanisms associated with inhibitory AhR-ER␣ cross talk in breast cancer cells include direct interactions of the AhR complex with iDREs in E2-responsive gene promoters and induction of proteasome-dependent degradation of ER␣ resulting in limiting levels of this protein (25, 54). It was not possible to evaluate the role of the latter pathway in AhRER␣/Sp1 cross talk because proteasome inhibitors directly inhibited CAD mRNA expression. Moreover, the proximal DRE in the CAD gene promoter was not functional (Fig. 4). Results of this study suggest a novel inhibitory mechanism where the liganded AhR disrupts formation of a transcriptionally active ER␣/Sp1 complex that remains bound to the E2-responsive region of the CAD gene promoter. ER␣/Sp1 and the AhR complexes are associated with the CAD promoter in the presence or absence of E2, TCDD or E2 plus TCDD as determined in a ChIP assay (Fig. 9). Results


of preliminary studies on interactions of corepressors/ coactivators with the CAD gene promoter suggest that their loss or recruitment does not correlate with the observed inhibitory AhR-ER␣/Sp1 cross talk (data not shown). However, FRET studies clearly demonstrate that the liganded AhR disrupts interactions of ER␣ and Sp1 and only the combination of both ChIP and FRET assay provides the necessary insights on this mechanism. These results highlight some of the early events (ⱕ2 h) associated with AhR-dependent inhibition of ER␣/Sp1 action. It is also likely that decreased gene expression is accompanied by redistribution of coactivators/corepressor interacting with the AhR/ER␣/ Sp1 complex, and these are currently being investigated. Future studies will also determine whether the mechanisms of inhibitory cross talk observed for the CAD gene promoter are similar to those for other E2-responsive genes regulated by ER␣/Sp proteins.

MATERIALS AND METHODS Cells, Chemicals, and Biochemicals MCF-7, ZR-75, and COS-1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). MCF-7 and COS-1 cells were cultured in DME/F12 (Sigma Chemical Co., St. Louis, MO) media supplemented with 5% fetal bovine serum (Intergen, Des Plains, IA; JRH Biosciences, Lenexa, KS; or Atlanta Biologicals, Inc., Norcross, GA), 1.5 g/liter sodium bicarbonate, and 10 ml/liter antibiotic-antimycotic solution (Sigma). ZR-75 cells were maintained in RPMI 1620 medium with phenol red and supplemented with 10% fetal bovine serum, 1.5 g/liter sodium bicarbonate, 2.38 g/liter HEPES, 4.5 g/liter dextrose, and 0.11 g/liter sodium pyruvate. Cells were maintained in 37 C incubators under humidified 5% CO2: 95% air. Dimethyl sulfoxide (Me2SO), PBS, and E2 were purchased from Sigma. MG132 was purchased from Calbiochem (EMD Biosciences, Inc., San Diego, CA). TCDD was prepared in this laboratory and was shown to be more than 99% pure by gas chromatographic analysis. ICI 182,780 was provided by Alan Wakeling (AstraZeneca Pharmaceuticals, Macclesfield, UK), and restriction enzymes, T4 DNA ligase, Reporter lysis buffer, and Luciferase Assay Reagent were purchased from Promega Corp. (Madison, WI) and/or Roche Molecular Biochemicals (Indianapolis, IN). ␤-Galactosidase activity was measured using Tropix Galacto-Light Plus Assay System (Tropix, Bedford, MA). Instant Imager and Lumicount microwell plate reader were purchased from Packard Instrument Co. (Downers Grove, IL). Anti-FLAG M2 antibody was purchased from Stratagene (La Jolla, CA), and all other antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Plasmids, Oligonucleotides, and Cloning Human ER␣ expression plasmid was originally provided by Dr. Ming-Jer Tsai (Baylor College of Medicine, Houston, TX) and was recloned into pcDNA3 in this laboratory. Basic pGL2 luciferase plasmid was purchased from Promega DRE-luciferase reporter construct was constructed in this laboratory and contains three tandem consensus DREs. PCI/hAhRFLAG and pcDNA3/hArnt-FLAG were kindly provided by Dr. Gary Perdew (Pennsylvania State University, State College, PA). All primers and oligonucleotides were prepared either by Genosys/Sigma (The Woodlands, TX) or Integrated DNA


Technologies (IDT, Coralville, IA) or Promega Corp. CAD promoter luciferase constructs pCAD1 (⫺90/⫹115) and pCAD2 (⫺90/⫹25) were kindly provided by Dr. Peggy Farnham (University of Wisconsin, Madison, WI). CAD promoter fragments were amplified by PCR as double-stranded DNA and inserted into the pGL2 basic luciferase reporter plasmid (Promega Corp.) vector between NheI and HindIII polylinker sites. pCAD1m1, pCAD1m2 and pCAD1m3 were made using pCAD1 as the template; pCAD1m4 was made using pCAD1m1 as the template, and pCAD1m5 was made using pCAD1m2 as the template. Forward primers used for PCR mutagenesis are listed in Table 1. pCADm-rev: 5⬘-CCA ACA GTA CCG GAA TGC CAA GCT TAC TTA GAT-3⬘ was the reverse primer used for PCR mutagenesis of all mutant constructs. All ligation products were transformed into competent Escherichia coli cells, and clones were confirmed by DNA sequencing (Gene Technologies Laboratory, Texas A&M University). Plasmid preparation kits were purchased from QIAGEN (Valencia, CA) or Bio-Rad Laboratories (Hercules, CA). pECFP-C1 and pEYFP-C1 mammalian expression vectors were obtained from BD Biosciences CLONTECH Laboratories, Inc. (Palo Alto, CA). CFP-hER␣, CFP-YFP chimera, CFP-Sp1, and YFP-hER␣ were constructed by Dr. Kyounghyun Kim in this laboratory (36). Full-length YFP-AhR was constructed using forward primer 5⬘-CAG TAG ATC TAT GAA CAG CAG CAG CGC CAA CAT-3⬘ and reverse primer 5⬘-CAG TGT CGA CTT ACA GGA ATC CAC TGG ATG TCA A-3⬘ and cloned in between BglII and Sal1 sites of pEYFP-C1 plasmid.


Transient Transfection Assays Cells were seeded in DMEM/F12 supplemented with 2.5% charcoal-stripped serum overnight in 12-well plates. Transfection was carried out either using calcium phosphate-DNA coprecipitation method or Fugene 6 transfection reagent (Roche) or lipofectamine 2000 (Invitrogen, Carlsbad, CA). The reporter plasmids were cotransfected with ER␣ expression vector using the calcium phosphate method for 5–6 h. After 5–6 h, cells were shocked with 0.5 ml of 25% glycerol for 1 min in a 12-well plate. Cells were then dosed for 36–48 h with different treatments. Transfection using Fugene 6 or lipofectamine 2000 was carried out according to the manufacturer’s protocol in absence of antibiotics in serum-free media. After 4 h, serum was then added or serum-free media were replaced with media containing 5% charcoal-stripped serum. Cells were then treated with Me2SO or other treatments. After 24–36 h, cells were washed with PBS and then harvested in 100 ␮l of cell lysis buffer (Promega Corp.). Cell lysate was frozen in liquid nitrogen for 30 sec and then thawed at room temperature in a water bath. Freeze-thaw cycles were repeated three times, and the samples were vortexed, centrifuged at 10,000 ⫻ g for 1 min, and supernatants were transferred to fresh tubes. Luciferase activities in the various treatment groups were performed on 20 ␮l of cell extract using the luciferase assay system (Promega Corp.) in a luminometer (Packard Instrument Co., Meriden, CT), and results were normalized to ␤-galactosidase enzyme activity that was also performed on 20 ␮l of cell extract. Coimmunoprecipitation

Northern Blot Analysis Cells were seeded in DMEM/F12 supplemented with 2.5% charcoal-stripped serum and then synchronized in serumfree media for 3 d. Cells were treated with various compounds for different time periods and RNA was extracted using RNAzol B (Tel-Test), following the manufacturer’s protocol; 15–20 ␮g of RNA were separated on a 1.2% agarose/1 M formaldehyde gel, and transferred onto nylon membrane for 48 h. RNA was cross-linked by exposing the membrane to UV light for 10 min, and the membrane was baked at 80 C for 2 h. The membrane was then prehybridized for 18 h at 60 C using ULTRAhyb-Hybridization Buffer (Ambion, Austin, TX) and hybridized in the same buffer for 24 h with the [␥32P]labeled cDNA probe. CAD cDNA probe used in this study has been described previously (26). The membrane was then washed in 2⫻ SSC [0.3 M sodium chloride, 0.03 M sodium citrate (pH 7)], and 0.5% sodium dodecyl sulfate (SDS) for 1 h, and then washed in 2⫻ SSC for 6–8 h. The resulting blots were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). ␤-Tubulin mRNAs were used as an internal control to normalize CAD mRNA levels.

MCF-7 cells were seeded in 10-mm plates in DMEM/F12 supplemented with 2.5% charcoal-stripped serum and were grown for 2 d. Cells were transfected with 3 ␮g of AhR-FLAG using Fugene 6 per the manufacturer’s protocol for 18 h and then treated with different compounds for 20 min. Cells were washed with ice-cold PBS (2⫻) and cell lysates were prepared in 800 ␮l of lysis buffer [50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, and protease inhibitor mixture (Sigma)] on ice for 45 min with intermittent vortexing followed by centrifugation at 13,000 ⫻ g for 15 min. Protein (500 ␮g) for each sample was used for immunoprecipitation; 20 ␮l of EZview Red ANTI-FLAG M2 agarose affinity gel (Sigma Chemical Co.) was added to the protein samples and immunoprecipitation was carried out for 6 h at 4 C on a rocking platform. In control samples, 1 ␮g of mouse IgG was added for 5 h followed by addition of 15 ␮l proteinL-agarose beads (Santa Cruz Biotechnology) for 1 h. The beads were washed with the lysis buffer (5⫻) for 15 min at 4 C followed by centrifugation at 8000 ⫻ g for 30 sec. After the final wash, beads were resuspended in 30 ␮l of 1⫻ sample buffer [50 mM Tris-HCl, 2% SDS, 0.1% bromphenol blue, 175

Table 1. Primers for pCAD Constructs Containing Point Mutations Plasmids

pCAD1m1 pCAD1m2 pCAD1m3 pCAD1m4 pCAD1m5

Primers Used

pCAD1m1-fwd: 5⬘-CAG TGC TAG CCA ACC TCA C-3⬘ pCAD1m2-fwd: 5⬘-CAG TGC TAG CCA ACC TCA C-3⬘ pCAD1m3-fwd: 5⬘-CAG TGC AGC GCC CCT AAA ACC GCC TG-3⬘ pCAD1m4-fwd: 5⬘-CAG TGC TAG CCA ACC TAA AACCGC CTG-3⬘ pCAD1m5-fwd: 5⬘-CAG TGC TAG CCA ACC TAA AACCGC CTG-3⬘

CCC GTG GCT CCG CGG ACC CGC CCC TTA CGT GCC CGG CCC CCC GTG GCT CCG CGG ACC CCA ACC TTA CGT GCC CGG CCC CCG TGG CTC CGC GGA CCC GCC CCT TAC GTG CCC GGC CCC CCC GTG GCT CCG CGG ACC CGC CCC TTA CGT GCC CGG CCC CCC GTG GCT CCG CGG ACC CCA ACC TTA CGT GCC CGG CCC

fwd, Forward primers; the reverse primers for all constructs are given in Materials and Methods.


mM ␤-mercaptoethenol), boiled for 5 min, separated on 7.5– 10% SDS-PAGE gel for 5 h at 130 V, and Western blot analysis was performed. Western Blot Analysis Protein samples were boiled in 1⫻ sample buffer for 5 min, separated on 7.5–10% gel, and then transferred to polyvinylidene difluoride membrane (Bio-Rad) overnight at 30 V. Membranes were blocked in Blotto—5% milk, Tris-buffered saline [10 mM Tris-HCl (pH 8.0), 150 mM NaCl], and 0.05% Tween 20—for 30 min and probed with primary antibodies ER␣ (H184), Sp1 (PEP2) or FLAG (M2) for 2–4 h. Membranes were washed for 30 min in 1⫻ Tris-buffered saline-Tween, probed with peroxidase-conjugated secondary antibody for 1–2 h, and then washed in 1⫻ Tris-buffered saline-Tween for 30 min. Ten milliliters of horseradish peroxidase substrate (Dupont-NEN, Boston, MA) were added and incubated for 1 min and visualized by autoradiography. Quantitation was performed using a Sharp JX-330 scanner (Sharp Electronics, Mahwah, NJ) and Zero-D Scanalytics software (Scanalytics Corp., Sunnyvale, CA).


based on data collected from each experiment. Cells that did not match the selection criteria were eliminated from the FRET analysis. Negative (CFP empty and YFP empty) and positive (CFP-YFP chimera) controls were used to calculate the approximate FRET efficiency in cells treated with different ligands; it was assumed that the signal from cells transfected with the positive CFP-YFP chimera construct would exhibit 50% FRET efficiency when compared with signals from cells transfected with CFP/YFP empty constructs. For identification of Region of Interest (ROI) and FRET analysis, MetaMorph software version 6.0 was used (Universal Imaging Corp., Downingtown, PA). Acceptor signal acquired with the FRET channel was corrected by subtracting the background signal as well as the donor bleed through signal. Fifteen to 20 images were collected from each sample and one to five cells per image captured were analyzed. Two to four experiments per each combination of transfected fusion constructs were conducted on different days. Student’s t test was used to analyze the statistical significance between control and ligand-treated cells at P ⬍ 0.05, and this analysis was performed using Prism software version 4.0 (GraphPad Software Inc., San Diego, CA). ChIP Assay

FRET To perform FRET, cells were seeded in two-well Labtek chamber slides without cover (Nalge Nunc International, Rochester, NY) in DMEM/F12 supplemented with 5% charcoal-stripped serum and grown for 36 h. Cells were then transfected with 750 ng of CFP-Sp1, 500 ng CFP-ER␣, 500 ng YFP-AhR, or 500 ng of YFP-ER␣ alone or in combination using Fugene 6 in absence of serum. After 4 h, 5% charcoalstripped serum was added, and after 14–18 h, cells were washed with PBS and then put on the stage of the Bio-Rad 2000MP system equipped with a Nikon T#300 inverted microscope with a ⫻60 (NA1.2) water immersion objective lens and a Titanium:Saphire pulsed laser tuned to 820 nm wavelength and a continuous wavelength Argon/Krypton laser tuned to 488 nm excitation (Table 2). Cells were pretreated with TCDD for 15 min, and images were acquired between 8 and 18 min after addition of Me2SO or E2. Images of cells transfected with CFP and YFP fusion construct alone or in combination, were collected using 2 photon-820 nM excitation wavelength. Both CFP and YFP were excited at 820 nm to effectively generate 410 nm for excitation of the CFP and FRET channels. Emission of CFP (CFP channel; donor signal) was collected using a 500 dichroic longpass filter and 450 nm /80 nm filter, whereas emission of YFP (FRET channel; acceptor signal) was collected using a 528 nm/50 nm filter. Donor bleed through signal to the FRET channel was calculated by measuring the FRET channel signal resulting from MCF-7 cells transfected only with the CFP fusion construct. Acceptor bleed through the FRET channel was calculated by measuring the FRET channel signal resulting from MCF-7 cells transfected with the YFP fusion construct alone. For determination of YFP-hER␣ localization in the YFP channel, YFP-hER␣ was excited at 488 nm with Argon/Krypton laser and emission of YFP-hER␣ was collected at 525/50 nm as described (36). To correct for variations in fluorophore expression resulting from different transfection efficiencies, minimum levels of YFP expression and maximum levels of CFP were selected

ZR-75 and MCF-7 cells (1 ⫻ 107) were treated with Me2SO (at time 0), E2 (10 nM), TCDD (10 nM), or TCDD ⫹ E2 (TE, 10 nM of each) for several time points. Cells were then fixed with 1.5% formaldehyde for 5 min, and the cross-linking reaction was stopped by addition of 125 mM glycine for 5 min. After washing twice with PBS, cells were scraped and pelleted. Collected cells were hypotonically lysed [5 mM piperazineN,N⬘-bis(2-ethanesulfonic acid) (pH 8.0), 85 mM KCl, 0.5% CA-630, plus protease inhibitors], and nuclei were collected by centrifugation, then dissolved in sonication buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)] and sonicated to desired chromatin length (500 bp ⬃ 1 kb). The chromatin was precleared by addition of protein A-conjugated beads (Pierce, Rockford, IL), and then incubation at 4 C for 1 h with gentle agitation. The beads were pelleted, and the precleared chromatin supernatants were immunoprecipitated with antibodies (1 ⬃ 2 ␮g per ChIP) specific to IgG, Sp1, ER␣, AhR, and Arnt (all from Santa Cruz Biotechnology) at 4 C overnight. The protein-antibody complexes were collected by addition of 5 ␮l of protein A-conjugated beads at room temperature for 1 h. The beads were extensively washed by low-salt wash buffer [0.1% SDS, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0)], high-salt buffer (500 mM NaCl instead), LiCl buffer [1% CA-630, 1% sodium deoxycholate, 250 mM or 500 mM LiCl, 1 mM EDTA, 100 mM TrisHCl (pH 8.0)], and TE buffer [0.1% Tween 20, 0.1% SDS, 2 mM EDTA, 50 mM Tris-HCl (pH 8.0)]. The protein-DNA crosslinks were eluted (1% SDS, 50 mM NaHCO3, 1.5 ␮g/ml of salmon sperm DNA) and reversed (5 ␮l of 5 N NaCl, 2 ␮l 10 ␮g/␮l ribonuclease for 100 ␮l eluent) at 65 C for 5–6 h. DNA was purified by Qiaquick Spin Columns (QIAGEN) followed by PCR amplification. The CAD primers are: 5⬘-CTT GGG GTG GGA GGG ACT-3⬘ (forward), and 5⬘-GCG GCA GCA GCA GAG ACT-3⬘ (reverse), which amplify a 158-bp region of the human CAD promoter containing GC-rich area. The CYP1A1 primers are: 5⬘-CAC CCT TCG ACA GTT CCT CTC-3⬘ (forward), and 5⬘-GCT AGT GCT TTG ATT GGC AGA G-3⬘ (reverse), which amplify a 381-bp region of human CYP1A1

Table 2. Excitation and Emission of Fluorescent Proteins Channel

Excitation (nm)

Emission (nm)

Laser

CFP channel FRET channel YFP channel

820 820 488

488 525 525

Titanium:Saphire Titanium:Saphire Argon/Krypton


enhancer containing DREs. The positive control primers are: 5⬘-TAC TAG CGG TTT TAC GGG CG-3⬘ (forward), and 5⬘TCG AAC AGG AGG AGC AGA GAG CGA-3⬘ (reverse), which amplify a 167-bp region of GAPDH gene. The negative control primers are: 5⬘-ATG GTT GCC ACT GGG GAT CT-3⬘ (forward), and 5⬘-TGC CAA AGC CTA GGG GAA GA-3⬘ (reverse), which amplify a 174-bp region of genomic DNA between the GAPDH gene and the CNAP1 gene. PCR products were resolved on a 2% agarose gel in the presence of 1:10 000 SYBR gold (Molecular Probes, Eugene, OR). Statistical Analysis Experiments were repeated two or more times, and data are expressed as mean ⫾ SE for at least three replicates for each treatment group. Statistical differences between treatment groups were determined using Super ANOVA and Scheffe´’s test. Treatments were considered significantly different from controls if P ⬍ 0.05.

Acknowledgments Received March 1, 2006. Accepted April 28, 2006. Address all correspondence and requests for reprints to: Stephen Safe, Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, Texas 77843-4466. E-mail: [email protected]. The financial assistance of the National Institutes of Health (ES09106 and CA76636), the Department of the Defense (DAMD17-03-1-0341), and the Texas Agricultural Experiment Station is gratefully acknowledged. Disclosure Statement: The authors have no conflicts of interest.

REFERENCES 1. Clark JH 1998 Female reproduction and toxicology of estrogens. In: Korach KS, ed. Reproductive and developmental toxicology. New York: Marcel Dekker; 259–275 2. Turner RT, Riggs BL, Spelsberg TC 1994 Skeletal effects of estrogen. Endocr Rev 15:275–300 3. Couse JF, Korach KS 1999 Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 20:358–417 4. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ␤. Proc Natl Acad Sci USA 95:15677–15682 5. Chow J, Tobias JH, Colston KW 1992 Estrogen maintains trabecular bone volume in rats not only by suppression of bone resorption but also by stimulation of bone formation. J Clin Invest 89:74–78 6. Farhat MY, Lavigne MC, Ramwell PW 1996 The vascular protective effects of estrogen. FASEB J 10:615–624 7. Clemons M, Goss P 2001 Estrogen and the risk of breast cancer. N Engl J Med 344:276–285 8. Hulka BS, Liu ET, Lininger RA 1994 Steroid hormones and risk of breast cancer. Cancer 74:1111–1124 9. MacGregor JI, Jordan VC 1998 Basic guide to the mechanisms of antiestrogen action. Pharmacol Rev 50: 151–196 10. Jordan VC 2003 Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 1. Receptor interactions. J Med Chem 46:883–908 11. Jordan VC 2003 Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 2. Clinical considerations and new agents. J Med Chem 46:1081–1111


12. Clarke R, Leonessa F, Welch JN, Skaar TC 2001 Cellular and molecular pharmacology of antiestrogen action and resistance. Pharmacol Rev 53:25–71 13. Safe S 2005 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related environmental antiestrogens: characterization and mechanism of action. In: Naz RK, ed. Endocrine disruptors. 2nd ed. Boca Raton, FL: CRC Press; 249–287 14. Safe S, McDougal A, Gupta MS, Ramamoorthy K 2001 Selective Ah receptor modulators (SAhRMs): progress towards development of a new class of inhibitors of breast cancer growth. J Women’s Cancer 3:37–45 15. McDougal A, Wormke M, Calvin J, Safe S 2001 Tamoxifen-induced antitumorigenic/antiestrogenic action synergized by a selective Ah receptor modulator. Cancer Res 61:3901–3907 16. Krishnan V, Porter W, Santostefano M, Wang X, Safe S 1995 Molecular mechanism of inhibition of estrogeninduced cathepsin D gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. Mol Cell Biol 15:6710–6719 17. Harper N, Wang X, Liu H, Safe S 1994 Inhibition of estrogen-induced progesterone receptor in MCF-7 human breast cancer cells by aryl hydrocarbon (Ah) receptor agonists. Mol Cell Endocrinol 104:47–55 18. Zacharewski TR, Bondy KL, McDonell P, Wu ZF 1994 Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-pdioxin on 17␤-estradiol-induced pS2 expression. Cancer Res 54:2707–2713 19. Lu Y-F, Sun G, Wang X, Safe S 1996 Inhibition of prolactin receptor gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 human breast cancer cells. Arch Biochem Biophys 332:35–40 20. Gillesby B, Santostefano M, Porter W, Wu ZF, Safe S, Zacharewski T 1997 Identification of a motif within the 5⬘-regulatory region on pS2 which is responsible for Ap1 binding and TCDD-mediated suppression. Biochemistry 36:6080–6089 21. Porter W, Wang F, Duan R, Qin C, Castro-Rivera E, Safe S 2001 Transcriptional activation of heat shock protein 27 gene expression by 17␤-estradiol and modulation by antiestrogens and aryl hydrocarbon receptor agonists: estrogenic activity of ICI 164,384. J Mol Endocrinol 26: 31–42 22. Wang W, Smith R, Safe S 1998 Aryl hydrocarbon receptor-mediated antiestrogenicity in MCF-7 cells: modulation of hormone-induced cell cycle enzymes. Arch Biochem Biophys 356:239–248 23. Duan R, Porter W, Samudio I, Vyhlidal C, Kladde M, Safe S 1999 Transcriptional activation of c-fos protooncogene by 17␤-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition. Mol Endocrinol 13:1511–1521 24. Wormke M, Stoner M, Saville B, Safe S 2000 Crosstalk between estrogen receptor ␣ and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteosomes. FEBS Lett 478:109–112 25. Wormke M, Stoner M, Saville B, Walker K, Abdelrahim M, Burghardt R, Safe S 2003 The aryl hydrocarbon receptor mediates degradation of the estrogen receptor ␣ through activation of proteasomes. Mol Cell Biol 23:1843–1855 26. Khan S, Abdelrahim M, Samudio I, Safe S 2003 Estrogen receptor/Sp1 complexes are required for induction of CAD gene expression by 17␤-estradiol in breast cancer cells. Endocrinology 144:2325–2335 27. Abdelrahim M, Samudio I, Smith R, Burghardt R, Safe S 2002 Small inhibitory RNA duplexes for Sp1 mRNA block basal and estrogen-induced gene expression and cell cycle progression in MCF-7 breast cancer cells. J Biol Chem 277:28815–28822 28. Safe S, Kim K 2004 Nuclear receptor-mediated transactivation through interaction with Sp proteins. Prog Nucleic Acid Res Mol Biol 77:1–36 29. Klinge CM, Bowers JL, Kulakosky PC, Kamboj KK, Swanson HI 1999 The aryl hydrocarbon receptor (AHR)/


30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements. Mol Cell Endocrinol 157:105–119 Kobayashi A, Sogawa K, Fujii-Kuriyama Y 1996 Cooperative interaction between AhR䡠Arnt and Sp1 for the druginducible expression of CYP1A1 gene. J Biol Chem 271: 12310–12316 Wang F, Hoivik D, Pollenz R, Safe S 1998 Functional and physical interactions between the estrogen receptor-Sp1 and the nuclear aryl hydrocarbon receptor complexes. Nucleic Acids Res 26:3044–3052 Tamrazi A, Carlson KE, Daniels JR, Hurth KM, Katzenellenbogen JA 2002 Estrogen receptor dimerization: ligand binding regulates dimer affinity and dimer dissociation rate. Mol Endocrinol 16:2706–2719 Bai Y, Giguere V 2003 Isoform-selective interactions between estrogen receptors and steroid receptor coactivators promoted by estradiol and ErbB-2 signaling in living cells. Mol Endocrinol 17:589–599 Llopis J, Westin S, Ricote M, Wang Z, Cho CY, Kurokawa R, Mullen TM, Rose DW, Rosenfeld MG, Tsien RY, Glass CK, Wang J 2000 Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription. Proc Natl Acad Sci USA 97:4363–4368 Weatherman RV, Chang CY, Clegg NJ, Carroll DC, Day RN, Baxter JD, McDonnell DP, Scanlan TS, Schaufele F 2002 Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer. Mol Endocrinol 16:487–496 Kim K, Barhoumi R, Burghardt R, Safe S 2005 Analysis of estrogen receptor ␣-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer. Mol Endocrinol 19:843–854 Beischlag TV, Perdew GH 2005 ER␣-AHR-ARNT proteinprotein interactions mediate estradiol-dependent transrepression of dioxin-inducible gene transcription. J Biol Chem 280:21607–21611 Matthews J, Wihlen B, Thomsen J, Gustafsson JA 2005 Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor ␣ to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters. Mol Cell Biol 25:5317–5328 Berry M, Nunez A-M, Chambon P 1989 Estrogen-responsive element of the human pS2 gene is an imperfectly palindromic sequence. Proc Natl Acad Sci USA 86:1218–1222 Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor dynamics and sufficiency in estrogen receptorregulated transcription. Cell 103:843–852


41. Shao W, Halachmi S, Brown M 2002 ERAP140, a conserved tissue-specific nuclear receptor coactivator. Mol Cell Biol 22:3358–3372 42. Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M, Gannon F 2003 Estrogen receptor-␣ directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751–763 43. Acevedo ML, Lee KC, Stender JD, Katzenellenbogen BS, Kraus WL 2004 Selective recognition of distinct classes of coactivators by a ligand-inducible activation domain. Mol Cell 13:725–738 44. Krieg AJ, Krieg SA, Ahn BS, Shapiro DJ 2004 Interplay between estrogen response element sequence and ligands controls in vivo binding of estrogen receptor to regulated genes. J Biol Chem 279:5025–5034 45. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145–156 46. O’Malley BW 2005 A life-long search for the molecular pathways of steroid hormone action. Mol Endocrinol 19: 1402–1411 47. Cowley SM, Hoare S, Mosselman S, Parker MG 1997 Estrogen receptors ␣ and ␤ form heterodimers on DNA. J Biol Chem 272:19858–19862 48. Pace P, Taylor J, Suntharalingam S, Coombes RC, Ali S 1997 Human estrogen receptor ␤ binds DNA in a manner similar to and dimerizes with estrogen receptor ␣. J Biol Chem 272:25832–25838 49. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ER␣ and ER␤ at AP1 sites. Science 277:1508–1510 50. Safe S, Wormke M 2003 Inhibitory aryl hydrocarbonestrogen receptor a crosstalk and mechanisms of action. Chem Res Toxicol 16:807–816 51. Eisman JA, Barkla DH, Tutton PJ 1987 Suppression of in vivo growth of human cancer solid tumor xenografts by 1,25-dihydroxyvitamin D3. Cancer Res 47:21–25 52. Muller P, Kietz S, Gustafsson JA, Strom 2002 A The anti-estrogenic effect of all-trans-retinoic acid on the breast cancer cell line MCF-7 is dependent on HES-1 expression. J Biol Chem 277:28376–28379 53. Ohtake F, Takeyama K, Matsumoto T, Kitagawa H, Yamamoto Y, Nohara K, Tohyama C, Krust A, Mimura J, Chambon P, Yanagisawa J, Fujii-Kuriyama Y, Kato S 2003 Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature 423: 545–550 54. Hoivik D, Willett K, Wilson C, Safe S 1997 Estrogen does not inhibit 2,3,7,8-tetraclorodibenzo-p-dioxin-mediated effects in MCF-7 and Hepa 1c1c7 cells. J Biol Chem 272:30270–30274

Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.