The Aryl Hydrocarbon Receptor Mediates Degradation of Estrogen Receptor {alpha} through Activation of Proteasomes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbonreceptor (AhR) ligands suppress 17ß-estradiol (E)-inducedresponses in the rodent uterus and mammary tumors and in humanbreast cancer cells. Treatment of ZR-75, T47D, and MCF-7 humanbreast cancer cells with TCDD induces proteasome-dependent degradationof endogenous estrogen receptor ${alpha}$ (ER ${alpha}$ ). The proteasome inhibitorsMG132, PSI, and PSII inhibit the proteasome-dependent effectsinduced by TCDD, whereas the protease inhibitors EST, calpaininhibitor II, and chloroquine do not affect this response. ER ${alpha}$ levels in the mouse uterus and breast cancer cells were significantlylower after cotreatment with E plus TCDD than after treatmentwith E or TCDD alone, and our results indicate that AhR-mediatedinhibition of E-induced transactivation is mainly due to limitinglevels of ER ${alpha}$ in cells cotreated with E plus TCDD. TCDD aloneor in combination with E increases formation of ubiquitinatedforms of ER ${alpha}$ , and both coimmunoprecipitation and mammalian two-hybridassays demonstrate that TCDD induces interaction of the AhRwith ER ${alpha}$ in the presence or absence of E. In contrast, E doesnot induce AhR-ER ${alpha}$ interactions. Thus, inhibitory AhR-ER ${alpha}$ crosstalk is linked to a novel pathway for degradation of ER ${alpha}$ in whichTCDD initially induces formation of a nuclear AhR complex whichcoordinately recruits ER ${alpha}$ and the proteasome complex, resultingin degradation of both receptors.

INTRODUCTION

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Introduction

Estrogenic hormones induce their tissue-specific responses throughbinding the estrogen receptor (ER), which is a ligand-activatedtranscription factor and a member of the nuclear receptor (NR)superfamily (3, 15). The two ER subtypes (ER ${alpha}$ and ERß)and other NRs exhibit modular structures containing N-terminalactivation function 1 (AF1) and C-terminal AF2, which also containsthe ligand-binding domain, a DNA-binding domain (DBD), and anadjacent hinge region. Most early-stage mammary tumors are ERpositive and are responsive to endocrine therapies which targetER ${alpha}$ and/or E biosynthesis (5, 13, 47). Selective ER modulators,such as tamoxifen, are extensively used for treating early-stagebreast cancer, and the primary modes of action of selectiveER modulators involve competitive binding to the ER and subsequentinhibition of one or more steps in ER-mediated transactivation.Several studies show that there are important mechanistic differencesamong antiestrogens, and this is consistent with their tissue-specificER antagonist-agonist activities (20, 52, 53). For example,the "pure" antiestrogens ICI 164,384 and/or ICI 182,780 notonly bind ER ${alpha}$ with high affinity but induce a rapid proteasome-dependentdegradation of the receptor, and this is observed in breastcancer cells and human tumors (41, 52). Rapid degradation ofER ${alpha}$ protein in cells or tumors treated with ICI 164,384 or 182,780may play an important role in the antiestrogenic activity ofthese compounds.

Studies in this laboratory have investigated inhibition of ERsignaling through cross talk with the ligand-activated arylhydrocarbon receptor (AhR) (reviewed in references 36 and 44),and selective AhR modulators are highly effective inhibitorsof mammary tumor growth in rodent models (32, 44). The environmentaltoxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potentAhR agonist, and treatment of hepatoma cells with TCDD inducesrapid proteasome-dependent degradation of the AhR (7, 30, 35,40). Similar results have been observed in T47D and MCF-7 humanbreast cancer cells treated with TCDD or selective AhR modulators,and this is also accompanied by proteasome-dependent degradationof ER ${alpha}$ (32, 54). This study further investigates the mechanismsof AhR-mediated degradation of ER ${alpha}$ protein in breast cancer cellsand the contributions of this pathway to inhibitory AhR-ER ${alpha}$ crosstalk. In breast cancer cells, TCDD alone and in combinationwith E induces rapid degradation of ER ${alpha}$ , and AhR interactionwith ER ${alpha}$ in a mammalian two-hybrid assay is dependent on TCDDbut not E. Interactions of ligand-bound AhR with ER ${alpha}$ are accompaniedby enhanced formation of ubiquitinated forms of ER ${alpha}$ , proteasome-dependentdegradation of ER ${alpha}$ , and decreased E-induced transactivation.Thus, ligand activation of the nuclear AhR coordinately recruitsboth ER ${alpha}$ and proteasomes which degrade both receptors and therebyenhance inhibitory AhR-ER ${alpha}$ cross talk in breast cancer cells.

MATERIALS AND METHODS

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Materials and Methods

Chemicals, constructs, cells, and animals. TCDD was prepared in this laboratory and shown to be >99%pure by gas chromatographic analysis. 17ß-Estradiol(E), chloroquine, cycloheximide, cell culture chemicals, andmedia were purchased from Sigma. Calpain inhibitor II (Cal II),EST, MG132, PSI, PSII, bis-indolylmaleimide I, manumycin A,U0126, PP2, wortmannin, and KT5720 were purchased from Calbiochem.Primary antibodies for human ER ${alpha}$ , AhR, Sp1, CYP1A1, c-Fos, andubiquitin proteins were purchased from Santa Cruz. Alexa Fluor594 goat anti-rabbit was purchased from Molecular Probes. Fluoresceinisothiocyanate-conjugated goat anti-rabbit was purchased fromZymed Laboratories. The rat c-Fos pSP65 expression vector waskindly provided by Tom Curran (Roche Research Center), and thehuman phuAhR expression vector was a kind gift from ChristopherBradfield (University of Wisconsin School of Medicine). ThepERE₃ reporter construct containing three tandem consensus EREsites linked to a luciferase gene was created by cloning anoligonucleotide containing this sequence into the BamHI-HindIII-cutpXP-2 plasmid (American Type Culture Collection, Manassas, Va.)30 bp upstream from a TATA box. The sequence of the ERE wasGGTCACAGTGACC. The ER ${alpha}$ -GAL4-AD and AhR-GAL4-DBD fusion proteinswere constructed using the mammalian Matchmaker two-hybrid kit(Clontech) pVP16 and pM fusion vectors. The expression plasmidsfor ER ${alpha}$ with deletions of amino acids 1 to 178 (ER ${alpha}$ ${Delta}$ AF1), 185 to252 (ER ${alpha}$ ${Delta}$ DBD), and 282 to 595 (ER ${alpha}$ ${Delta}$ AF2) were kindly provided byPierre Chambon (Institute de Genetique et Biologie Moleculaireet Cellulaire). T47D, MCF-7, and ZR-75 human breast cancer cellswere obtained from the American Type Culture Collection andcultured as previously described (54). B6C3F1 mice were obtainedfrom Charles River Laboratories. The mice were kept in a temperature-controlledconditioned room with a 14-h light and 10-h dark photocycle.Rodent chow and water was supplied ad libitum.

Protein isolation and Western analysis. Cells were seeded into 35-mm six-well tissue culture platesin phenol red-free medium (Dulbecco's modified Eagle's medium[DMEM] Ham F-12) containing 2.5% charcoal-stripped fetal bovineserum (FBS). After 24 h, cells were treated and harvested atdesignated time points and lysed in ice-cold lysis buffer (50mM HEPES [pH 7.5], 500 mM NaCl, 10% [vol/vol] glycerol, 1% TritonX-100, 1.5 mM MgCl₂, 1 mM EGTA) supplemented with protease inhibitorcocktail (Sigma). Equal amounts of protein from each treatmentgroup were boiled in 1x Laemmli buffer (50 mM Tris-HCl, 2% sodiumdodecyl sulfate [SDS], 0.1% bromphenol blue, 175 mM ß-mercaptoethenol),separated by SDS-10% polyacrylamide gel electrophoresis (SDS-PAGE),and electrophoresed to polyvinylidene difluoride (PVDF) membrane.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) andprobed with primary antibodies ER ${alpha}$ G-20 (1:1,000), AhR N-19 (1:200),Sp1 PEP2 (1:5,000), CYP1A1 G-18 (1:1,000), and c-Fos H-125 (1:1,000).Following incubation with peroxidase-conjugated secondary antibody,immunoglobulins were visualized using the ECL detection system(NEN). Quantitation was performed using a Sharp JX-330 scannerand Zero-D Scanalytics software (Scanalytics Corp.). The humanER ${alpha}$ , human phuAhR, and rat c-Fos pSP65 expression vectors wereused to in vitro translate standards for Western blotting usingthe TNT T7 quick-coupled transcription/translation system (Promega).Human recombinant Sp1 protein (Promega) was added to the invitro-translated mixtures as the Sp1 standard.

Transfection assays. ZR-75 cells were seeded onto 12-well plates at a concentrationof 2.75 x 10⁵ cells per well in phenol red-free DMEM Ham F-12supplemented with 2.5% charcoal-stripped FBS. After 18 h, cellswere transfected by the calcium phosphate method with 500 ngof pERE₃ luciferase reporter plasmid and 250 ng of pCDNA3.1-ß-gal(Invitrogen) as the control vector. Cells were treated for 36h and assayed for luciferase (Promega) and ß-galactosidase(Tropix) activity in a Packard luminometer according to themanufacturer's instructions. Protein-protein interactions betweenER ${alpha}$ and AhR were examined in ZR-75 cells by using the ER ${alpha}$ -GAL4-AD(vpER) and AhR-GAL4-DBD (pmAhR) fusion constructs. ZR-75 cellswere transfected by the calcium phosphate method with 500 ngof 5XGAL-luciferase, 250 ng of pmAhR, 100 ng of vpER, 250 ngof pCDNA3.1-ß-gal as the control vector, or emptypM and pVP vectors as DNA mass balance controls. Cells weretreated for 36 h, harvested, and assayed as described above.The effects of TCDD on proteasome-dependent degradation of ER ${alpha}$ mutants were determined in T47D cells. Cells were seeded onto35-mm six-well culture plates at a concentration of 6 x 10⁵cells per well in DMEM Ham F-12 without phenol red, supplementedwith 2.5% charcoal-stripped FBS. After 18 h, cells were transfectedby the calcium phosphate method with 500 ng of ER ${alpha}$ ${Delta}$ AF1, ER ${alpha}$ ${Delta}$ DBD,or ER ${alpha}$ ${Delta}$ AF2 expression plasmids. After a 12-h recovery period,cells were treated with dimethyl sulfoxide (DMSO; control) or10 nM TCDD for 6 h. Cells were collected for transfected andendogenous ER ${alpha}$ protein analysis using Western blotting buffersas described in "Protein isolation and Western analysis." Fifty-microgramprotein aliquots were electrophoresed for each treatment andtransfection group.

Nuclear extract preparation and EMSA. MCF-7 and ZR-75 cells were seeded at a density of 5 x 10⁶ cells/platein 60-mm tissue culture plates using DMEM Ham F-12 without phenolred, supplemented with 2.2 g of sodium bicarbonate/liter andantibiotic-antimycotic solution, and 2.5% charcoal-strippedFBS, pH 7.4. After 24 h, cells were treated for 3 h with 10nM E or 10 nM E plus 10 nM TCDD following a 30-min pretreatmentwith DMSO, 10 µM MG132, or 10 µM Cal II. Nuclearextracts were obtained using the NE-PER nuclear and cytoplasmicextraction kit (Pierce) according to the manufacturer's instructions.Three micrograms of nuclear protein from each treatment groupwas incubated for 10 min at 25°C with 500 ng of poly(dI-dC)in 30 µl of HEGDK⁺ (25 mM HEPES, 1.5 mM EDTA, 10% glycerol,1 mM DTT, 100 mM KCl) and ³²P-end-labeled ERE probe (5'-GTCCAA AGT CAG GTC ACA GTG ACC TGA AAG TT-3'). ER ${alpha}$ D-12 antibodyand normal mouse immunoglobulin G (IgG) were used for supershiftcontrols. Samples were electrophoresed on a 5% polyacrylamidegel at 120 V in 90 mM Tris, 90 mM borate, 2 mM EDTA (pH 8.0),dried, and visualized by autoradiography.

Northern blot analysis. T47D cells were seeded into 100-mm tissue culture plates and,after 24 h, cells were treated for 6 h with DMSO or 10 nM TCDDfollowing a 30-min pretreatment with DMSO, 10 µM MG132,or 10 µM PSI. Thirty micrograms of total RNA from eachtreatment was loaded onto a 1.2% agarose, 10% formaldehyde geland electrophoresed in 1x morpholinepropanesulfonic acid (MOPS)buffer. Separated RNA was transferred to Zeta-Probe GT (Bio-Rad)membrane by capillary action in 1x MOPS for 48 h, UV cross-linkedfor 15 min, dried at 80°C for 1 h, and probed at 42 to 65°Caccording to the Zeta-Probe GT protocol. Membranes were scannedon a STORM 860 PhosphorImager (Molecular Dynamics). Band intensitiesof AhR, Arnt, and ER ${alpha}$ were normalized to values obtained forthe ß-tubulin loading control. Expression vectorsused for generating RNA probes for Northern blot analysis weregenerously supplied by the following: AhR (Kristy Dolwick, NorthwesternUniversity Medical School), Arnt (Rosally Agbunag, Universityof California), hER ${alpha}$ (Ming-Jer Tsai, Baylor College of Medicine),and ß-tubulin (Masahito Negishi, National Instituteof Environmental Health Sciences, National Institutes of Health).

Kinase inhibitors and cycloheximide studies. ZR-75 cells were seeded as described in the Western immunoblottingprotocol above and treated for 3 h with DMSO, E, TCDD, or ETfollowing a 30-min pretreatment with DMSO, 10 µM PP2,1 µM bis-indolylmaleimide I, 1 µM KT5720, 2 µMmanumycin A, 5 µM U0126, 400 nM wortmannin, methanol (MeOH),or 25 µM cycloheximide. Cells were harvested and immunoblottedas described above in the "Protein isolation and Western analysis"protocol.

Coimmunoprecipitation. MCF-7 and ZR-75 cells were seeded into 150-mm tissue cultureplates in maintenance medium and allowed to grow to approximately90% confluence. Cells were treated with DMSO or 10 nM TCDD for30 min, and nuclear extracts for each treatment group were obtainedusing the NE-PER nuclear and cytoplasmic extraction kit withthe addition of protease inhibitor cocktail. Duplicate aliquotsof 550 µg (MCF-7) and 525 µg (ZR-75) of equal volumewere used for the experiments. The nuclear protein was dilutedfivefold in ice-cold phosphate-buffered saline (PBS) containingprotease inhibitor cocktail to a final volume of 1 ml, followedby the addition of 30 µl of protein A/G PLUS-agarose beads(Santa Cruz). The reactions were placed on a rocker at 4°Cfor 3 h, followed by centrifugation at 600 x g at 4°C for5 min. A 900-µl aliquot of supernatant was removed fromeach sample and placed into a new Eppitube on ice. Mouse monoclonalanti-ER ${alpha}$ D-12 (1 µg) or normal mouse IgG (1 µg) wasadded to either replicate treatment set, followed by the additionof 30 µl of protein A/G PLUS-agarose beads. The sampleswere then placed on a rocker at 4°C for 12 h, followed bycentrifugation at 600 x g at 4°C for 5 min. The supernatantwas removed by aspiration, and the pellets were washed oncewith 1 ml of ice-cold PBS followed by centrifugation at 600x g at 4°C for 5 min. The agarose pellet was then resuspendedin 50 µl of 1x Laemmli buffer, boiled, and centrifuged.The supernatant was separated by SDS-10% PAGE, electrophoresedto PVDF membrane, and visualized by ECL as described above.

Ubiquitinated ER ${alpha}$ immunoprecipitation. ZR-75 cells were seeded as described in the coimmunoprecipitationprotocol above and treated with DMSO or 10 nM E, 10 nM TCDDor ET, or 10 nM ICI 182,780 for 6 h. Duplicate aliquots of 300µg were immunoprecipitated with anti-Sp1 PEP2 (1 µg)or anti-ER ${alpha}$ D-12 (1 µg) as described above. Immunoprecipitateswere washed with two cycles of 1 ml of ice-cold radioimmunoprecipitationassay buffer followed by 1 ml of ice-cold PBS using centrifugationat 600 x g at 4°C for 5 min. The agarose pellet was resuspendedin 50 µl of 1x Laemmli buffer, boiled, and centrifuged,The supernatant was separated by SDS-10% PAGE, electrophoresedto PVDF membrane, blotted with anti-ubiquitin P4D1 (Sigma),and visualized by ECL as described above. The membrane was thenstripped using stripping buffer (62.5 mM Tris-HCl, 112 mM 2-mercaptoethanol,20% SDS [wt/vol]; pH 6.8) at 60°C for 1 h and reprobed withanti-ER ${alpha}$ D-12 and anti-Sp1 PEP2 consecutively.

Effects of siRNA for the AhR. ZR-75 cells were cultured in DMEM Ham F-12 containing 5% FBSin six-well plates until 50 to 60% confluent. Based on resultsof ongoing studies, a maximal decrease in the AhR protein wasobserved using 7 µl of a 20 µM solution of the smallinhibitory RNA (siRNA), and this amount was transfected intoZR-75 cells using oligofectamine reagent (Invitrogen, Carlsbad,Calif.). The final concentration of siRNAs in each well was140 nM. Thirty-six hours after transfection, cells were treatedwith DMSO, 10 nM E2, or 10 nM TCDD for 5 h, and nuclear extractswere obtained and analyzed by Western blot analysis for AhR,ER ${alpha}$ , and Sp1 proteins essentially as described elsewhere (1).Replicate (three) experiments were carried out to quantitatethe effects of siRNA for the AhR on TCDD-induced downregulationof ER ${alpha}$ . The siRNA oligonucleotides for the AhR and scrambledsiRNA were as follows: scramble siRNA, 5'-GCG CGC UUU GUA GGAUUC G TT and TT CGC GCG AAA CAU CCU AAG C-5'; siRNA for AhR,5'-UAC UUC CAC CUC AGU UGG C TT and TT AUG AAG GUG GAG UCA ACCG-5'; siRNA for lamin A/C, 5'-CUG GAC UUC CAG AAG AAC A TT andTT GAC CUG AAG GUC UUC UUG U-5'.

Immunofluorescence. For uterine immunohistochemistry, 25-day-old mice were injectedintraperitoneally with 200 ng of E in 100 µl of corn oil,1 µg of TCDD in 100 µl of corn oil, ET, or cornoil alone. Twelve hours after treatment, mice were euthanizedby CO₂ asphyxiation. Uteri were removed, fixed in 4% paraformaldehydeovernight, washed with 70% ethanol, paraffin embedded, and sectionedat a 5-µm thickness onto positively charged slides and,after subsequent processing, slides were immunostained withER ${alpha}$ H-184 antibodies and analyzed by immunofluorescence as indicatedbelow.

For immunocytochemistry, ZR-75 cells were seeded onto four-wellglass chamber slides at a density of 75,000 cells per well inRPMI maintenance medium. After 24 h, cells were treated withDMSO, 10 nM E, 10 nM TCDD, or ET for 24 h. Slides were thenfixed for 10 min in -20°C MeOH, air dried, and washed for5 min in PBS-0.3% Tween. Slides were blocked for 1 h with 5%goat serum in antibody dilution buffer (1% bovine serum albumin-PBS-0.3%Tween-31%glycerol [vol/vol] [pH to 8.0] with 0.5 M Na₂CO₃ [pH 9.5]).A 1:100 dilution of anti-ER ${alpha}$ H-184-5% goat serum-antibody dilutionbuffer, or 5% goat serum-antibody dilution buffer alone (control)was added to the samples and placed in a humidified chamberovernight at 4°C. Slides were then washed three times for30 min in PBS-Tween and blocked again for 1 h with 5% goat serum-antibodydilution buffer. Alexa Fluor 594 goat anti-rabbit secondaryantibody was added at a 1:1,000 dilution in 5% goat serum-antibodydilution buffer to all samples for 1 h at room temperature.Slides were washed three times for 30 min in PBS-Tween and oncefor 15 min in deionized water and mounted as above. Immunofluorescencepreparations were evaluated with a Zeiss Axioplan2 microscope(Carl Zeiss) fitted with a Hamamatsu-C5810 chilled 3CCD colorcamera (Hamamatsu Corporation). Images of at least three differentfields from three different sections per treatment group containinguterine luminal epithelium and stromal cells were captured usingidentical settings. Fluorescence intensity measurements of ERin both epithelial and stromal cells were obtained followingsubtraction of background staining determined from the controlprepared without primary antibody. Values of mean fluorescenceintensity ± the standard error (SE) were analyzed statistically.

Statistics. All quantitative data were analyzed by an analysis of variancefollowed by Fisher's protected least significant differencetest for significance (P < 0.05). Data are expressed as means± SE (n $>=$ 3).

RESULTS

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Results

TCDD induces proteasome-dependent degradation of ER ${alpha}$ in breast cancer cell lines. ZR-75 human breast cancer cells express ER ${alpha}$ and the AhR, andresults in Fig. 1A summarize the effects of treatment with DMSO(D), 10 nM TCDD (T), and 10 nM E alone and in combination (ET)on levels of ER ${alpha}$ , AhR, and Sp1 proteins in the presence of solvent(DMSO), proteasome (MG132), or protease (Cal II) inhibitors.E induced degradation of ER ${alpha}$ (but not AhR or Sp1), and this responsewas inhibited by proteasome but not protease inhibitors. Incells treated with T or ET, there was coordinate degradationof both ER ${alpha}$ and the AhR (but not Sp1) proteins which was blockedby proteasome but not protease inhibitors. Most significantly,there was a decrease in ER ${alpha}$ levels in all three cell lines cotreatedwith ET compared to that of treatment with E alone. The effectsof the D, E, T, and ET treatments are also illustrated in atime course study on ER ${alpha}$ degradation in ZR-75 cells (Fig. 1B).There was a rapid and sustained degradation of ER ${alpha}$ by TCDD andsignificantly (>50%) lower ER ${alpha}$ levels in cells treated withET compared to cells treated with E alone, and comparable resultswere obtained in MCF-7 and T47D breast cancer cells. These datacomplement results of previous studies in MCF-7 and T47D celllines, which also express AhR and ER ${alpha}$ proteins (54). Thus, inthree ER-positive breast cancer cell lines, TCDD activationof AhR results in proteasome-dependent degradation of both AhRand ER ${alpha}$ proteins, whereas E induces degradation of ER ${alpha}$ but notthe AhR; cotreatment with ET enhanced ER ${alpha}$ degradation, whereasSp1 levels were unchanged in all treatment groups. The effectsof TCDD and E on degradation of ER ${alpha}$ mutants with deletions ofthe DBD (ER ${alpha}$ ${Delta}$ DBD), AF1 (ER ${alpha}$ ${Delta}$ AF1), and AF2 (ER ${alpha}$ ${Delta}$ AF2) were investigatedby transient transfection in T47D cells (Fig. 1C). TCDD- andE2-induced degradation of endogenous wild-type ER ${alpha}$ served asa positive control. The results showed that in cells treatedwith TCDD, deletion of the DBD did not affect degradation, whereasdeletion of AF1 or AF2 eliminated this response. In contrast,E2 induced degradation of wild-type ER ${alpha}$ , ER ${alpha}$ ${Delta}$ DBD, ER ${alpha}$ ${Delta}$ AF1, and ER ${alpha}$ ${Delta}$ AF2,showing that in T47D cells, multiple domains of ER ${alpha}$ are targetedfor degradation by proteasomes. In contrast, degradation ofER ${alpha}$ induced by TCDD was not observed for mutants with deletionsof AF1 or AF2, suggesting that TCDD and E2 induce distinct patternsof ER ${alpha}$ degradation. A previous study showed that E2-induced degradationof ER ${alpha}$ mutants by proteasomes in HeLa cells was DBD dependent(52), indicating that cell context is also important for ER ${alpha}$ degradation by proteasomes. siRNAs can be used for gene silencingin mammalian cells (10, 16, 17), and studies in this laboratoryhave applied this technique for study of ER ${alpha}$ /Sp1 and AhR actionin breast cancer cells (1; M. Abdelrahim, unpublished results).Transfection of ZR-75 cells with siRNA for the AhR inhibitedTCDD-induced, but not E2-induced, degradation of ER ${alpha}$ proteincompared to control (solvent-treated) cells or cells transfectedwith scrambled siRNA (Fig. 1D and E). Sp1 was used as a control,and levels of this protein were unaffected by any of the treatments.Results illustrated in Fig. 1E show that siRNA for the AhR induceddegradation of the AhR but not Sp1 protein or lamin A/C (datanot shown), whereas siRNA for lamin A/C did not affect AhR proteinbut decreased lamin A/C protein (data not shown). Similar resultswere obtained in MCF-7 cells (data not shown). Thus, AhR expressionin ZR-75 cells is required for TCDD-induced degradation of ER ${alpha}$ ,and this has also been previously observed in AhR-deficientMCF-7 cells (54).

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FIG.1. Activated AhR causes degradation of ER ${alpha}$ in multiple breast cancer cell lines through the proteasome pathway. (A) ZR-75 cells were treated for 3 h with DMSO (D), 10 nM E, 10 nM TCDD (T), or their combination (ET) following a 30-min pretreatment with vehicle control (EtOH or DMSO), proteasome (MG132), or protease (Cal II) inhibitors at 10 µM concentrations for each. Whole-cell extracts from the different treatment groups were analyzed by Western blot analysis for ER ${alpha}$ (G-20), AhR (N-19), and Sp1 (PEP2) proteins as described in Materials and Methods. Relative ER ${alpha}$ protein levels for each cell line are illustrated in bar graphs. (B) Time course degradation of ER ${alpha}$ in ZR-75 cells. Cells were treated with DMSO, 10 nM E, 10 nM TCDD, or 10 nM ET for the designated times, and whole-cell extracts from the different treatment groups (D, T, E, and ET) were analyzed for ER ${alpha}$ and Sp1 protein by Western blot analysis as described in Materials and Methods. In vitro-translated ER ${alpha}$ , AhR, and pure Sp1 protein (i.v.) were used as controls. (C) Degradation of wild-type and variant ER ${alpha}$ in T47D cells. Cells were transfected with ER ${alpha}$ variants treated with 10 nM E or 10 nM T, and levels of endogenous ER ${alpha}$ and transfected ER ${alpha}$ variants were determined by Western blot analysis as described in Materials and Methods. Results are expressed as means ± SE for three replicate determinations, and levels of wild-type or variant ER ${alpha}$ significantly (P < 0.05) lower than the controls (DMSO) are indicated with an asterisk. Western blot analysis of these proteins is indicated (upper left). (D) Effects of siRNA for the AhR on degradation of ER ${alpha}$ . siRNA or scrambled siRNA was transfected in ZR-75 cells and the effects of 10 nM TCDD or E2 on ER ${alpha}$ protein were determined by Western blot analysis of whole-cell extracts as described in Materials and Methods. TCDD and E2 induced degradation of ER ${alpha}$ in control cells and cells transfected with scrambled siRNA, whereas siRNA for AhR blocked the effects of TCDD but not E2. These data were observed in duplicate experiments. (E) Degradation of ER ${alpha}$ and AhR. Cells were treated with DMSO, E, or T and siRNAs for the AhR or lamin A/C, and levels of ER ${alpha}$ /Sp1 (right) or AhR/Sp1 (left) protein were determined in replicate (three) experiments. TCDD alone significantly (P < 0.05) decreased levels of ER ${alpha}$ , and TCDD plus siRNA for AhR (iAhR) significantly (P < 0.05) reversed the effects of TCDD. siRNAs for the AhR (but not lamin A/C) significantly (P < 0.05) decreased AhR levels. Results are expressed as means ± SE relative to control (DMSO) protein levels.

The effects of E and ET treatment on binding of nuclear extractsfrom MCF-7 cells to [³²P]ERE were investigated in gel mobilityshift assays (Fig. 2A). Nuclear extracts from cells treatedwith ET in solvent alone or in combination with Cal II gavea retarded band with decreased intensity compared to the bandobtained using extracts from cells treated with E alone, andthese data corresponded to results of immunoblot analysis ofER ${alpha}$ protein in the same extracts (Fig. 2B). In contrast, theproteasome inhibitor MG132 blocked ER ${alpha}$ protein degradation incells treated with E or ET, and nuclear extracts exhibited increasedretarded band intensities (compared to DMSO) (Fig. 2A) and increasedimmunoreactive ER ${alpha}$ protein (Fig. 2B). Antibody supershift experimentswith ER ${alpha}$ antibody or nonspecific IgG confirmed that the retardedband complex contained ER ${alpha}$ protein (Fig. 2A). The effects ofthe various treatments on Sp1 protein were also determined asa control experiment, since levels of Sp1 are relatively constantin low-passage breast cancer cells (54). The results (Fig. 2)show that E, ET, and the proteasome and protease inhibitorsdid not affect Sp1 protein expression, indicating specificityfor the AhR-ER ${alpha}$ interactions.

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FIG. 2. Decreased ER ${alpha}$ protein-DNA binding parallels diminished ER ${alpha}$ protein levels. (A) Gel mobility shift assay. MCF-7 cells were treated for 3 h with 10 nM E or 10 nM E plus 10 nM T (ET) following a 30-min pretreatment with DMSO, 10 µM MG132, or 10 µM Cal II, and binding of extracts to [³²P]ERE was analyzed in a gel mobility shift assay as described in Materials and Methods. Antibody supershift (SS) experiments used ER ${alpha}$ antibody (D-12) or nonspecific mouse IgG. ns, nonspecific binding. (B) Immunoreactive ER ${alpha}$ or Sp1 proteins in nuclear extracts. The same nuclear extracts from the treated cells from panel A were also analyzed by Western blot analysis as described in Materials and Methods. i.v., in vitro-translated ER ${alpha}$ and pure Sp1 protein control lane.

The effects of treatments on cellular ER ${alpha}$ levels were investigatedby immunostaining ZR-75 cells with ER ${alpha}$ antibodies (Fig. 3A).In cells treated with DMSO, there was widespread staining ofER ${alpha}$ , which was located in the nuclei. Treatment of the cellswith 10 nM E, 10 nM T, or their combination for 24 h causeda decrease in immunostaining of ER ${alpha}$ and, in the cotreatment group,only low levels of nuclear ER ${alpha}$ were detected. These results confirmthat treatment of MCF-7 cells with ET not only decreased immunoreactiveER ${alpha}$ protein but also decreased binding of nuclear extracts to[³²P]ERE, suggesting that limiting levels of nuclear ER ${alpha}$ in cellstreated with ET may contribute to inhibition of E-induced geneand reporter gene expression by TCDD (reviewed in reference44). We also investigated the effects of E and ET in the immaturefemale mouse uterus, which is also responsive to inhibitoryAhR-ER ${alpha}$ cross talk (51). There was extensive swelling of uterineepithelial and stromal cells after treatment with E for 12 h(Fig. 3B); however, in animals cotreated with ET, the swellingresponse was decreased and expression of ER ${alpha}$ protein was lowerin both stromal and epithelial cells. This response in vivoparalleled the effects observed in ZR-75 cells. Quantificationof ER ${alpha}$ fluorescence intensity revealed that treatment with E,T, or cotreatment with ET results in levels of ER ${alpha}$ which aresignificantly lower than those observed after treatment withcorn oil alone (P < 0.001). The mean ER ${alpha}$ fluorescence intensityvalues obtained from uterine sections were 56.41 ± 5.12,30.26 ± 5.42, 27.88 ± 5.06, and 13.46 ±3.48 for corn oil control, E, T, and ET, respectively.

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FIG. 3. AhR-mediated ER ${alpha}$ degradation occurs in situ. (A) ZR-75 cells were treated with DMSO (D), 10 nM E, 10 nM TCDD (T), or ET for 24 h, and ER ${alpha}$ was immunostained as described in Materials and Methods. Field width is 150 µm. (B) Immature (25-day-old) B6C3F1 mice were injected intraperitoneally with corn oil (C), 200 ng of E, or 1 µg of TCDD (T) or ET for 12 h. Uteri were fixed and immunostained as described in Materials and Methods. Field width is 240 µm. Control fields represent background without ER ${alpha}$ H184 antibody.

Inhibitory AhR-ER ${alpha}$ cross talk: role of proteasome-dependent degradation of ER ${alpha}$ . TCDD inhibits transactivation of several E-induced genes inbreast cancer cells (9, 14, 18, 21, 25, 38), and different mechanismsof inhibitory AhR-ER cross talk have been proposed (36, 44).The results summarized in Fig. 4A show that E induces luciferaseactivity in ZR-75 cells transfected with pERE₃ alone and, incells cotreated with E and different concentrations of TCDD,there was a concentration-dependent decrease in hormone-inducedactivity. In a parallel experiment, treatment of ZR-75 cellswith 10 nM E alone decreased ER ${alpha}$ levels to 30% of those observedin control (DMSO) cells (Fig. 4B), and this was associated withmaximal transactivation (Fig. 4A). These results are consistentwith previous reports and reflect the notion of spare ER ${alpha}$ levelscoupled with proteasome-dependent activation of ER ${alpha}$ (2, 11, 29,49). However, in ZR-75 cells treated with E plus different concentrationsof TCDD, there was a further decrease in ER ${alpha}$ protein below levelsobserved in cells treated with E alone (Fig. 3B). Figure 4Cillustrates the concentration-dependent effects of TCDD on levelsof ER ${alpha}$ protein as a percentage of ER ${alpha}$ levels in cells treatedwith 10 nM E alone and on luciferase activity as a percentageof the E-induced response. These parallel curves suggest thatin the cells cotreated with ET, inhibition of E-induced transactivationby TCDD was related to the enhanced AhR-mediated degradationof ER ${alpha}$ . These data suggest that cotreatment with ET results inlimiting levels of ER ${alpha}$ , as observed in cells treated with theantiestrogen ICI 182,780, which also induces proteasome-dependentdegradation of ER ${alpha}$ (41, 52). Previous studies have demonstratedthat TCDD inhibits induction of c-fos gene expression by E andinduction of E-responsive constructs containing c-fos gene promoterinserts, as shown in Fig. 4A for pERE₃ (9). The results in Fig.5A demonstrate that TCDD also inhibits induction of c-Fos proteinby E, and this was observed in the presence of a solvent control(MeOH) or after addition of the protease inhibitor EST. TCDDalone did not affect c-Fos protein levels. The pattern of ER ${alpha}$ protein levels in these same treatment groups (Fig. 5B) alsoshowed that in cells treated with ET, both c-Fos and ER ${alpha}$ proteinlevels were maximally decreased. The importance of ER ${alpha}$ proteinlevels in regulation of c-Fos was confirmed in cells treatedwith the proteasome inhibitor PSI, where inhibition of E-inducedc-Fos protein by TCDD was not observed (Fig. 5A), and this correlatedwith high ER ${alpha}$ levels which were maintained in the presence ofPSI (Fig. 5B). In contrast, levels of Sp1 protein were unaffectedby the different treatments (Fig. 5C). The results in Fig. 5Aindicate that the proteasome inhibitor PSI increases Fos proteinlevels in the various treatment groups compared to levels observedin solvent (MeOH) or protease-treated cells. These data suggestthat c-Fos protein is regulated by proteasomes in MCF-7 cells,and this is consistent with the reported regulation of c-Fosprotein in other cells lines by the ubiquitin-proteasome pathway(48, 50). Thus, inhibition of E-induced c-fos gene and reportergene expression by TCDD (9) is also accompanied by decreasedc-Fos protein levels, and these responses at the protein (andgene) level correlated with the limiting ER ${alpha}$ levels observedin breast cancer cells after cotreatment with ET. Therefore,inhibitory AhR-ER ${alpha}$ cross talk in breast cancer cells is dependenton ET-induced degradation of ER ${alpha}$ protein by activation of proteasomes.

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FIG. 4. ER ${alpha}$ levels determine the magnitude of hormone-induced transactivation. (A) ZR-75 cells were transfected with pERE₃ treated with DMSO (D), 10 nM E, or E plus decreasing concentrations of TCDD (ET) for 36 h, and luciferase activity was determined as described in Materials and Methods. Results are expressed as means for duplicate determinations for each treatment group. (B) In parallel duplicate experiments, whole-cell extracts were collected, and ER ${alpha}$ protein levels in the various treatment groups described in the legend for panel A were analyzed by Western blot analysis as described in Materials and Methods. i.v., in vitro-translated ER ${alpha}$ and pure Sp1 control lane. Results are expressed as means for duplicate determinations for each treatment group. (C) TCDD cotreatment with E mediates a parallel decrease in ER ${alpha}$ protein and transactivation. The data from panels A and B were used to plot the effects of increasing concentrations of TCDD on the percent decrease of ER ${alpha}$ protein (•) and transactivation ( ${circ}$ ) below that observed in cells treated with E alone (set at 100%).

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FIG. 5. ER ${alpha}$ levels determine regulation of c-Fos protein expression. MCF-7 cells were pretreated for 30 min with methanol vehicle (MeOH), 10 µM PSI, or 10 µM EST. Cells were pretreated with T for 3 h prior to addition of 10 nM E (total DMSO did not exceed 0.03%), and c-Fos, ER ${alpha}$ , and Sp1 proteins were detected by Western blot analysis from the same PVDF membrane, as described in Materials and Methods. i.v., in vitro-translated rat c-Fos control. Results are expressed as means ± SE for three determinations for each treatment group. Significant induction of E-induced c-Fos protein is indicated by the letter a above the bar. Significant inhibition of E-induced c-Fos protein and decreased ER ${alpha}$ levels by ET (compared to E) are indicated by the letter b above the bar (P < 0.05).

TCDD-induced ubiquitination of ER ${alpha}$ . Proteasome-dependent degradation of proteins including ER ${alpha}$ isa multistep process involving the 26S proteasome complex, inwhich proteins targeted for degradation are first conjugatedwith ubiquitin. Results illustrated in Fig. 6 summarize studiesthat investigated ubiquitination of ER ${alpha}$ in ZR-75 cells by usingimmunoprecipitation and Western blot analysis of whole-celllysates or nuclear extracts from cells treated with solvent(DMSO), 10 nM E, 10 nM TCDD or ET, or 10 nM ICI 182,780. Resultsof previous studies show that cells treated with ICI 182,780undergo a high level of ubiquitination and protein degradation(52), and this treatment group served as a positive control,whereas Sp1 protein levels are relatively constant and servedas an additional negative control protein for this study. Duplicatealiquots of nuclear extracts from different treatment groupswere immunoprecipitated with ER ${alpha}$ or Sp1 antibodies and then analyzedfor ubiquitinated proteins by Western blot analysis using ubiquitinantibodies (Fig. 6A). In vitro-translated ER ${alpha}$ and human recombinantSp1 protein and 15-µg aliquots of nuclear extract servedas controls for this experiment. Minimal to nondetectable levelsof ubiquitinated proteins were detected in the lanes immunoprecipitatedwith Sp1 antibodies. However, a series of ubiquitinated bands(>84 kDa) were detected in the lanes immunoprecipitated withER ${alpha}$ antibodies, and increased staining of ubiquitinated bandswas observed in cells treated with E, TCDD, ET, and ICI 182,780compared to DMSO (control). The in vitro and nuclear extractcontrol lanes show multiple ubiquitinated bands as expected,given the propensity of this regulatory protein. The same PVDFmembrane was then stripped and reprobed with ER ${alpha}$ antibodies (Fig.6B). The results confirm that ER ${alpha}$ was immunoprecipitated, andthe pattern of ER ${alpha}$ protein intensities was comparable to thoseobserved in the nuclear extracts. Higher-molecular-weight bandsare also visible in this panel, and some of these may correspondto the ubiquitinated bands observed in Fig. 6A. This same membranewas then reprobed with Sp1 antibodies (Fig. 6C), and Sp1 proteinlevels were similar in all treatment groups; this was also observedin the nuclear extract control. These data confirm that proteasome-dependentdegradation of ER ${alpha}$ induced by the ligand (TCDD)-activated AhRgave enhanced ubiquitination of ER ${alpha}$ ; moreover, the higher-molecular-weightubiquitinated ER ${alpha}$ bands are also observed in cells after treatmentwith E or ICI 182,780.

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FIG. 6. AhR-mediated ubiquitination of ER ${alpha}$ . ZR-75 cells were treated with DMSO (D), 10 nM E, 10 nM TCDD (T) or ET, or 10 nM ICI 182,780 for 6 h, and nuclear extracts (300-µg aliquots) were immunoprecipitated with Sp1 (PEP2) or ER ${alpha}$ (D-12) antibodies. In vitro-translated ER ${alpha}$ and pure Sp1 protein (i.v.) and 15 µg of nuclear extract from each treatment group were loaded as controls. Western blot analysis was performed as described in Materials and Methods. (A) The PVDF membrane probed with ubiquitin antibody (P4D1). (B) The same membrane, stripped and reprobed with ER ${alpha}$ antibody (D-12). (C) The same membrane reprobed with Sp1 antibody (PEP2). Loading controls using heavy chain, nonspecific, and light chain IgG are also shown.

AhR-mediated degradation of ER ${alpha}$ : modulation of gene expression, effects of protein synthesis, and kinase inhibitors. Since TCDD activates proteasome-dependent degradation of bothAhR and ER ${alpha}$ protein, we also investigated the effects of 10 nMTCDD alone (DMSO) and in combination with proteasome inhibitors(MG132 and PSI) on ER ${alpha}$ , AhR, Arnt, and ß-tubulin mRNAlevels (Fig. 7A). The results show that ER ${alpha}$ mRNA levels weresimilar in solvent control (D) or TCDD (T)-treated in cellsin the presence or absence of proteasome inhibitors, and thiswas confirmed in four separate experiments. Moreover, AhR, Arnt,and ß-tubulin mRNA levels were also comparable inthe different treatment groups, confirming that TCDD does notdownregulate ER ${alpha}$ (or the AhR) protein by decreasing mRNA levels.The role of protein synthesis in activation of proteasomes byTCDD was investigated in ZR-75 cells treated with solvent (D)or 10 nM TCDD (T) alone (MeOH) or in the presence of 25 µMcycloheximide (Fig. 7B). The results show that TCDD inducesdegradation of AhR and ER ${alpha}$ in the presence or absence of cycloheximide.In contrast, cycloheximide inhibits induction of CYP1A1 proteinby TCDD, and this is consistent with previous studies on thisAh-responsive gene in which TCDD induces both mRNA and proteinlevels. These results suggest that activation of proteasome-dependentdegradation of AhR and ER ${alpha}$ by TCDD does not involve new proteinsynthesis.

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FIG. 7. Proteasome-dependent degradation of ER ${alpha}$ ; role of mRNA levels, protein synthesis, and kinase inhibitors. (A) mRNA levels. T47D cells were treated for 6 h with DMSO (D) or TCDD (T) following a 30-min pretreatment with DMSO, 10 µM MG132, or 10 µM PSI, and ER ${alpha}$ , AhR, Arnt, and ß-tubulin mRNA levels were determined as described in Materials and Methods. (B) Effects of cycloheximide. ZR-75 cells were treated for 3 h with DMSO (D) or 10 nM TCDD (T) following a 30-min pretreatment with MeOH or 25 µM cycloheximide, and whole-cell lysates were analyzed for AhR, ER ${alpha}$ , and CYP1A1 proteins by Western blot analysis as described in Materials and Methods. ns, nonspecific protein. (C) Effects of kinase inhibitors. ZR-75 cells were treated with DMSO (D), 10 nM E, 10 nM TCDD (T), and ET for 3 h following a 30-min pretreatment with 10 µM PP2, 1 µM bis-indolylmaleimide 1, 1 µM KT5720, 2 µM manumycin A, 5 µM U0126, or 400 nM wortmannin. ER ${alpha}$ and Sp1 proteins were determined by Western blot analysis as described in Materials and Methods.

Activation of kinase pathways is required for proteasome-dependentdegradation of some proteins, and the results in Fig. 7C summarizeeffects of several kinase inhibitors on ER ${alpha}$ and Sp1 protein levels.Cells were treated with DMSO (D), 10 nM E, 10 nM TCDD or ET,and several inhibitors (kinases), including 10 µM PP2(src), 1 µM bis-indolylmaleimide 1 (protein kinase C),1 µM KT 5720 (protein kinase A), 2 µM manumycinA (ras), 5 µM U0126 (mitogen-activated protein kinasekinase [MAPKK]), or 400 nM wortmannin (phosphatidylinositol3-kinase), for 3 h and whole-cell extracts were analyzed forER ${alpha}$ and Sp1 by Western blot analysis. The pattern of downregulationof ER ${alpha}$ by TCDD was unaffected by the kinase inhibitors, and similareffects were observed in cells treated with E alone and ET,although the magnitude of the response was decreased in cellscotreated with the ras inhibitor manumycin A. The MAPKK inhibitorPD98059 blocked activation of proteasomes by TCDD (data notshown); however, this compound and related 3'-methoxy-substitutedflavonoids inhibit nuclear translocation and activation of theAhR by TCDD (39), suggesting that this response is also requiredfor AhR-mediated activation of proteasomes.

Ligand activation of the AhR is required for AhR-ER ${alpha}$ interactions. Klinge and coworkers (21) first showed that a glutathione S-transferase(GST)-ER ${alpha}$ chimeric protein interacted with the AhR (but not Arnt)in a pull-down assay, and this interaction was AhR ligand (ß-naphthoflavone)dependent. The results in Fig. 8 show that ER ${alpha}$ and AhR antibodies(but not IgG) immunoprecipitated both the AhR and ER ${alpha}$ proteinin nuclear extracts from MCF-7 (Fig. 8A) or ZR-75 (Fig. 8B)cells treated with 10 nM TCDD. There was also evidence for someinteractions of ER ${alpha}$ and the AhR in solvent (DMSO)-treated ZR-75cells; however, a more intense band was observed in the extractfrom cells treated with TCDD. Interaction of these proteinswas further investigated in a mammalian two-hybrid assay usingan expression plasmid containing the DBD of the yeast GAL4 protein(amino acids 1 to 147) fused to full-length AhR (pm-AhR), anda plasmid expressing the VP16 (amino acids 411 to 455) activationdomain fused to ER ${alpha}$ ; empty vectors (pm and vp) served as controls.The GAL4-responsive construct (pGAL4) contains five tandem yeastGAL4 response elements linked to a luciferase reporter gene.In cells treated with DMSO (solvent control) or E and cotransfectedwith pmAhR plus vp (empty vector) or vpER, comparable activitieswere observed, indicating that the high activity was primarilydue to unliganded pmAhR, and this was not enhanced by vpER inthe absence or presence of E. The high basal activity of pmAhRin ZR-75 cells is in contrast to the lower response observedfor pmAhR (mouse) in COS cells, which was similar to the activityof pm alone (19). This may be due, in part, to breast cancercell expression of cofactors such as p300, steroid receptorcoactivators, and TATA binding protein, which interact withthe AhR (26, 33, 43). The activity of pm-AhR plus vp was notincreased in cells treated with TCDD alone or ET; however, asignificant increase (over pmAhR plus vp) was observed whencells were cotransfected with pmAhR plus vpER. Thus, ER-AhRinteractions in the mammalian two-hybrid assay were dependenton TCDD, and these results were consistent with interactionsof these proteins in the pull-down assay (21). The pattern ofvpER degradation in transfected cells (Fig. 8D) was similarto that observed for wild-type ER ${alpha}$ (Fig. 1). Previous studiesshowed that E induced degradation of transfected vpER and ER ${alpha}$ in HeLa cells, and this study confirms that TCDD alone or incombination with E also induces degradation of both proteinsin breast cancer cells. Thus, TCDD-induced degradation of ER ${alpha}$ (and vpER) is linked to physical interactions of AhR and ER ${alpha}$ .

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FIG. 8. Activated AhR interacts with ER ${alpha}$ . MCF-7 (A) and ZR-75 (B) cells were treated with DMSO (D) or TCDD (T) for 30 min. Nuclear extracts were isolated and immunoprecipitated with nonspecific mouse or goat IgG, anti-ER ${alpha}$ (D12), or the AhR. AhR and ER ${alpha}$ proteins were analyzed by Western blot analysis as described in Materials and Methods. In vitro-translated AhR and ER ${alpha}$ were also used as markers. (C) Mammalian two-hybrid interactions of pm-AhR and vp-ER in ZR-75 breast cancer cells. ZR-75 cells were transfected with pGAL4, pm plus vp (empty vectors), pm plus vpER, pmAhR plus vp, or pmAhR plus vpER, treated with DMSO (D), 10 nM E (E), 10 nM (TCDD), or E plus TCDD (ET), and luciferase activity was determined as described in Materials and Methods. Results are expressed as means ± SE for three separate determinations for each treatment group. Luciferase activity significantly (P < 0.05) higher than that observed in cells transfected with pmAhR plus vp is indicated with an asterisk. (D) Levels of vpER were also determined by Western blot analysis in cells transfected with pmAhR and vpER. ns, nonspecific.

DISCUSSION

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Discussion

The ubiquitin-proteasome pathway targets proteins for degradationand plays a critical role in multiple biochemical processes,including differentiation, cell cycle regulation, transport,embryogenesis, immune responses, apoptosis, and control of signaltransduction (8, 12, 24, 34). Proteins targeted for degradationby proteasomes are first ubiquitinated by enzyme-catalyzed formationof polyubiquitin-protein conjugates, which are substrates forATP-dependent proteolysis by the 26S proteasome complex. Proteasomesplay an important role in regulating cellular expression ofER ${alpha}$ and other NR proteins; however, there is considerable variabilityin proteasome activation and modulation of these nuclear transcriptionfactors (2, 4, 6, 11, 23, 27-29, 45, 46, 49, 55). For example,both the androgen and vitamin D receptors are degraded by proteasomesto maintain basal levels of these proteins, and receptor agonistsor proteasome inhibitors increase receptor levels through inhibitionof proteasome-dependent degradation in several cell lines (28,45, 46). In contrast, synthetic or endogenous ligands for theprogesterone receptor, thyroid hormone receptor, retinoic acidreceptor (RAR), and ER ${alpha}$ activate proteasome-dependent receptordegradation (4, 6, 23, 27, 28, 45, 46, 55).

Ligands for the AhR also activate proteasome-dependent degradationof ER ${alpha}$ protein, and this process is not observed in benzo(a)pyrene-resistantMCF-7 cells that do not express AhR (54); AhR antagonists thatinhibit formation of the nuclear AhR complex also block AhR-mediateddegradation of ER ${alpha}$ (data not shown). siRNA oligonucleotides canbe used for gene silencing in mammalian cells (10, 16, 17),and this technique has been used for investigating ER ${alpha}$ /Sp1-mediatedtransactivation and inhibitory AhR-ER ${alpha}$ cross talk in breast cancercells (1; M. Abdelrahim, unpublished results). Transfectionof siRNA for the AhR induces a 50 to 70% decrease in AhR levelsin MCF-7 cells and also inhibits degradation of ER ${alpha}$ by TCDD butnot E2 (Fig. 1D and E) These data complement results of previousstudies in AhR-deficient MCF-7 cells, where TCDD did not inducedegradation of ER ${alpha}$ (54). Results shown in Fig. 1 to 3 confirmthat the AhR agonist TCDD alone or in combination with E inducesrapid degradation of endogenous ER ${alpha}$ in at least three ER ${alpha}$ -positivebreast cancer cell lines (T47D, ZR-75, and MCF-7), and theseresponses are blocked by proteasome inhibitors but not proteaseinhibitors. This represents a novel interaction between ER ${alpha}$ andthe basic helix-loop-helix AhR protein, in which ligands forthe AhR activate proteasome-dependent degradation of both AhRand ER ${alpha}$ , whereas ER ${alpha}$ agonists induce proteolysis of ER ${alpha}$ but notthe AhR. Interestingly, we also observed a punctate patternof nuclear ER ${alpha}$ staining in cells treated with E, TCDD, and ET(Fig. 3A), and this has only been observed in previous studiesusing ER ${alpha}$ -specific ligands (49). The effects of ET in the mouseuterus (Fig. 3B) showed that there was a significant decreasein E-induced swelling of epithelial and stromal cells, and ETalso decreased ER ${alpha}$ staining in epithelial and stromal cells.These responses in vivo paralleled results in cell culture (Fig.3A) and are consistent with inhibitory AhR-ER ${alpha}$ cross talk previouslyobserved in the rodent uterus (42, 51).

This study investigates the mechanism and role of AhR-dependentdegradation of endogenous ER ${alpha}$ protein in mediating inhibitionof E-induced gene and reporter gene and protein expression inbreast cancer cells. E activates degradation of ER ${alpha}$ , and thisis accompanied by high transactivation (Fig. 4) (29). Sinceboth E and TCDD induce proteasome-dependent degradation of ER ${alpha}$ ,cells cotreated with ET express <20% of ER ${alpha}$ protein levelsobserved in untreated cells and lower levels than in cells treatedwith E alone. Both ET and the direct-acting pure antiestrogenICI 182,780 induce substantial degradation and ubiquitinationof ER ${alpha}$ (Fig. 6), suggesting that cellular levels of ER ${alpha}$ are limitingand are insufficient for hormone-induced transactivation (Fig.4 and 5). Thus, inhibitory AhR-ER ${alpha}$ cross talk in these cellsis linked to activation of proteasome-dependent degradationof ER ${alpha}$ by ET.

Preliminary studies in this laboratory did not observe E- orTCDD-induced degradation of transfected wild-type or mutantER ${alpha}$ in HeLa cells (data not shown). Moreover, our data showedthat E increased transfected ER ${alpha}$ levels, indicating that transfectedER ${alpha}$ may behave differently from endogenous ER ${alpha}$ . Therefore, thisstudy used E-responsive breast cancer cell lines which expressfunctional AhR and ER ${alpha}$ as models for studying the mechanismsof AhR-dependent degradation of ER ${alpha}$ through activation of proteasomes.Proteasome-dependent degradation of ER ${alpha}$ is dependent on multiplefactors, including ligand structure, cell context, and the formof ER ${alpha}$ expressed (i.e., wild type, chimeric, or mutant) (2, 11,29, 52, 54). For example, one study reported that ligand-mediateddownregulation of ER ${alpha}$ is linked to coactivator recruitment andcoordinate degradation of both receptor and coactivator proteinsin HeLa cells transfected with ER ${alpha}$ or mutant ER ${alpha}$ expression plasmids(29). In contrast, another study indicated that mutations whichblock AF2-interacting coactivator recruitment did not blockproteasome-dependent degradation of mutant ER ${alpha}$ or mutant chimericvp-ER ${alpha}$ protein in HeLa cells (52). Moreover, it was also reportedthat E2 did not induce proteasome-dependent degradation of mutantER ${alpha}$ containing a DBD deletion in HeLa cells (52), whereas ourresults show that TCDD- and E2-induced degradation of ER ${alpha}$ isDBD independent and that the response for TCDD requires bothAF1 and AF2 of ER ${alpha}$ (Fig. 1C). These results clearly distinguishbetween activation of proteasomes by TCDD or E2 through theirrespective receptors, since different domains of ER ${alpha}$ are required.These results also show that the effects of E2 on degradationof ER ${alpha}$ are dependent on cell context (53). Current studies areinvestigating specific regions within AF1 and AF2 of ER ${alpha}$ whichare required for AhR-dependent degradation of ER ${alpha}$ by proteasomes.

TCDD induces kinase activities in some tissues and cells (31);however, results of ongoing studies indicate that TCDD doesnot activate the src-MAPKK or -phosphatidylinositol 3-kinasepathways in ER-positive breast cancer cells (data not shown).Therefore, the results in Fig. 7 showing that a series of kinaseinhibitors did not affect AhR-mediated degradation of ER ${alpha}$ suggestthat activation of kinase-dependent phosphorylation of ER ${alpha}$ byTCDD is not required for degradation of ER ${alpha}$ by proteasomes. Incontrast, MAPK-dependent phosphorylation of S294 of the progesteronereceptor is required for proteasome degradation of this receptorin T47D cells (27), whereas RAR ${alpha}$ 1 degradation by proteasomesis inhibited by phosphorylation (23).

The AhR and ER ${alpha}$ can be coimmunoprecipitated in both MCF-7 andZR-75 cells (Fig. 8), and this interaction was enhanced by TCDD,suggesting a possible mechanism of AhR-induced degradation ofER ${alpha}$ that involves initial ligand (TCDD)-dependent interactionof AhR with ER ${alpha}$ . These interactions were further investigatedin a mammalian two-hybrid assay using pmAhR [GAL4(DBD)-AhR fusionprotein] and vpER ${alpha}$ (VP16-ER ${alpha}$ fusion protein) and treated withsolvent (D), E, or TCDD alone or in combination. Interactionsof AhR and ER ${alpha}$ were dependent on TCDD but not E. This was similarto results of previous studies using GST-AhR and ER ${alpha}$ in pull-downassays (21) and suggests an explanation for the unidirectionalligand-dependent activation of proteasomes in AhR-ER ${alpha}$ cross talk.E does not induce AhR-ER ${alpha}$ interactions in the mammalian two-hybridassay (Fig. 8) and, therefore, activates proteasome-dependentdegradation of ER ${alpha}$ but not the AhR (Fig. 1). Moreover, sincethe AhR and ER ${alpha}$ are cytosolic and nuclear proteins, respectively,addition of E cannot induce colocalization of both proteinsin the nucleus. In contrast, TCDD induces nuclear localizationof the AhR, AhR-ER ${alpha}$ interactions, and proteasome-dependent degradationof the receptors. These results suggest a mechanism (Fig. 9)in which ligand binding to AhR induces formation of the nuclearAhR, which recruits both ER ${alpha}$ and proteasomes, resulting in ubiquitinationof AhR and ER ${alpha}$ and degradation of both proteins. This processis highly specific, since Sp1 protein is not affected by TCDD,even though Sp1 binds to both AhR and ER ${alpha}$ (22, 37). Currently,we are using this model to further investigate AhR-mediateddegradation of specific domains of ER ${alpha}$ and to determine the roleof protein-protein interactions in activating the proteasomepathway for degradation of one or both interacting proteins.

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FIG. 9. Proposed model for AhR-mediated degradation of ER ${alpha}$ through activation of the proteasome pathway. Cytosolic AhR is activated by TCDD ligand, which induces disassociation of the heat shock protein 90 (hsp 90), AhR-interacting protein (AIP), and pp60^Src kinase complex. Activated AhR translocates to the nucleus, which enables direct interaction with nuclear ER ${alpha}$ , subsequent ubiquitination, and proteolysis by the 26S proteasome complex. Both ER ${alpha}$ ubiquitination and proteolysis are increased in cells cotreated with ET. E alone induces degradation of nuclear ER ${alpha}$ but not the cytosolic AhR, and this may be due to lack of receptor colocalization.

ACKNOWLEDGMENTS

The financial assistance of the National Institutes of Health(ES04176 and ES09106), the Texas Agricultural Experiment Station,and the Sid Kyle Endowment are gratefully acknowledged.

FOOTNOTES

* Corresponding author. Mailing address: Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466 TAMU, College Station, TX 77843-4466. Phone: (979) 845-5988. Fax: (979) 862-4929. E-mail: ssafe{at}cvm.tamu.edu.

REFERENCES

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