Suppression of β-Amyloid Precursor Protein Signaling into the ...

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MOLECULAR AND CELLULAR BIOLOGY, Feb. 2007, p. 1321–1333 Vol. 27, No. 4 0270-7306/07/$08.000 doi:10.1128/MCB.01280-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved. Suppression of -Amyloid Precursor Protein Signaling into the Nucleus by Estrogens Mediated through Complex Formation between the Estrogen Receptor and Fe65 Junying Bao, 1,3 Chuanhai Cao, 2 Xiaohui Zhang, 1,3 Feng Jiang, 1,3 Santo V. Nicosia, 1,3 and Wenlong Bai 1,3 * Departments of Pathology and of Cell Biology and Molecular Oncology, USF College of Medicine, 1 and Programs of Molecular Oncology and Drug Discovery, H. Lee Moffitt Cancer Center, 3 Tampa, Florida 33612-4799, and Department of Medical Microbiology and Johnnie B. Byrd, Sr., Alzheimer’s Center & Research Institute, USF College of Medicine, Tampa, Florida 33612-4799 2 Received 13 July 2006/Returned for modification 15 August 2006/Accepted 16 November 2006 The C-terminal fragment of the -amyloid precursor protein produced after cleavage by -secretase, namely, APPct or AICD, has been shown to form a multimeric complex with the adaptor protein Fe65 and to regulate transcription through the recruitment of the histone acetyltransferase Tip60. The present study shows that 17-estradiol inhibits the transcriptional and apoptotic activities of the APPct complex by a process involving the interaction of estrogen receptor alpha (ER) with Fe65. ER-Fe65 complexes were detected both in vitro and in the mouse brain, and recruitment of ER to the promoter of an APPct target gene (KAI1) was demonstrated. Our studies reveal a novel mechanism of estrogen action, which may explain the well-known neuroprotective functions of estrogens as well as the complex role of this female hormone in the pathogenesis of neuronal degeneration diseases. Estrogens are pleiotropic hormones that regulate the growth and differentiation of many diverse tissues. The actions of estrogens are mediated through estrogen receptors alpha and beta (ER and ER, respectively), which belong to the steroid/ thyroid nuclear receptor superfamily, a group of ligand-regu- lated, zinc finger-containing transcription factors (11, 45). The two ERs share a similar structural organization composed of an amino-terminal A/B region containing activation function 1 (AF-1), a central DNA binding domain (DBD), and a carboxyl- terminal ligand-binding domain (LBD) containing activation function 2 (AF-2). While AF-1 of the ER is constitutively active, the activity of AF-2 is strictly ligand dependent. In response to ligand activation, the ER forms a homodimer that binds estrogen response elements (EREs) and recruits multi- ple transcriptional coactivator complexes (23), leading to tran- scriptional stimulation. Besides their well-established role in the female reproduc- tive system, estrogens also play an important role in regulating the neuronal activities of the pituitary gland, the hypothala- mus, and other specific brain regions. It is generally believed that estrogens are neuroprotective and that the decrease in their abundance after menopause contributes to the develop- ment of neurodegenerative diseases such as Alzheimer’s disease (AD). Multiple animal experiments and molecular analyses overwhelmingly support a neuroprotective role for estrogens (14, 31, 46). For example, estrogens have been shown to pro- tect neurons from ischemic stroke, an activity that depends on ER (9). 17-Estradiol was shown to exert neurotrophic ef- fects in tissue explants derived from the developing mouse hypothalamus and preoptic area (44). Synaptogenic effects of 17-estradiol were demonstrated to occur in adult brain re- gions, such as the hippocampus, that are important for cogni- tive function (17, 51). For various models of neuronal damage, such as that caused by ischemic stroke, estrogens were re- ported to enhance neuronal survival (31, 46, 50). The -amyloid precursor protein (APP) plays important roles in AD pathogenesis. It is a membrane-spanning protein with classical features of an orphan G protein-coupled recep- tor that is physiologically processed by cleavages with - or - and -secretase. After cleavage by -secretase, APP is further cleaved in the transmembrane region by -secretase. The re- sulting extracellular amyloid -peptide (A) is deposited in the senile plaques in the brains of AD patients, which are consid- ered the hallmark of AD. Besides its role in neurodegenera- tion, APP has multiple cellular functions, which include cell adhesion and motility (37), synaptic transmission and plasticity (38), and memory (7). It is also important that APP is ubiqui- tously expressed and not restricted to the brain. Increased expression of APP protein has been described for oral squa- mous cell carcinoma (24), suggesting additional biological functions for APP in nonneuronal and cancer cells. Perhaps one of the most interesting findings about APP in recent times is the demonstration of its potential role in tran- scriptional regulation (4). Besides A, -cleavage of APP re- leases its intracellular C-terminal fragment, or APPct, which moves to the nucleus with Fe65 (20, 22). Fe65 binds Tip60 (34), a histone acetyltransferase that is involved in regulating higher-order chromosome structure and consequential gene expression (21). In the present study, we demonstrate that estrogens inhibit the transcriptional activity of and apoptosis induced by the APPct transcriptional complex. Mechanistic * Corresponding author. Mailing address: Department of Pathology, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., MDC 11, Tampa, FL 33612-4799. Phone: (813) 974- 0563. Fax: (813) 974-5536. E-mail: [email protected]. † Present address: Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306-4300. Published ahead of print on 27 November 2006. 1321 on March 12, 2018 by guest http://mcb.asm.org/ Downloaded from

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MOLECULAR AND CELLULAR BIOLOGY, Feb. 2007, p. 1321–1333 Vol. 27, No. 40270-7306/07/$08.00�0 doi:10.1128/MCB.01280-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Suppression of �-Amyloid Precursor Protein Signaling into the Nucleusby Estrogens Mediated through Complex Formation between

the Estrogen Receptor and Fe65�

Junying Bao,1,3 Chuanhai Cao,2 Xiaohui Zhang,1,3 Feng Jiang,1,3†Santo V. Nicosia,1,3 and Wenlong Bai1,3*

Departments of Pathology and of Cell Biology and Molecular Oncology, USF College of Medicine,1 and Programs of Molecular Oncology andDrug Discovery, H. Lee Moffitt Cancer Center,3 Tampa, Florida 33612-4799, and Department of Medical Microbiology and

Johnnie B. Byrd, Sr., Alzheimer’s Center & Research Institute, USF College of Medicine, Tampa, Florida 33612-47992

Received 13 July 2006/Returned for modification 15 August 2006/Accepted 16 November 2006

The C-terminal fragment of the �-amyloid precursor protein produced after cleavage by �-secretase, namely,APPct or AICD, has been shown to form a multimeric complex with the adaptor protein Fe65 and to regulatetranscription through the recruitment of the histone acetyltransferase Tip60. The present study shows that17�-estradiol inhibits the transcriptional and apoptotic activities of the APPct complex by a process involvingthe interaction of estrogen receptor alpha (ER�) with Fe65. ER�-Fe65 complexes were detected both in vitroand in the mouse brain, and recruitment of ER� to the promoter of an APPct target gene (KAI1) wasdemonstrated. Our studies reveal a novel mechanism of estrogen action, which may explain the well-knownneuroprotective functions of estrogens as well as the complex role of this female hormone in the pathogenesisof neuronal degeneration diseases.

Estrogens are pleiotropic hormones that regulate the growthand differentiation of many diverse tissues. The actions ofestrogens are mediated through estrogen receptors alpha andbeta (ER� and ER�, respectively), which belong to the steroid/thyroid nuclear receptor superfamily, a group of ligand-regu-lated, zinc finger-containing transcription factors (11, 45). Thetwo ERs share a similar structural organization composed ofan amino-terminal A/B region containing activation function 1(AF-1), a central DNA binding domain (DBD), and a carboxyl-terminal ligand-binding domain (LBD) containing activationfunction 2 (AF-2). While AF-1 of the ER is constitutivelyactive, the activity of AF-2 is strictly ligand dependent. Inresponse to ligand activation, the ER forms a homodimer thatbinds estrogen response elements (EREs) and recruits multi-ple transcriptional coactivator complexes (23), leading to tran-scriptional stimulation.

Besides their well-established role in the female reproduc-tive system, estrogens also play an important role in regulatingthe neuronal activities of the pituitary gland, the hypothala-mus, and other specific brain regions. It is generally believedthat estrogens are neuroprotective and that the decrease intheir abundance after menopause contributes to the develop-ment of neurodegenerative diseases such as Alzheimer’s disease(AD). Multiple animal experiments and molecular analysesoverwhelmingly support a neuroprotective role for estrogens(14, 31, 46). For example, estrogens have been shown to pro-tect neurons from ischemic stroke, an activity that depends on

ER� (9). 17�-Estradiol was shown to exert neurotrophic ef-fects in tissue explants derived from the developing mousehypothalamus and preoptic area (44). Synaptogenic effects of17�-estradiol were demonstrated to occur in adult brain re-gions, such as the hippocampus, that are important for cogni-tive function (17, 51). For various models of neuronal damage,such as that caused by ischemic stroke, estrogens were re-ported to enhance neuronal survival (31, 46, 50).

The �-amyloid precursor protein (APP) plays importantroles in AD pathogenesis. It is a membrane-spanning proteinwith classical features of an orphan G protein-coupled recep-tor that is physiologically processed by cleavages with �- or �-and �-secretase. After cleavage by �-secretase, APP is furthercleaved in the transmembrane region by �-secretase. The re-sulting extracellular amyloid �-peptide (A�) is deposited in thesenile plaques in the brains of AD patients, which are consid-ered the hallmark of AD. Besides its role in neurodegenera-tion, APP has multiple cellular functions, which include celladhesion and motility (37), synaptic transmission and plasticity(38), and memory (7). It is also important that APP is ubiqui-tously expressed and not restricted to the brain. Increasedexpression of APP protein has been described for oral squa-mous cell carcinoma (24), suggesting additional biologicalfunctions for APP in nonneuronal and cancer cells.

Perhaps one of the most interesting findings about APP inrecent times is the demonstration of its potential role in tran-scriptional regulation (4). Besides A�, �-cleavage of APP re-leases its intracellular C-terminal fragment, or APPct, whichmoves to the nucleus with Fe65 (20, 22). Fe65 binds Tip60(34), a histone acetyltransferase that is involved in regulatinghigher-order chromosome structure and consequential geneexpression (21). In the present study, we demonstrate thatestrogens inhibit the transcriptional activity of and apoptosisinduced by the APPct transcriptional complex. Mechanistic

* Corresponding author. Mailing address: Department of Pathology,University of South Florida College of Medicine, 12901 Bruce B.Downs Blvd., MDC 11, Tampa, FL 33612-4799. Phone: (813) 974-0563. Fax: (813) 974-5536. E-mail: [email protected].

† Present address: Department of Biomedical Sciences, FloridaState University College of Medicine, Tallahassee, FL 32306-4300.

� Published ahead of print on 27 November 2006.

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investigations reveal that the transcriptional repression is dueto the “tethering” of ER� to the APPct complex through Fe65.The potential functional significance of this inhibition and itsrelevance to the well-documented neuroprotective activities ofestrogens and to their suspected role in preventing AD arediscussed.

MATERIALS AND METHODS

Materials. 17�-Estradiol was purchased from Sigma (St. Louis, MO). AlexaFluor 594–goat anti-mouse immunoglobulin G (IgG [heavy plus light chains]),Alexa Fluor 488–goat anti-rabbit IgG, and Alexa Fluor 594–chicken anti-goatIgG (heavy plus light chains) were obtained from Molecular Probes (Eugene,OR). Anti-hemagglutinin (anti-HA; 12CA5) antibody was obtained from Roche.Gal4 DBD antibody was purchased from BD Biosciences Clontech (Palo Alto,CA). Anti-FE65 (clone 3H6) was purchased from Upstate (Charlottesville, VA).The following antibodies were obtained from Santa Cruz Biotechnology (SantaCruz, CA): anti-ER� (F-10 and H-184), -Gal4 (DBD) (RK5C1), -c-Myc (9E-10and A-14), -KAI1 (C-16), -�-amyloid (D-17), and -amyloid A4 (N-18). Anti-APPC-terminal antibody (CT-15) has been described previously (25). Lipofectaminewas purchased from Gibco-BRL Life Technologies (Rockville, MD). The ECLreagents for immunoblotting were obtained from Amersham Pharmacia BiotechInc. (Piscataway, NJ). Luciferase assay systems were obtained from PromegaCorporation (Madison, WI), and 4�,6�-diamidino-2-phenylindole (DAPI) mount-ing medium was obtained from Vector Laboratories (Burlingame, CA). All otherreagents were reagent grade.

Expression vectors for APP, Tip60, and Fe65 were gifts from Thomas C.Sudhof (4). pRST7-ER�, ER282G, ER179C, and ER-TAF1 were gifts fromDonnald P. McDonnell (47). pLEN-hER�, pLEN-hER�Y537A (26, 49),pLEN�gal, EREe1bLuc (26), and Galluc (53) were described previously.

Tissue preparation and sectioning. For immunofluorescence and chromatinimmunoprecipitation (CHIP) studies, 5-month-old wild-type and B6C3 APPswe/PSEN1(A246E) transgenic mice were deeply anesthetized with pentobarbital (50mg/kg of body weight) and perfused transcardially with 0.1 M phosphate-buff-ered saline (PBS; pH 7.4) followed by ice-cold phosphate-buffered 4% paraform-aldehyde. Brains were taken out of the skulls, fixed by submersion in 4% para-formaldehyde in 0.1 M phosphate buffer for 24 h at 4°C, and stored at 4°C inphosphate-buffered sucrose (30%). Tissues were sectioned serially at 30 �m ona freezing microtome and then mounted onto aminopropyltriethoxysilane-coatedglass slides.

Transfections and reporter assays. HeLa and ER-positive Ishikawa cells wereplated on Dulbecco’s modified Eagle’s medium containing 10% fetal bovineserum at 1.5 � 105 cells/well in six-well plates. One day after plating, transfec-tions were performed with Lipofectamine. After transfection, cells were placedin Dulbecco’s modified Eagle’s medium containing 1% charcoal-stripped fetalbovine serum and treated with vehicle and estrogens. At 48 h posttransfection,cellular extracts were prepared by directly adding lysis buffer containing 25 mMTris-phosphate (pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N�,N�,N�,N�-tetra-acetic acid, 10% glycerol, and 0.2% Triton X-100 to the cellson ice. Luciferase activity was determined with luciferase assay systems followingthe company’s protocol. �-Galactosidase activity was determined as previouslydescribed (27, 28).

Immunoprecipitation and immunoblotting analyses. To prepare cells for assayof the interaction of ectopically expressed proteins, cells were plated in Dulbec-co’s modified Eagle’s medium containing 10% fetal bovine serum at 1.0 � 106

cells per 100-mm dish. One day after plating, transfections were performed withLipofectamine. Transfected cells were placed in Dulbecco’s modified Eagle’smedium containing 1% charcoal-stripped fetal bovine serum and treated withdifferent reagents. Seventy-two hours later, cellular extracts were prepared in abuffer containing 20 mM Tris-HCl (pH 7.5), 0.5% NP-40, 250 mM NaCl, 3 mMEDTA, 3 mM EGTA, 10 �g of aprotinin per ml, 10 �g of leupeptin per ml, 10mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride.

For assay of the interaction of endogenous proteins, mouse brain tissue wasremoved and homogenized in a lysis buffer containing 50 mM Tris (pH 7.4), 1mM dithiothreitol, and 0.1 mM EDTA for 10 strokes on ice with a Polytronhomogenizer. The homogenate was then sonicated for 15 seconds on ice.

For immunoprecipitations, cellular and mouse brain extracts were then incu-bated with 4 �g antibodies overnight at 4°C and subsequently with proteinG-agarose beads for an additional hour. The agarose beads were then washedthree times, and immunoprecipitates were heated in sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis sample buffer.

For immunoblotting, cellular extracts or immunoprecipitates were separated

in SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, andprobed with different antibodies. Proteins were detected with ECL reagents.

GST pull-down assays. ER� and Fe65 were translated in vitro from PRST-ER� and HA-Fe65, respectively, using T7 polymerase and a coupled transcrip-tion/translation kit (Promega, Madison, WI). GST-APPct protein was producedin and purified from Escherichia coli strain BL21(DE3). Glutathione S-trans-ferase (GST) pull-down assays were performed with glutathione beads as de-scribed previously (29). The proteins in the precipitates were detected by immu-noblotting analysis.

Confocal and deconvolution immunofluorescence imaging. Cells grown oncoverslips and frozen sections of mouse brain mounted on glass slides werewashed with PBS three times and fixed in 2% paraformaldehyde for 15 min atroom temperature. After additional washing with PBS, fixed cells were madepermeable with 1% Triton X-100 and 1% bovine serum albumin for cells or withgoat serum for tissue. The slides were then incubated with primary antibodies for2 h at room temperature or overnight at 4°C, followed by incubation withsecondary antibodies, i.e., IgG conjugated to Alexa Fluor 594 for red or tofluorescein isothiocyanate for green, for another 1 to 2 h at room temperature.The slides were then washed three times in PBS and stained with DAPI inantifade mounting medium. Fluorescent images were obtained with a confocallaser scanning microscope or a Leitz Orthoplan 2 microscope.

CHIP assays. For CHIP assays, mouse brain tissues were prepared as de-scribed above. Cells were plated in Dulbecco’s modified Eagle’s medium con-taining 10% fetal bovine serum at 1.0 � 106 cells per 100-mm dish. One day afterplating, transfections were performed with Lipofectamine. Transfected cellswere placed in phenol red-free Dulbecco’s modified Eagle’s medium containing1% charcoal-stripped fetal bovine serum and treated with ethanol (EOH) orestradiol (E2). Seventy-two hours later, cells were washed with PBS and cross-linked with 1% formaldehyde. After being washed twice with cold PBS, cellswere lysed in buffer containing 5 mM PIPES [piperazine-N,N�-bis(2-ethanesul-fonic acid) (pH 8.0)], 85 mM KCl, 0.5% Nonidet P-40, and protease inhibitors.Cell nuclei were pelleted and resuspended in a buffer containing 50 mM Tris-Cl(pH 8.1), 10 mM EDTA, 1% SDS, and protease inhibitors.

Soluble chromatin was prepared by sonication of transfected cells or braintissue, with an average size of sheared fragments of about 400 bp, and diluted inbuffer containing 16.7 mM Tris-Cl (pH 8.1), 0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 167 mM NaCl, and protease inhibitors. Immunoprecipitates wereprepared as described above, and DNA fragments were detected by PCR, using1 �l of a 50-�l DNA extract as the template for 25 cycles. The sequences of thePCR primers for the human KAI1 gene promoter were 5�-GGATGGGGTGGGCTCGAAG-3� (upstream) and 5�-CGCCCCCAGAAGACACGC-3� (down-stream). For the mouse KAI1 gene promoter, the sequences of the primers were5�-ACCGTTAGGCAGCGCCGTGAG-3� (upstream) and 5�-CTTGGGAAGGCGGTGCGCTC-3�. The amplified DNA fragment corresponds to positions�413 to �69 of the KAI1 gene promoter, containing an NF-B site (2, 8, 39).

Cell survival and apoptosis assays. Transfected cells were washed with phos-phate-buffered saline and fixed in 3.7% formaldehyde. The viability of trans-fected cells in each well was determined by counting the total number of greencells in each well under a fluorescence microscope. For the detection of apoptoticcells, fixed cells were stained with DAPI, and cells positive for fluorescence wereobserved with a Leitz Orthoplan 2 microscope. Representative micrographs werecaptured by a charge-coupled device camera with the Smart Capture program(Vysis, Downers Grove, IL). The apoptotic indices of transfected cells weredetermined by scoring 300 green fluorescent protein (GFP)-positive cells forchromatin condensation and apoptotic body formation.

Caspase-3 activity assay. Caspase-3 activity was determined by use of acaspase-3 fluorometric assay kit (R&D Systems), following a protocol from thevender. Absorption at 405 nm was measured in an MRX microplate reader(Dynex Technologies, Chantilly, VA). Caspase-3 activity is expressed in opticaldensity units (at 405 nm) per �g of protein.

RESULTS

17�-Estradiol inhibits the Fe65-dependent transcriptionalactivity of the APPct protein fused to Gal4 DNA binding do-mains. To test the effect of estrogens on the activity of theAPPct complex, ER-negative HeLa cells were transfected withFe65 and Gal4-APPct together, with or without ER�. Theactivity of the cotransfected Gal4 reporter was determined inthe presence or absence of 17�-estradiol. As shown in Fig. 1A,cells transfected with a mutant APPct that does not interact

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with Fe65, APPct� (4), contained no detectable reporter activ-ity, whereas those with wild-type APPct contained largeamounts of activity, confirming the transcriptional activity as-sociated with APPct and its dependency on Fe65 (4). In cellsexpressing ER�, 17�-estradiol reduced the activity of theAPPct complex (Fig. 1A). Inhibition by 17�-estradiol was notobserved in cells lacking ER� expression, and the degree ofinhibition depended on the amount of ER� (Fig. 1A and B).17�-Estradiol did not affect Gal4-APPct or Fe65 expression(Fig. 1B) and thus apparently inhibits the specific activity ofGal4-APPct per molecule. As expected, 17�-estradiol inducedthe activity of an ERE-based reporter, and the degree of in-duction depended on the amount of ER� (Fig. 1C). Ectopi-cally expressed ER� is known to have constitutive transcrip-tional activity, probably explaining why the expression of ER�also decreased the activity of the APPct complex to a smallextent in the absence of 17�-estradiol (Fig. 1A).

To rule out the possibility that the inhibition of APPct ac-tivity by estrogens is an artifact of ectopic ER� expression inER-negative cells, the transcriptional activity of the APPctcomplex was measured in ER-positive Ishikawa cells in thepresence or absence of 17�-estradiol. As shown in Fig. 1D,17�-estradiol significantly reduced the activity of the APPctcomplex. The degree of reduction increased as the time of

treatment was prolonged. The data suggest that endogenousER� is sufficient to mediate the inhibition of the APPct com-plex by estrogens.

ER� forms a complex with the Fe65-APPct complex. ER�interacts with the transcriptional coactivator Tip60 (3), as doesthe APPct complex (4). Thus, ER� may inhibit the activity ofthe complex by sequestering Tip60. To test this possibility, weasked whether ectopic expression of Tip60 would negate theinhibitory effects of 17�-estradiol on APPct activity in cellsexpressing APPct and ER�. As shown in Fig. 2A, 17�-estradiolinhibited the activity of the APPct complex in cells cotrans-fected with Tip60. The degree of inhibition was comparable tothat in cells lacking ectopic Tip60 expression (Fig. 1A). Thisfinding suggests that activated ER� does not inhibit APPctactivity by competing with the APPct complex for the limitedpool of Tip60. Ectopic Tip60 decreased APPct activity, likelydue to the “squelching” of other cofactors away from theAPPct transcriptional complex.

To define the mechanism underlying the estrogen inhibitionof APPct activity, coimmunoprecipitation analyses were per-formed to test whether ER� forms a complex with Fe65, theadaptor essential for APPct to regulate transcription (4). InHeLa cells coexpressing HA-Fe65 and ER�, both proteinswere present in anti-HA immunoprecipitates, regardless of

FIG. 1. Inhibition of transcriptional activity of Gal4-APPct by estrogens. (A) HeLa cells were transfected with 0.5 �g of 3�17merLuc, 0.1 �gof pLEN�Gal, 0.3 �g of HA-Fe65, 0.3 �g of Gal4-APPct� (APPct�) or Gal4-APPct (APPct), and the indicated amounts of pLENhER� (ER�)and treated with 10�8 M 17�-estradiol (E2) or ethanol (EOH) as a vehicle control for 48 h. Luciferase activity was determined and normalizedwith cognate �-galactosidase activity. (B) Cells were transfected and treated as described for panel A. Immunoblotting analyses were performedwith the indicated antibodies. (C) HeLa cells were transfected with 0.5 �g EREe1bLuc, 0.5 �g pLEN�Gal, and the indicated amounts ofpLENhER�. Cells were treated and luciferase activity determined as described for panel A. (D) ER-positive Ishikawa cells were transfected asdescribed for panel A, but without pLENhER�. Cells were treated, and luciferase activity was determined.

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whether cells received vehicle or 17�-estradiol (Fig. 2B, leftpanels). Because 17�-estradiol decreased the level of totalER� protein in the cells, the data in Fig. 2B suggest that thehormone enhances the specific interaction between ER� andFe65, even though the ER� signals are comparable betweenanti-HA immunoprecipitates prepared from cells treated withvehicle and 17�-estradiol. Anti-HA antibody did not coprecipi-tate ER� in the absence of HA-Fe65 and thus does not cross-react with ER�. The protein detected in anti-HA precipitatesof cotransfected cells was indeed ER�, as it was only detectedwhen ER� was expressed. Reciprocal coimmunoprecipitationanalyses showed that anti-ER� also precipitated Fe65 togetherwith ER�, confirming the complex formation (Fig. 2B, rightpanels).

Since Fe65 forms a complex with both APPct and ER�, it isvery likely that ER� and APPct coexist in the same complex,using Fe65 as an intermediate. Consistent with this idea, ourimmunological analyses detected little interaction (Fig. 3A) ofectopically expressed ER� and Gal4-APPct in HeLa cells with-out Fe65. In cells cotransfected with HA-Fe65, however, ER�

and Gal4-APPct were able to interact, as indicated by thepresence of Gal4-APPct in anti-ER� precipitates (Fig. 3A).This interaction occurred in the presence of either vehicle or17�-estradiol.

To clearly show the Fe65-dependent association of ER� andAPPct, pull-down assays were performed using GST-APPctand recombinant Fe65 and ER� produced by in vitro tran-scription-coupled translation reactions. When all three pro-teins were included in the assay, GST-APPct precipitated bothER� and Fe65 (Fig. 3B). GST alone had no effect, and GST-APPct did not precipitate ER� in the absence of Fe65. Coo-massie blue staining showed that equal amounts of GST-APPctprotein were used for the pull-down reactions.

Using GST pull-down assays, we further mapped the domainof ER� that interacts with Fe65 and APPct (Fig. 4A). Inreaction mixtures containing Fe65 in vitro, similar amounts offull-length ER� protein and truncated ER� protein lacking theamino-terminal A/B region (ER179C) were specifically precip-itated by GST-APPct. Consistently, the ER� protein lackingthe carboxyl-terminal LBD (ERN282G) did not interact withFe65 and APPct in vitro. More importantly, GST-APPct failedto precipitate a full-length ER� protein that was mutated atthree amino acids essential for AF-2 activity (ER-TAF1) (47).Consistent with the binding analyses, reporter assays showedthat 17�-estradiol inhibited APPct activity in cells expressingN-terminally truncated ER� but did not inhibit APPct activityin cells expressing C-terminally truncated ER� or the AF-2mutant (Fig. 4B). Overall, the analyses show that the AF-2region is essential, whereas the amino-terminal A/B region,including AF-1, is expendable for the interaction of ER� withFe65 and APPct. They also show that the region required forinteraction is also required for inhibition of the activity of theAPPct complex by estrogens.

Interaction and colocalization between endogenous ER�,Fe65, and APP in mouse brains. Since full-length APP inter-acts with Fe65 through its carboxyl-terminal region, it shouldalso interact with ER� in the same way as does Gal4-APPct.Indeed, anti-ER� antibody coprecipitated APP from ex-tracts of HeLa cells cotransfected with ER�, APP, and Fe65

FIG. 2. Complex formation between ectopic ER� and Fe65 inHeLa cells. (A) Cells were transfected as described in the legend toFig. 1A, with or without 0.3 �g of HA-Tip60, and treated for 48 h.Luciferase activity was determined and normalized with �-galactosi-dase activity. (B) Cells were transfected with 2 �g of pLENhER�, withor without 4 �g of HA-Fe65, and treated with EOH or 10�8 M E2 for72 h. Immunoprecipitation (IP) analyses were performed, followed byimmunoblotting (IB) with the indicated antibodies.

FIG. 3. Complex formation between ER� and APPct mediatedthrough Fe65. (A) HeLa cells were transfected with 4 �g of Gal4-APPct and pLENhER�, with or without 4 �g of HA-Fe65, and treatedfor 72 h. Coimmunoprecipitations were performed with the indicatedantibodies. (B) ER� protein, either alone (lanes 3 and 4) or togetherwith Fe65 protein (lanes 1 and 2), was produced by in vitro transcrip-tion-coupled translation in the absence or presence of E2. GST pull-down assays were performed, and the precipitated proteins were de-tected by immunoblotting. An image of Coomassie blue (C.B.) stainingwas included to show that similar amounts of GST and GST-APPctwere used for the pull-down assays.

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(Fig. 5A). ER� did not interact with APP in the absence ofectopic Fe65 or with APP containing a mutation in its Fe65binding site (APP�). These data demonstrate the Fe65 depen-dence of the APP-ER� interaction.

Although transcriptional activity associated with APPct hasbeen demonstrated nicely in transfection assays, endogenousAPPct in neuronal cells is difficult to detect at the protein level,presumably due to its instability after cleavage by �-secretase(20). Since ER� interacts with full-length APP in cotransfec-tion assays (Fig. 5A), we performed coimmunoprecipitationsto determine whether endogenous ER�, Fe65, and APP inter-act in the brain tissues of control mice and transgenic mice thatexpress APP with the “Sweden” mutation and presenilin 1 witha deletion of exon 9, a mutation that corresponds to a form ofearly-onset AD (19). This transgenic mouse strain was in-cluded in the analyses because it contains increased APP and,presumably, increased APPct due to increased APP as well as�-secretase activity as a result of increased presenilin 1 expres-sion. As shown in Fig. 5B, anti-Fe65 antibody, but not rabbitIgG, coimmunoprecipitated endogenous ER� and Fe65 frombrain homogenates of control mice. In reciprocal analyses,anti-ER� antibody coprecipitated both endogenous Fe65 andAPP (Fig. 5C). More Fe65 and APP were coprecipitated fromtransgenic than from wild-type mice. The levels of Fe65 andER� expression were comparable among wild-type and trans-genic mice, and as expected, the level of APP expression washigher in transgenic than in nontransgenic mice (Fig. 5D). Itappears that the interaction between Fe65 and ER� is greaterin transgenic than in wild-type mice. The exact reason is un-known, but it is possible that greater APP expression somehowenhances the interaction through feedback.

Colocalization analyses with immunofluorescence, using sec-tions cut through the hippocampus area (Fig. 5E1), were per-formed to confirm the interaction between ER�, Fe65, andAPP in vivo. In this experiment, endogenous Fe65 and ER�(Fig. 5E2) were coexpressed in the majority of the neurons,and confocal imaging showed that the colocalization mainlyoccurred in the nucleus. In contrast to ER�, which is predom-inantly localized in the nucleus, APP is predominantly local-ized in the cytoplasm (Fig. 5E3). However, there were neuronsin which cytoplasmic ER� or nuclear APP was detected (Fig.5E3). In those cells, colocalization with ER� in the cytoplasmwas also detected by confocal imaging. Overall, these immu-nological analyses show that the interaction between ER�,Fe65, and APP demonstrated in cell lines occurs in vivo inmouse brains.

Suppression of the ability of the APPct complex to induceKAI1 expression by estrogens via recruitment of ER� to thepromoter. Since the transcriptional activity of the APPct com-plex has just recently been realized, only a few target genes areknown. These include the gene encoding KAI1, or tetraspanin(2, 32), a cell surface molecule acting as a tumor metastasissuppressor. Therefore, we tested the effects of estrogens andthe APPct complex on the activity of the KAI1 gene promoterin ER�-positive N2a neuroblastoma cells. Using a GFP vectoras a marker, we first showed that 40% of N2a cells weresusceptible to transfection under our conditions (data notshown). We next showed that ectopic expression of APPct,Fe65, and Tip60 increased the expression of KAI1 and thattreatment of cells with 17�-estradiol suppressed the induction(Fig. 6A). This experiment showed that the hormone-mediatedinactivation of the APPct complex demonstrated in reporter

FIG. 4. Defining the domain of ER� required for interaction with and inhibition of the APPct complex. (A) Full-length ER� (lanes 1), ER�with a truncated N-terminal A/B region (ER179C) (lanes 2), ER� with a truncated C-terminal LBD (ER282G) (lanes 3), and an ER� mutant withinactive AF-2 (ER-TAF1) (lanes 4) were produced together with Fe65 by in vitro transcription/translation reactions in the presence of E2. GSTpull-down assays were performed. Precipitated proteins were detected by immunoblotting analyses with anti-ER� (C-terminal, top panel),anti-ER� (N-terminal, middle panel), and anti-Fe65 (lower panel) antibodies. Coomassie blue (C.B.) staining data were included to show thatsimilar amounts of GST and GST-APPct were used for the pull-down assays. (B) HeLa cells were transfected and treated as described in the legendto Fig. 1A, except that 0.3 �g of PRST7-hER� or a deletion mutant was used for transfections. Luciferase activity was determined and normalizedwith cognate �-galactosidase activity.

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assays can be translated into the suppression of endogenousgene expression in neuronal cells. The estrogen suppressionwas reversed by cotreatment with the antiestrogen ICI 182,780,showing that the estrogen effect is mediated through the en-dogenous ER.

To test whether ER� is recruited to the promoter of theKAI1 gene, 293 cells were transfected with Fe65, Tip60, andAPP together, with or without ER�, and the presence of ER�,Fe65, Tip60, and APP on the promoter was analyzed by CHIPassays. Mutant APP (APP�) that lacks the ability to interactwith Fe65 was included as a control. As shown in Fig. 6B,Tip60 was recruited to the promoter of the KAI1 gene in cellsexpressing wild-type APP but not mutant APP. Interestingly,ER� was recruited to the KAI1 gene promoter, which wasenhanced by treatment of cells with 17�-estradiol (Fig. 6B).Because 17�-estradiol decreases total amounts of ER� (datanot shown), the estrogen regulation of the recruitment is moreprofound than what appears in Fig. 6B. Concomitant with theincreased ER� recruitment in 17�-estradiol-treated cells, theamounts of Tip60 on the promoter were decreased. Immuno-

blotting analysis showed that 17�-estradiol did not alter thelevels of APP, Fe65, and Tip60 expression in the transfectedcells (data not shown). These data suggest that the inhibition ofthe transcriptional activity of the APPct complex by estrogensis due to the interference of the recruitment of coactivators topromoters by ER�.

Further CHIP analyses showed that treatment with 17�-estradiol for 45 min increased ER� and reduced Tip60 recruit-ment to the KAI1 gene promoter, suggesting that the estrogeneffect does not require long estrogen treatments (Fig. 6C).More importantly, in cells lacking Fe65 expression, ER� wasnot recruited to the KAI1 gene promoter. This demonstratesthat ER� occupancy depends on the presence of the APPct-Fe65 complex. In mouse brains, ER� was recruited to thepromoter of the KAI1 gene together with other components ofthe APPct complex (Fig. 6D), indicating that the CHIP dataobtained with transfected proteins truly reflect what happensin vivo in mouse brains.

Estrogen suppression of neuronal cell apoptosis induced bythe APPct complex. To determine whether inhibition of APPct

FIG. 5. Interaction and colocalization of endogenous ER�, Fe65, and APP in brains of APP and presenilin 1 double-transgenic (TG) andnontransgenic (non-TG) mice. (A) HeLa cells were transfected with 2 �g of pLENhER� and 4 �g of HA-Fe65, together with 4 �g of APP or APPmutant (APP�), and treated for 72 h. The interaction between full-length APP and ER� was determined by coimmunoprecipitation. (B to D)Coimmunoprecipitations were performed with homogenized brain tissues of female mice. The levels of endogenous proteins in mouse brain tissuesor the precipitates were determined by immunoblotting. Extracts of HeLa cells transfected with control or ER�, Fe65, and APP expression vectorswere included as controls. One mouse brain tissue was used per assay, and the data were reproduced twice. (E) Imaging analyses of female mousebrain sections through the area of the hippocampus. (E1) Coronal section through the hippocampus, with high-intensity Fe65 signal. (E2) Confocalimages of tissue sections of a selected hippocampus area (see the white square in panel E1) after double staining for endogenous Fe65 (red) andER� (green). (E3) Confocal images of tissue sections of the same hippocampus area after double staining for endogenous APP (red) and ER�(green).

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activity by estrogen had biological consequences, we per-formed the following experiment. N2a cells were transfectedwith GFP, APPct, Fe65, and either Tip60 or vector alone. Thetransfected cells were then treated with ethanol or 17�-estra-diol, and the number of GFP-positive cells in each well wascounted. Ectopic expression of APPct and Fe65 reduced thenumber of N2a cells, and coexpression of Tip60 slightly accen-tuated this effect (Fig. 7A). Treatment of cells with 17�-estra-diol prevented the decrease in cell number induced by theAPPct-Fe65-Tip60 complex.

To determine whether the decrease in the number of GFP-positive cells by the APPct complex involves apoptosis, trans-fected N2a cells were fixed and stained with DAPI, and thenuclear morphology of green cells was examined for features ofapoptosis under a fluorescence microscope. As shown by rep-resentative micrographs in Fig. 7B, cells transfected withempty vector displayed a normal morphology similar to thatof surrounding nontransfected cells. Cells transfected withAPPct, Fe65, and Tip60 and treated with ethanol frequentlydisplayed nuclear condensation or fragmentation, an apoptoticmorphology. In contrast, cells transfected with APPct, Fe65,and Tip60 and treated with 17�-estradiol showed a normalmorphology similar to that of cells transfected with emptyvector. The data suggest that apoptosis is at least part of the

mechanism responsible for the decrease in cell number in-duced by the APPct complex.

The apoptotic index was then determined by calculating thepercentage of apoptotic cells in 300 randomly selected greencells. As shown in Fig. 7C, apoptotic indices for EOH-treatedcells were 8% for control cells (vector only), 20% for cellsreceiving APPct and Fe65, and 28% for cells receiving APPct,Fe65, and Tip60. Apoptotic indices for 17�-estradiol-treatedcells were similar to those for control cells, regardless ofwhether they expressed APPct and Fe65 or APPct, Fe65, andTip60. These analyses show that the APPct complex inducesthe apoptosis of N2a cells and that this function is suppressedby estrogen treatment.

To test whether the estrogen suppression of apoptosis in-duced by the APPct complex also occurs in differentiated neu-rons, N2a cells were induced to differentiate by serum with-drawal in combination with cyclic AMP treatment (Fig. 7D).Cells were then transfected with APPct, Fe65, and Tip60 andassayed for apoptosis in the presence of 17�-estradiol or vehi-cle. In the absence of 17�-estradiol, Fe65 and Tip60 did notinduce the apoptosis of differentiated N2a cells, but APPct wasslightly apoptotic; 17�-estradiol treatment had little effect onthe basal level of apoptosis (Fig. 7E). Transfection of APPctwith Fe65 and with both Fe65 and Tip60 increased the per-

FIG. 6. Induction of KAI1 by the APPct complex and its suppression by 17�-estradiol. (A) N2a cells were transfected with 2 �g of controlvector, Gal4-APPct, HA-Fe65, and HA-Tip60, as indicated. After treatment with EOH, 10�8 M E2, or 10�8 M E2 plus 10�7 M ICI 182,780 for48 h, the expression of KAI1 and �-actin was determined by immunoblot analyses. The �-actin blot was included as a loading control. (B) 293 cellswere transfected with 4 �g of HA-Fe65, Myc-Tip60, pLENhER� or control vector, and pCMV-APP or pCMV-APP�. After treatment with EOHor 10�8 M E2 for 48 h, CHIP assays were performed with the indicated antibodies. (C) Cells were transfected as described for panel B, but withor without HA-Fe65. Forty-eight hours later, the cells were treated with EOH or E2 for 45 min, and CHIP assays were performed. (D) Brain tissueswere dissected from female APP and presenilin 1 double-transgenic mice, and CHIP assays were performed with the indicated antibodies.

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FIG. 7. Estrogen protection of cells from APPct complex-induced apoptosis and its dependency on the ER. (A) N2a cells were transfected with 0.3�g of pEGFP, Gal4-APPct, and HA-Fe65 together, with or without Myc-Tip60. After treatment with EOH or 10�8 M E2 for 72 h, the number oftransfected (green) cells in each well was counted. Triplicate samples were analyzed for each data point, and the data were reproduced. (B) N2a cellstransfected and treated as described for panel A were fixed and stained with DAPI. Representative micrographs are shown. (C) N2a cells were transfectedand treated as described for panel A. The apoptotic index of transfected cells was determined. Triplicate samples were analyzed per data point, and thegraph represents three independent experiments. (D) Control (upper panels) or differentiated (lower panels) N2a cells were transfected with GFP andstained with DAPI. Neurofilament (green) was visualized by immunofluorescence with anti-MAP2 antibody. cAMP, cyclic AMP. (E) Differentiated N2acells were transfected with 0.3 �g of APPct, Fe65, and Tip60 and treated with EOH or E2. The apoptotic index of GFP-positive cells was determinedas described for panel C. (F) N2a cells were transfected with 4 �g of plasmids, as indicated. Forty-eight hours later, cells were treated for 45 min, andcaspase-3 activity was determined. Triplicate samples were analyzed for each data point, and the data were reproduced. (G) HeLa cells were transfectedand treated as described for panel A. The only difference is that 0.3 �g of pLENhER� or a control vector was included in the transfection mix. Thenumber of transfected cells was determined. (H) Cells were transfected as described for panel A and stained with DAPI. Representative micrographsare shown. (I) HeLa cells were transfected and treated as described for panel A. The apoptotic index of transfected cells was determined.

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centage of apoptotic cells four- and fivefold, respectively.Treatment of cells with 17�-estradiol prevented apoptosis in-duced by APPct, Fe65, and Tip60. Consistent with the effectof estrogens on apoptosis induced by the APPct complex,

caspase-3 activation by the APPct complex was also inhibitedby 17�-estradiol (Fig. 7F). The effect was observed in cellstreated with the hormone for either 45 min (Fig. 7F) or 48 h(data not shown). The caspase-3 analyses show that the apop-tosis induced by the APPct complex is likely through the in-trinsic apoptotic pathway that involves caspases. The suppres-sion by a short estrogen treatment indicates that the estrogeneffect is rather direct. Overall, these analyses show that theinduction of apoptosis by the APPct complex and the protec-tion by estrogens also occur in differentiated neurons.

Because N2a cells express ER�, it is impossible to tellwhether the suppressive effect of estrogens on apoptosis in-duced by the APPct complex is mediated through the ER. Toaddress the issue of ER dependency, ER-negative HeLa cellswere transfected with or without ER�, and the effect of estro-gens on apoptosis induced by the APPct complex was exam-ined. As shown in Fig. 7G, 17�-estradiol had no effect on thenumber of transfected cells in the absence of ectopic ER� ex-pression. After cotransfection with ER�, 17�-estradiol blockedboth the decrease in cell number (Fig. 7G) and the increasein apoptotic index (Fig. 7H and I) induced by the APPctcomplex. These findings demonstrate that the estrogen ef-fect on apoptosis induced by the APPct complex is mediatedthrough the ER.

Essential role of complex formation between ER� and Fe65in mediating estrogen suppression of the activity and functionof the APPct complex. Our analyses so far have shown that theestrogen suppression of the transcriptional and biological ac-tivities of the APPct complex correlates very well with theability of the ER� to form a complex with Fe65, suggesting acause-and-effect relationship. However, the data have notruled out the possibility that the estrogen suppression is indi-rectly mediated through an estrogen target gene whose expres-sion is regulated by the activated ER� through its binding to anERE. A mutant ER� that does not bind Fe65 but is active onan ERE-based reporter gene would be helpful to distinguishbetween these two possibilities.

Previous studies have shown that mutation of tyrosine-537 toalanine produces a mutant ER� that is constitutively active onERE-based reporter genes (49), a phenotype we confirmed inreporter analyses (Fig. 8A). Because tyrosine-537 is located inthe AF-2 region that is critical for Fe65 interaction (Fig. 4), wetested the ability of this mutant ER� to bind Fe65 and inhibitthe activity of the APPct complex. As shown in Fig. 8, thismutant ER� failed to interact with Fe65 (Fig. 8B) and tosuppress the ability of the APPct complex to induce transcrip-tional activation (Fig. 8C) or apoptosis (Fig. 8D). As controls,the wild-type and mutant ER� proteins were expressed tocomparable levels, and they both did not decrease the level ofectopic expression of APPct or Fe65 (data not shown). Theseanalyses link the inhibitory effect of estrogens on the APPctcomplex to the ability of ER� to bind Fe65 and separate theeffect from the classic nuclear actions of the hormone on tran-scription mediated through EREs.

DISCUSSION

While the molecular mechanism underlying estrogen actionin the reproductive system is well defined, little is known aboutestrogen action in neuronal cells. The present study reveals a

FIG. 7—Continued.

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FIG. 8. Effect of tyrosine-537 mutation on ER�-mediated suppression of the transcriptional activity of the Fe65 complex and its ability to induceapoptosis. (A) HeLa cells were transfected with 0.5 �g of EREe1bLuc, 0.5 �g of pLEN�gal, and 0.3 �g of pLENhER� or pLENhER�Y537A, asindicated. Cells were treated with 10�8 M E2 or EOH for 48 h, and the luciferase activity was determined. (B) HeLa cells were transfected with 4 �gof HA-Fe65 and 2 �g of pLENhER� or pLENhER�Y537A and treated with 10�8 M E2 for 72 h. The interaction between Fe65 and wild-type or mutantER� was determined by coimmunoprecipitation. (C) HeLa cells were transfected with 0.5 �g 3�17merLuc, 0.1 �g pLEN�Gal, 0.3 �g Gal4-APPct� orGal4-APPct, 0.3 �g HA-Fe65, and 0.3 �g pLENhER� or ER�Y537A, as indicated. Cells were treated with 10�8 M E2 or EOH for 48 h, and luciferaseactivity was determined. (D) HeLa cells were transfected with 0.3 �g of pEGFP, APPct, Fe65, ER�, or ER�Y537A, as indicated, and treated with EOHor E2. The apoptotic index of GFP-positive cells was determined. (E) Estrogen action through “tethering” of the ER to the Fe65-APP complex. APPforms a complex with Fe65 on the plasma membrane through its carboxyl terminus. After cleavage by �-secretase, Fe65 moves to the nucleus, recruitsTip60, and induces neuronal cell apoptosis by activating the transcription of target genes. The activated ER binds to the Fe65 transcriptional complexand suppresses gene activation and cell death by decreasing the amount of Tip60 coactivator in the complex. Using Fe65 as an adaptor, the ER may alsointeract with full-length APP in the cytoplasm to regulate its processing by proteolytic enzymes.

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novel molecular mechanism by which physiological levels ofestrogens protect neuronal cells from apoptosis triggered by�-secretase-mediated cleavage of APP, a pathological processcharacteristic of neurological disorders such as AD. This mech-anism involves the “tethering” of ER� to the Fe65 transcrip-tional complex on gene promoters (Fig. 8E). In contrast to thetypical genomic action of estrogens mediated through ER�sitting on ERE-containing promoters, which uses helix-12 as amain activator to recruit coactivators, this novel mode of es-trogen action involves the use of the helix-12 region as aninteraction motif to bind an adaptor molecule, leading to adecrease in the recruitment of a transcriptional coactivator.Multiple lines of evidence are presented here to support thismechanism. First, 17�-estradiol inhibited the transcriptionalactivity of the APPct complex, and the endogenous ERs inIshikawa cells were sufficient to mediate this action. Second,ER� formed a complex with APPct and full-length APP viaFe65 as an adaptor both in vitro and in vivo. Complex forma-tion was shown for both ectopic proteins and endogenousproteins in the mouse brain, and the amounts of complex weregreater in brain tissues of transgenic mice expressing mutantpresenilin 1 and APP than in brain tissues of wild-type mice.Third, the transcriptional inhibition observed in reporter as-says translated into estrogen suppression of the expression ofendogenous genes induced by the APPct complex (for exam-ple, the KAI1 gene). CHIP assays detected ER� bound to theKAI1 gene promoter, which was increased by 17�-estradioltreatment. Fourth, apoptotic assays showed that 17�-estradiolimpaired the ability of the APPct complex to induce apoptosisin transformed and differentiated neuronal cells and that suchan impairment depended on ER�. Finally, the alanine-537 ERmutant was constitutively active on an ERE-based reporter butwas unable to bind Fe65 or mediate the suppressive effects ofestrogens on the APPct complex.

The analysis with amino-terminally truncated ER shown inFig. 4 suggested that the N terminus of ER� is expendable, butit did not rule out the potential involvement of AF-1 in medi-ating the inhibition of the APPct complex by estrogens in thecontext of the full-length receptor. Similarly, the analysis withthe AF-2 mutants did not prove that AF-2 is essential forinhibition because the mutant receptor failed to interact withFe65. The accurate assessment of the differential involvementof AFs is hindered by the lack of full-length ER mutants thatare capable of binding to Fe65 but selectively deficient in theAFs. It is also important that the reduction in the activity ofthe APPct complex caused by ectopically expressed ER� in theabsence of estrogens (Fig. 1) does not necessarily mean thatthe inactive receptor has the ability to suppress the activity ofthe APPct complex. Ectopically expressed ER� is known todisplay constitutive activity in transient reporter assay systems,presumably due to estrogenic compounds present in the me-dium or produced by the cells. Furthermore, many nonsteroidcompounds that do not bind ER� cause ligand-independentactivation of the receptor (41). Interestingly, one such com-pound is dopamine (36, 41). It will be interesting to find outwhether dopamine or other neurotransmitters protect neuronsfrom APPct-induced apoptosis by stimulating complex forma-tion between Fe65 and ligand-independently activated ER�.

Consistent with the constitutive effect of ectopic ER� onAPPct activity, complex formation also occurred between ec-

topically expressed ER� and Fe65 in the absence of estrogens.The inhibition of APPct activity by 17�-estradiol is also con-sistent with the enhancement of complex formation by thehormone. Even though binding assays did not detect an in-crease in the amount of ER� protein present in anti-HA–Fe65immunoprecipitates after estrogen treatment, the specificinteraction per molecule of ER� was clearly increased by es-trogens. This is because the total level of ER� protein isdecreased by estrogens (Fig. 1B and 2B), likely due to degra-dation of ER� by the ubiquitin-proteasome pathway duringestrogen-induced transcriptional activation (30, 35). Consis-tent with the above assessment, complex formation betweenendogenous ER� and Fe65 in mouse brains is most likelyestrogen regulated. This is suggested by the reduced complexformation detected in male and old mice (data not shown), asthey contain fewer endogenous estrogens than do female andyoung mice.

Although the data in Fig. 4 and 8 show that complex forma-tion between ER� and Fe65 is required for suppression of theactivity of the APPct complex by estrogens, complex formationdoes not always lead to suppression. Complex formation alsooccurs in cells treated with tamoxifen, which increases ratherthan decreases the activity of the APPct transcriptional com-plex (data not shown). It is reasonable to conclude that com-plex formation only provides a platform for the subsequentER�-mediated changes in the recruitment of transcriptionalcofactors to the promoter, a process dependent on the prop-erties of the ligand. This idea is supported by the decreasedrecruitment of the Tip60 coactivator to the KAI1 gene pro-moter caused by 17�-estradiol, which was associated with aconcomitant increase in the recruitment of ER� protein to thepromoter (Fig. 6B and C). Overall, the data suggest thatcomplex formation between ER� and APPct mediatedthrough Fe65 is required but may not be sufficient for thesuppression of the transcriptional activity of the APPct com-plex and that the promoter context-specific displacement oftranscriptional cofactors induced by ligands might be anadditional determinant.

Fe65 belongs to a family of WW domain proteins, amongwhich many have been shown to be involved in transcriptionalregulation (42, 43). For example, the Yes kinase-associatedprotein binds to the polyoma enhancer binding protein 2 tran-scription factor through its WW domain and serves as a coac-tivator (52), and PQBP-1, a polyglutamine tract binding pro-tein with a WW domain, inhibits transcription activation byBrn-2 (48). In addition, the WW domains of Ess1/Pin1 PPIase(43), Yes kinase-associated protein (12), and Rsp5/Nedd4ubiquitin ligase (5) bind the carboxyl-terminal domain of RNApolymerase II, suggesting a fundamental role of WW domainsin transcription. It will be interesting to find out whether theinteraction between ER� and Fe65 can be extended to otherWW proteins and whether the interaction represents a com-mon means for transcriptional regulation by activated ER�.

Previous studies have shown that estrogens control the ac-tivation of kinases involved in neuronal death (15) or survival(1) through nongenomic actions or function as antioxidants(6). Some of these activities were demonstrated with high con-centrations of estrogens, and the ER was found to not alwaysbe required. The suppression of APPct complex-inducedapoptosis was clearly ER dependent and happened with 17�-

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estradiol at physiological concentrations. Moreover, the dem-onstration of the existence of ER�, Fe65, and APP in the sameprotein complex suggests a role of estrogens in regulating thepathogenesis of AD that is more direct than what earlier stud-ies may have suggested. Because full-length APP formed acomplex with the receptor via Fe65, it is possible that otherbiological processes involving APP are also regulated by estro-gens. For example, estrogens have been shown to regulate thenonamyloidogenic processing of APP by �-secretase, whichleads to the suppression of A� production by �-secretase (33).This effect could also be mediated through complex formationbetween ER� and Fe65 that takes place outside the nucleus.Our confocal immunofluorescence analyses of mouse brainsections detected cytoplasmic signals of ER� in certain neu-rons (Fig. 5E), suggesting that it is possible for ER� to interactwith APP outside the nucleus. It has also been shown thatestrogens suppress the transport of proteins such as NF-B(13) and glucocorticoid receptor (16) from the cytoplasm tothe nucleus. The cytoplasmic APP-Fe65-ER� complex mayalso regulate the intracellular transport of neuronal proteins.

In contrast to animal experiments that consistently demon-strated a neuroprotective activity for estrogens, the Women’sHeath Initiative (WHI) studies have indicated that estrogens,alone or in combination with synthetic progesterone, exert nosignificant benefit for AD and may even have an adverse effect(10, 40). Because the ages of women in the WHI study areabout 65 years or older and the age of menopause is from 45to 55, it is arguable that the use of estrogens in older womenmay exacerbate existing problems. Literature information hassuggested that estrogens may protect women against the de-velopment of AD if applied early but are of little or no valueonce the damage has been done, i.e., after the disease hasprogressed to a clinically detectable state (18). Since the cur-rent study shows that estrogens suppress the transcriptionalactivation of genes by the APPct complex, the suppressionobviously happens at early stages of the apoptotic process. Thefindings from the present study are thus more relevant to thepreventive effect of estrogens on AD than to the cure of pre-existing AD by estrogens. Continued investigation of the bio-logical significance of the ER� and APP interaction may pro-vide a better understanding of the opposite effects of estrogensobserved in the WHI studies and in animal models. Alongthese lines, it is important that the expression of KAI1 iscontrolled by NF-B and that the effect of the APPct complexon the expression of KAI1 is mediated through the NF-B site(2). Like the ER, NF-B plays complex roles in AD. It isantiapoptotic, so its transcriptional activity may be needed forneuronal survival. On the other hand, NF-B is a proinflam-matory transcriptional factor that may facilitate the progres-sion of AD. It is important to identify the subset of NF-Btarget genes suppressed by the APPct complex and those geneswhose suppression by the complex is relieved by estrogens.

ACKNOWLEDGMENTS

We thank Thomas C. Sudhof for providing the various APP, Fe65,and Tip60 expression vectors used in the studies and for helpful dis-cussions. We also thank Edward H. Koo for the CT-15 anti-APPctantibody, Donald P. McDonnell for the mutant ER� expression vec-tors, and Benita S. Katzenellenbogen for the ER�Y537A mutant. Weare grateful to Nancy Olashaw for a critical reading of the manuscriptand to Hui Zheng, David Morgan, and Jun Tan for helpful discussions.

Confocal imaging was performed in the Imaging Core Facility at H.Lee Moffitt Cancer Center and Research Institute.

This work was supported by grants from the National Institutes ofHealth (CA79530) (W.B.) and from the Department of Defense(DAMD-17-03-1–0177) (W.B.).

REFERENCES

1. Alexaki, V. I., I. Charalampopoulos, M. Kampa, H. Vassalou, P. Theodoro-poulos, E. N. Stathopoulos, A. Hatzoglou, A. Gravanis, and E. Castanas.2004. Estrogen exerts neuroprotective effects via membrane estrogen recep-tors and rapid Akt/NOS activation. FASEB J. 18:1594–1596.

2. Baek, S. H., K. A. Ohgi, D. W. Rose, E. H. Koo, C. K. Glass, and M. G.Rosenfeld. 2002. Exchange of N-CoR corepressor and Tip60 coactivatorcomplexes links gene expression by NF-kappaB and beta-amyloid precursorprotein. Cell 110:55–67.

3. Brady, M. E., D. M. Ozanne, L. Gaughan, I. Waite, S. Cook, D. E. Neal, andC. N. Robson. 1999. Tip60 is a nuclear hormone receptor coactivator. J. Biol.Chem. 274:17599–17604.

4. Cao, X., and T. C. Sudhof. 2001. A transcriptionally active complex of APPwith Fe65 and histone acetyltransferase Tip60. Science 293:115–120.

5. Chang, A., S. Cheang, and X. Espanel. 2000. Rsp5 WW domains interactdirectly with the carboxyl-terminal domain of RNA polymerase II. J. Biol.Chem. 275:20562–20571.

6. Chiueh, C., S. Lee, T. Andoh, and D. Murphy. 2003. Induction of antioxi-dative and antiapoptotic thioredoxin supports neuroprotective hypothesis ofestrogen. Endocrine 21:27–31.

7. Dodart, J. C., C. Mathis, and A. Ungerer. 2000. The beta-amyloid precursorprotein and its derivatives: from biology to learning and memory processes.Rev. Neurosci. 11:75–93.

8. Dong, J. T., W. B. Isaacs, J. C. Barrett, and J. T. Isaacs. 1997. Genomicorganization of the human KAI1 metastasis-suppressor gene. Genomics41:25–32.

9. Dubal, D. B., H. Zhu, J. Yu, S. W. Rau, P. J. Shughrue, I. Merchenthaler,M. S. Kindy, and P. M. Wise. 2001. Estrogen receptor alpha, not beta, is acritical link in estradiol-mediated protection against brain injury. Proc. Natl.Acad. Sci. USA 98:1952–1957.

10. Espeland, M. A., S. R. Rapp, S. A. Shumaker, R. Brunner, J. E. Manson,B. B. Sherwin, J. Hsia, K. L. Margolis, P. E. Hogan, R. Wallace, M. Dailey,R. Freeman, and J. Hays. 2004. Conjugated equine estrogens and globalcognitive function in postmenopausal women. JAMA 291:2959–2968.

11. Evans, R. M. 1988. The steroid and thyroid receptor superfamily. Science240:889–895.

12. Gavva, N. R., R. Gavva, K. Ermekova, M. Sudol, and C. K. J. Shen. 1997.Interaction of WW domains with hematopoietic transcription factor p45/NF-E2 and RNA polymerase II. J. Biol. Chem. 272:24105–24108.

13. Ghisletti, S., C. Meda, A. Maggi, and E. Vegeto. 2005. 17�-Estradiol inhibitsinflammatory gene expression by controlling NF-B intracellular localiza-tion. Mol. Cell. Biol. 25:2957–2968.

14. Goodenough, S., M. Schafer, and C. Behl. 2003. Estrogen-induced cell sig-nalling in a cellular model of Alzheimer’s disease. J. Steroid Biochem. Mol.Biol. 84:301–305.

15. Goodenough, S., D. Schleusner, C. Pietrzik, T. Skutella, and C. Behl. 2005.Glycogen synthase kinase 3beta links neuroprotection by 17beta-estradiol tokey Alzheimer processes. Neuroscience 132:581–589.

16. Guibao, G., and R. B. Simerly. 1994. Hormonal regulation of opioid peptideneurons in the anteroventral periventricular nucleus. Horm. Behav. 28:503–511.

17. Hao, J., W. G. Janssen, Y. Tang, J. A. Roberts, H. McKay, B. Lasley, P. B.Allen, P. Greengard, P. R. Rapp, J. H. Kordower, P. R. Hof, and J. H.Morrison. 2003. Estrogen increases the number of spinophilin-immunore-active spines in the hippocampus of young and aged female rhesus monkeys.J. Comp. Neurol. 465:540–550.

18. Henderson, V. W. 1997. The epidemiology of estrogen replacement therapyand Alzheimer’s disease. Neurology 5(Suppl.):S27–S35.

19. Jankowsky, J. L., D. J. Fadale, J. Anderson, G. M. Xu, V. Gonzales, N. A.Jenkins, N. G. Copeland, M. K. Lee, L. H. Younkin, S. L. Wagner, S. G.Younkin, and D. R. Borchelt. 2004. Mutant presenilins specifically elevatethe levels of the 42 residue beta-amyloid peptide in vivo: evidence foraugmentation of a 42-specific gamma secretase. Hum. Mol. Genet. 13:159–170.

20. Kimberly, W. T., J. B. Zhang, S. Y. Guenette, and D. J. Selkoe. 2001. Theintracellular domain of �-amyloid precursor protein is established by Fe65and translocates to the nucleus in a notch-like manner. J. Biol. Chem.276:40288–40292.

21. Kimura, A., and M. Horikoshi. 1998. Tip60 acetylates six lysines of a specificclass in core histones in vitro. Genes Cells 3:789–800.

22. Kinoshita, A., C. M. Whelan, O. Berezovska, and B. T. Hyman. 2002. Directvisualization of the gamma secretase-generated carboxyl-terminal domain ofthe amyloid precursor protein: association with Fe65 and translocation to thenucleus. J. Neurochem. 82:839–847.

23. Klinge, C. M., S. C. Jernigan, K. A. Mattingly, K. E. Risinger, and J. Zhang.

1332 BAO ET AL. MOL. CELL. BIOL.

on March 12, 2018 by guest

http://mcb.asm

.org/D

ownloaded from

Page 13: Suppression of β-Amyloid Precursor Protein Signaling into the ...

2004. Estrogen response element-dependent regulation of transcriptionalactivation of estrogen receptors alpha and beta by coactivators and core-pressors. J. Mol. Endocrinol. 33:387–410.

24. Ko, S. Y., S. C. Lin, K. W. Chang, Y. K. Wong, C. J. Liu, C. W. Chi, and T. Y.Liu. 2004. Increased expression of amyloid precursor protein in oral squa-mous cell carcinoma. Int. J. Cancer 111:727–732.

25. Koo, E. H., and S. L. Squazzo. 1994. Evidence that production and release ofamyloid beta-protein involves the endocytic pathway. J. Biol. Chem. 269:17386–17389.

26. Lee, H., F. Jiang, J. Q. Wang, S. V. Nicosia, J. Yang, B. Su, and W. Bai. 2000.MEKK1 activation of human estrogen receptor alpha and stimulation of theagonistic activity of 4-hydroxytamoxifen in endometrial and ovarian cancercells. Mol. Endocrinol. 14:1882–1896.

27. Lee, H., and W. Bai. 2002. Inhibition of estrogen receptor nuclear export byligand-induced and p38-mediated receptor phosphorylation. Mol. Cell. Biol.22:5835–5845.

28. Li, P., S. V. Nicosia, and W. Bai. 2001. Antagonism between PTEN/Mmac1/Tep-1 and androgen receptor in growth and apoptosis of prostate cancercells. J. Biol. Chem. 276:20444–20450.

29. Li, P., H. Lee, S. Guo, T. G. Unterman, G. Jenster, and W. Bai. 2003.AKT-independent protection of prostate cancer cells from apoptosis medi-ated through complex formation between the androgen receptor and FKHR.Mol. Cell. Biol. 23:104–118.

30. Lonard, D. M., Z. Nawaz, C. L. Smith, and B. W. Malley. 2000. The 26Sproteasome is required for estrogen receptor-alpha and coactivator turnoverand for efficient estrogen receptor-alpha transactivation. Mol. Cell 5:939–948.

31. MacLusky, N. J. 2004. Estrogen and Alzheimer’s disease: the apolipoproteinconnection. Endocrinology 145:3062–3064.

32. Maecker, H. T., S. C. Todd, and S. Levy. 1997. The tetraspanin superfamily:molecular facilitators. FASEB J. 11:428–442.

33. Manthey, D., S. Heck, S. Engert, and C. Behl. 2001. Estrogen induces a rapidsecretion of amyloid � precursor protein via the mitogen-activated proteinkinase pathway. Eur. J. Biochem. 268:4285–4291.

34. McAllister, D., X. Merlo, and J. Lough. 2002. Characterization and expres-sion of the mouse tat interactive protein 60 kD (TIP60) gene. Gene 289:169–176.

35. Nawaz, Z., D. M. Lonard, A. P. Dennis, C. L. Smith, and B. W. O’Malley.1999. Proteasome-dependent degradation of the human estrogen receptor.Proc. Natl. Acad. Sci. USA 96:1858–1862.

36. Olesen, K. M., H. M. Jessen, C. J. Auger, and A. P. Auger. 2005. Dopami-nergic activation of estrogen receptors in neonatal brain alters progestinreceptor expression and juvenile social play behavior. Endocrinology 146:3705–3712.

37. Sabo, S. L., A. F. Ikin, J. D. Buxbaum, and P. Greengard. 2001. The Alzheimeramyloid precursor protein (APP) and FE65, an APP-binding protein, regu-late cell movement. J. Cell Biol. 153:1403–1414.

38. Seabrook, G. R., D. W. Smith, B. J. Bowery, A. Easter, T. Reynolds, S. M.Fitzjohn, R. A. Morton, H. Zheng, G. R. Dawson, D. J. S. Sirinathsinghji,C. H. Davies, G. L. Collingridge, and R. G. Hill. 1999. Mechanisms contrib-

uting to the deficits in hippocampal synaptic plasticity in mice lacking amyloidprecursor protein. Neuropharmacology 38:349–359.

39. Shinohara, T., T. Miki, N. Nishimura, H. Nokihara, H. Hamada, N. Mukaida,and S. Sone. 2001. Nuclear factor-B-dependent expression of metastasis sup-pressor KAI1/CD82 gene in lung cancer cell lines expressing mutant p53. Can-cer Res. 61:673–678.

40. Shumaker, S. A., C. Legault, L. Kuller, S. R. Rapp, L. Thal, D. S. Lane, H.Fillit, M. L. Stefanick, S. L. Hendrix, C. E. Lewis, K. Masaki, and L. H.Coker. 2004. Conjugated equine estrogens and incidence of probable de-mentia and mild cognitive impairment in postmenopausal women. JAMA91:2947–2958.

41. Smith, C. L., O. M. Conneely, and B. M. O’Malley. 1993. Modulation of theligand-independent activation of the human estrogen receptor � by hormoneand antihormone. Proc. Natl. Acad. Sci. USA 90:6120–6124.

42. Sudol, M., K. Sliwa, and T. Russo. 2001. Functions of WW domains in thenucleus. FEBS Lett. 490:190–195.

43. Sudol, M., and T. Hunter. 2000. NeW wrinkles for an old domain. Cell103:1001–1004.

44. Toran-Allerand, C. D. 1976. Sex steroids and the development of the new-born mouse hypothalamus and preoptic area in vitro: implication for sexualdifferentiation. Brain Res. 189:413–427.

45. Tsai, M. J., and B. W. O’Malley. 1994. Molecular mechanisms of action ofsteroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63:451–486.

46. Turgeon, J. L., D. P. McDonnell, K. A. Martin, and P. M. Wise. 2004.Hormone therapy: physiological complexity belies therapeutic simplicity.Science 304:1269–1273.

47. Tzukerman, M. T., A. Esty, D. Santiso-Mere, P. Danielian, M. G. Parker,R. B. Stein, J. W. Pike, and D. P. McDonnell. 1994. Human estrogen receptortransactivational capacity is determined by both cellular and promoter con-text and mediated by two functionally distinct intramolecular regions. Mol.Endocrinol. 8:21–30.

48. Waragai, M., C. H. Lammers, S. Takeuchi, I. Iamfuku, Y. Udagawa, I.Kanazawa, M. Kawabata, M. M. Mouradian, and H. Okazawa. 1999.PQBP-1, a novel polyglutamine tract-binding protein, inhibits transcriptionactivation by Brn-2 and affects cell survival. Hum. Mol. Genet. 8:977–987.

49. Weis, K. E., K. Ekena, J. A. Thomas, G. Lazennec, and B. S. Katzenellen-bogen. 1996. Constitutively active human estrogen receptors containingamino acid substitutions for tyrosine 537 in the receptor protein. Mol. En-docrinol. 10:1388–1398.

50. Wise, P. M. 2005. Estrogens and cerebrovascular stroke: what do animalmodels teach us? Ann. N. Y. Acad. Sci. 1052:225–232.

51. Woolley, C. S., and B. S. McEwen. 1992. Estradiol mediates fluctuation inhippocampal synapse density during the estrous cycle in the adult rat. J. Neuro-sci. 12:2549–2554.

52. Yagi, R., L. F. Chen, K. Shigesada, Y. Murakami, and Y. Ito. 1999. A WWdomain-containing yes-associated protein (YAP) is a novel transcriptionalco-activator. EMBO J. 18:2551–2562.

53. Yang, J., L. New, Y. Jiang, J. Han, and B. Su. 1998. Molecular cloning andcharacterization of a human protein kinase that specifically activates c-JunN-terminal kinase. Gene 212:95–102.

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