Cellular Signalling 20 (2008) 1368–1374

Contents lists available at ScienceDirect

Cellular Signalling

j ourna l homepage: www.e lsev ie r.com/ locate /ce l l s ig

Akt stabilizes estrogen receptor α with the concomitant reduction in itstranscriptional activity

Sungwoo Park a, Jieun Song a, Cheol O Joe b, Incheol Shin a,⁎a Department of Life Science, Hanyang University, Seoul 133-791, Republic of Koreab Department of Life Science, KAIST, Daejon 305-701, Republic of Korea

a r t i c l e i n f o

Abbreviations: AF, activation function; E2, estradiol;dnGSK3β, dominant-negative GSK3β; ER, estrogen recERαS167A, S167A ERα; ERE, estrogen response elemGSK3β, glycogen synthase kinase 3β; MAPK, mitogen-Akt1, myristoylated Akt1; MEK1, mitogen-activated protregulated kinase kinase 1.⁎ Corresponding author. Department of Life Scienc

Hanyang University, 17 Haengdang-dong, Seongdong-gKorea. Tel.: +82 2 2290 2562; fax: +82 2 2298 2562.

E-mail address: incheol@hanyang.ac.kr (I. Shin).

0898-6568/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.cellsig.2008.03.004

a b s t r a c t

Article history:Received 10 February 2008Received in revised form 10 March 2008Accepted 10 March 2008Available online 19 March 2008

We have investigated the effect of Akt on estrogen receptor (ER) α protein level and its transcriptional activity.Transient transfection studies revealed that constitutively active Akt1 up-regulated ERα at the post-transcriptional level. Studies using Akt inhibitor and dominant-negative Akt1 showed that Akt1 kinase activityis required for the up-regulation of ERα. Cycloheximide decay assays and studies with proteasome inhibitorindicated that Akt1-mediated up-regulation of ERα was maintained by inhibiting proteasome-mediateddegradation of ERα. When Akt consensus phosphorylation site mutant, ERαS167Awas tested for Akt1-mediatedup-regulation, increase of ERαS167A by Akt1 was significantly impaired as compared to wild type ERα. Inaddition, dominant-negative glycogen synthase kinase (GSK) 3β and LiCl could also partially up-regulate ERαprotein level, suggesting that concerted action of Akt1-mediated phosphorylation on S167 and kinase activity ofAkt-downstreamGSK3β could affect ERα protein level. Paradoxically, co-expression of Akt1 could down-regulatetranscriptional activity of ERα. The inhibitory effect of Akt1 on ERα transcriptional activitywas not attributable tochanges in subcellular distribution of ERα. Transfection studies using increasing amount of Akt1 and ERαindicated that the transcriptional activity of ERαwasnegatively regulated by ERα protein quantities at higher ERαconcentrations. Chromatin immunoprecipitation assays revealed that at Akt1 concentration high enough toinduce up-regulation of ERα, association of ERα to promoter region of ERα target pS2 gene was impaired. Takentogether, these data suggest that Akt1 could increase ERα protein level with simultaneous reduction in itstranscriptional activity, possibly by modulating association of ERα to the target gene promoters.

© 2008 Elsevier Inc. All rights reserved.

Keywords:AktEstrogen receptor (ER) αGlycogen synthase kinase (GSK) 3βTranscriptional activity

1. Introduction

Estrogen receptor (ER), a ligand-activated transcription factor witha specific high-affinity to estradiol (E2) primarily controls reproduc-tive function and regulates target cell proliferation and gene tran-scription through direct interaction with estrogen response elements(EREs) in the target gene promoters [1–4]. ER belongs to nuclearreceptor superfamily and can be subdivided into two isoforms, ERαand ERβ that have structural similarity but distinct functions [5]. ERαis predominantly expressed in the breast [6], and plays a crucial role inthe formation of breast cancer [7]. Recent works suggest that theintracellular level of ERα is tightly regulated by ubiquitin-proteasome

dnAkt, dominant-negative Akt;eptor; ERαWT, wild type ERα;ent; FBS, fetal bovine serum;activated protein kinase; myr-ein kinase/extracellular signal-

e, College of Natural Science,u, Seoul, 133-791, Republic of

l rights reserved.

pathway [8–12] and post translational modification such as phos-phorylation is involved in ERα degradation in proteasome [13]. Indeed,it was reported that many kinases including curcumin sensitive kinase,mitogen-activated protein kinase (MAPK), pp90rsk1 and serine/threo-nine kinase Akt [1,14–16] phosphorylate ERα.

Akt is a central factor in tumorigenesis by promoting cell survival[17,18]. All three subtypes of Akt, Akt1, Akt2 and Akt3 contain an amino-terminal pleckstrin homology domain and activated by multistep pro-cessing through membrane translocation and phosphorylation at Thr-308/309 and Ser-473/474 [17]. Activated Akt promotes cell survival byphosphorylating and modulating the activity of apoptosis-relatedproteins such as Bad [19], Caspase 9 [20] and Forkhead transcriptionfactors [21]. It was also reported that Akt can also phosphorylate ERα atSer-167 and this phosphorylation can affect transcription activity of ERα[16,22]. Furthermore, Akt activation followedby loss of phosphatase andtensin homolog results in the activation of ERα-dependent pathwaysinvolved in the neoplastic process in vivo [23]. Interestingly, by a non-genomic mechanism, ERα can also activate Akt by binding to p85regulatory subunit of PI3K [24] and up-regulation of PI3K/Akt signalingstimulates growth of breast cancer cells [25].

Based on these observations, we have investigated the effect ofAkt on ERα protein level and its transcriptional activity to gain further

1369S. Park et al. / Cellular Signalling 20 (2008) 1368–1374

insight into the interplay between Akt and ERα. Transient transfectionstudies using Akt and ERα expression plasmids in ERα negative 293Tcells revealed that kinase activity of Akt can up-regulate ERα proteinlevel with concomitant down-regulation of its ligand-dependent tran-scriptional activity.

2. Materials and methods

2.1. Plasmids and site-directed mutagenesis

HA-tagged ERα (HA-ERα) was produced by subcloning of ERα cDNA from pHEG0,kindly provided by Dr. Stallcup (University of Southern California) into BamH1 and EcoRIsite of pcDNA3.1. HA-tagged myristoylated constitutively activated Akt1 (HA-myr-Akt1),HA-tagged kinase-dead dominant-negative Akt1 (HA-dnAkt1), constitutively activatedmitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1(CA-MEK1) were described previously [26], Akt2 and Akt3 isoform expression constructswere also described elsewhere [27]. pGLB-MERE was from Dr. El-Ashry (GeorgetownUniversity). HA-taggedwild typeGSK3β (HA-GSK3β) and kinase-dead dominant-negativeGSK3β (HA-dnGSK3β)were gifts fromDr. Jho E.H. (University of Seoul). S167Amutant ERαconstruct was produced using Quickchange kit (Stratagene, Cedar Creek, TX) withfollowing mutagenic primers: Fwd, 5′-GGCAGAGAAAGATTGGCCGCTACCAATGACAAGGG-3′, and Rev, 5′-CCCTTGTCATTGGTAGCGGCCAATCTTTCTCTGCC-3′.

2.2. Cell culture and transfection

293T human kidney cell line (ATCC) was routinely maintained in Dulbecco's modi-fied Eagle's medium (DMEM) with 50 U/mL penicillin, 50 μg/mL streptomycin, and 10%fetal bovine serum (FBS) (GIBCO, Carlsbad, CA) at 37 °C under 5% CO2. Calcium phosphatetransfection was performed as described [28].

2.3. Western blots

Whole cell lysates were obtained 48 h post-transfection in a cell lysis buffer (20 mMTris–Cl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 20 mM NaF, 1 mM Na3VO4, 1×proteaseinhibitor (Roche, Indianapolis, IN), 1% NP-40, 0.1% triton X-100, 0.1% SDS) and proteinconcentrations were determined with the BCA protein assay kit (Pierce, Rockford, IL).Equal amount of proteins (30 μg/sample) were boiled in 1×SDS sample buffer, subjectedto SDS-PAGE and transferred onto 0.2 μm nitrocellulose membrane (Whatman GmbH,Dassel, Germany). After blocking themembranewith 5% non-fat dry milk in TBST (20mMTris–Cl, pH 7.6, 140 mM NaCl, 0.05% Tween-20), the membrane was incubated withappropriate dilutions of specific primary antibodies: HA-probe (Y-11) and Actin (C-2)antibodies were from Santa Cruz (Santa Cruz, CA). Akt, p-Ser473 Akt, p-Ser167 ERαand c-Jun antibodies were obtained from Cell Signaling Technology (Beverly, MA). Afterovernight of incubationwith primary antibody solution, the membrane was washed withTBST 3 times for 5 min and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Amersham Biosciences, Little Chalfont, Buckingham-shire, UK). Immunoreactive protein bands were detected with West-zol Plus system(Intron Biotechnology, Seoul, Korea).

2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR)

RNA was extracted using TRI reagent (Molecular Research Center, Cincinnati OH).Subsequent RT-PCR was performed using RNA PCR core kit (Roche). Expression of ERαwas assessed using the following primers: Fwd, 5′-CTACTACCTGGAGAACGAGCC-3′, Rev,5′-CAATGGTGCACTGGTTGGTGGCGT-3′. Quantitation of β-Actin expression was per-formed as internal control using RT-PCR Primer and Control Set (Invitrogen, Carlsbad,CA).

2.5. Cycloheximide decay assays

ERα construct was transfected into 293T cells with or without Akt constructs. At48 h post-transfection, 20 μg/mL cycloheximide (Sigma-Aldrich, St. Louis, MO) wastreated to block further protein synthesis. The cells were harvested at each indicatedtime points and subjected to western blot analyses.

2.6. Dual luciferase assays

Cells were transfected with pGLB-MERE reporter, ERα construct and pCMV-RL as aninternal control. The medium was replaced at 24 h post-transfection with phenol redfree Improved Minimum Essential Medium (IMEM, Gibco) supplemented with 10%Charcoal/Dextran treated FBS, 50 U/mL penicillin and 50 μg/mL streptomycin. After 24 hof hormone starvation, 10 nM E2 (Sigma-Aldrich) was treated for 24 h and dualluciferase assays were performed according to manufacturer's protocol (Promega,Madison WI).

2.7. Immunostaining

Cells were grown on 0.1% gelatin-coated coverslip for 24 h and transfected bycalcium phosphate methods. At 48 h post-transfection, cells were fixed with −20 °C

methanol and permeabilized with 0.05% triton X-100 in PBS. After blocking with 3%non-fat dry milk in PBS, the fixed cells were incubated with ERα antibody (H-184)(Santa Cruz), followed by incubation with Oregon green conjugated anti-rabbit IgG(Molecular Probes, Eugene, OR). The nuclei were counterstained with 1 μg/mL Hoechst33340 (Invitrogen).

2.8. Cell fractionation

Cells were harvested and incubated in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl,0.1mMEDTA, 0.1mM EGTA,1mMdithiothreitol (DTT), 1×protease inhibitor) for 15minon ice. The cells were then incubated for additional 10min after the addition of 0.5% NP-40 (v/v). The cell lysates were centrifuged for 1 min at 14,000 g. The resulting super-natants were used as the cytoplasmic extracts. The pellets were resuspended in buffer B(20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1×proteaseinhibitor) and the homogenates were incubated for 15 min on ice. Nuclear debris wasexcluded by centrifugation for 10 min at 14,000 g and the resulting supernatants wereused as the nuclear extracts.

2.9. Chromatin immunoprecipitation (ChIP)

Cells were cross-linked with 1% formaldehyde and lysed in a cell lysis buffer(10 mM Tris, pH 8.0, 10 mM NaCl, 0.2% NP-40, 1×protease inhibitor).The samples werecentrifuged for 5 min at 1000 g. Pelleted nuclei were treated with 200 U of MicrococcalNuclease (New England Biolabs, Hitchin, Hertfordshire, UK) and resuspended in nucleilysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 1% SDS, 1×protease inhibitor), dilutedin IP dilution buffer (20 mM Tris, pH 8.0, 150mMNaCl, 2 mM EDTA, 0.01% SDS,1% TritonX-100, 1×protease inhibitor), then subjected to sonication (2 pulses, 10 s on/50 s off at70% amplitude with Dr. Hielscher, GmbH). Digested chromatin was immunoprecipi-tated with ERα antibody and protein A sepharose (Amersham Biosciences) and washedsequentially with washing buffer 1 (20 mM Tris, pH 8.0, 150 mMNaCl, 2 mM EDTA, 0.1%SDS, 1% Triton X-100) twice, washing buffer 2 (10 mM Tris, pH 8.0, 0.25 M LiCl, 1 mMEDTA,1% NP-40,1% deoxycholate) once, 0.1×TE buffer (1 mM Tris, pH 7.6, 0.1 mM EDTA)once, and elution buffer (0.1 M NaHCO3, 1% SDS) twice for 5 min in each step. Elutedchromatin was reverse cross-linked in 0.25 M NaCl for 4 h at 65 °C and DNA extractionwas performed using phenol/chloroform and ethanol precipitation and amplified byPCR with pS2 primers: Fwd, 5′-GACGGAATGGGCTTCATGAGC-3′, and Rev, 5′-CTGAGA-CAATAATCTCCACTG-3′.

3. Results

3.1. Akt1 up-regulated ERα at post-transcriptional level

To analyze the effect of ectopic Akt1 on ERα stability, a series ofconstructs were transfected to 293T cells as shown in Fig. 1A. ActiveAkt1 increased ERα protein level but dnAkt1 failed to increase ERαprotein level. Constitutive active versions of other Akt isoforms (Akt2and Akt3) could also increase ERα protein while dominant-negativeisoforms could not (Fig. 1A insert). Activation of MAPK signaling by co-transfection of constitutive active MEK1 did not increase ERα protein,suggesting that the effect on ERα protein level is Akt-specific. SinceAkt can regulate the other protein synthesis or affect transcriptionlevel by phosphorylation and subsequent modulation of transcriptionfactor activities [29,30], we performed semi quantitative RT-PCR insame sets of the transfected cells to examine whether increase of ERαoccurs at transcriptional level or post-transcriptional level (Fig. 1B). Toavoid saturation of PCR products, the amplified DNA bands within alinear range of increment in intensities during PCR cycles werecollected and quantified as shown in Fig. 1C. There was no significantdifference in the amounts of ERαmRNA by Akt co-transfection as wellas constitutive active MEK co-transfection. These results suggest thatthe kinase activity of Akt1 led to an increase of ERα protein at post-transcriptional level.

3.2. Akt1 affected ERα protein level by S167 phosphorylation andmodulation of downstream effector GSK3β

Since ERα has an Akt consensus phosphorylation site on S167 [22]in activation function (AF)-1 domain, we set out to investigatewhetherphosphorylation on this particular residuemay be responsible for Akt-dependent increase in ERα protein level. To check this possibility, weproduced S167Amutant ERα (ERαS167A) by site-directedmutagenesisas described inMaterials andmethods (Fig. 2A). Cells were transfected

Fig. 1. Effect of Akt1 on protein level of ERα. (A) Active Akt1 increases ERα protein level. 293T cells were transfected with HA-ERα, myrAkt1, dnAkt and CA-MEK1 and subjected towestern blots as described inMaterials andmethods. Insert: 293Tcells were transfectedwith ERα and the series of Akt isoforms as indicated. (B, C) RT-PCR profiles of ERα and internalcontrol β-actin. Oligonucleotide bands in the lanes in the linear range during PCR amplification cycles were collected (B) and the bands were quantified using 1D scan EX Eval. ERαbands of the 24th cycles(⁎) were normalized by β-actin bands of the 18th cycles(⁎⁎) (C).

1370 S. Park et al. / Cellular Signalling 20 (2008) 1368–1374

as shown in Fig. 2B, subjected towestern blot analyses. Co-transfectionof myr-Akt1 significantly increased wild type ERα (ERαWT) in whichS167 consensus phosphorylation remained intact but only led to amodest increase of ERαS167A. These results suggested that the effect ofAkt1 on ERα protein level was mainly caused by phosphorylation onS167. When basal levels of ERα expression between ERαWT andERαS167Awere compared (Fig. 2B,1st row, lane 1 vs. lane 4), ERαS167Ashowed a relatively decreased expression as compared to ERαWT (43%reduction), indicating that the phosphorylation on S167 is responsiblefor up-regulation of ERα protein level. However, the series of repeatedwestern blots revealed that ERαS167A protein level could also beslightly up-regulated (57% of control to 77% of control, Fig. 2B bottom)by Akt1 co-expression. To account for this small increase in ERαS167Aby Akt1, we investigated downstream effectors of Akt-mediatedsignaling, namely glycogen synthase kinase (GSK) 3β. Recent worksuggests that GSK3β regulates ERα stability and transcriptional activity[31]. GSK3β is a serine/threonine kinase regulated by phosphorylation,the unphosphorylated form being an enzymatically active form [32]and PI3K induced activation of Akt results in GSK3β phosphorylationand subsequent inactivation of the kinase [33]. To investigate thepossible involvement of Akt-downstream kinase, GSK3β on ERαprotein level, 293T cells were transfected with GSK3β expressionplasmids as shown in Fig. 2C. Western blot experiments revealed thatkinase-dead, dominant-negative form of GSK3β (dnGSK3β) could alsoeffectively increase ERαWTas well as ERαS167A protein level whereasco-transfection of GSK3β led to a small decrease in ERα protein level.These results suggest that kinase activity of GSK3β could also be

involved in the modulation of ERα regardless of Akt phosphorylationon S167. The involvement of GSK3β in modulation of ERα protein levelwas also confirmed by experiments with LiCl, a GSK3β inhibitor(Fig. 2D), showing the dosage-dependent increase of ERα protein levelby LiCl. The data in Fig. 2E also indicated that co-expression of myr-Akt1 and dnGSK3β could additively increase ERα. The results in Fig. 2indicated that Akt1 directly up-regulated ERα protein level by directphosphorylation on S167 and Akt down-stream kinase GSK3β partiallysupplemented the function of Akt1.

3.3. Inhibition of proteasome-mediated ERα degradation by Akt

Several studies recently described the involvement of the protea-some-dependent ERα degradation pathway in the regulation of ERαprotein in cells [8–12]. From the results shown in Fig. 1, we hypoth-esized that Akt1 may be involved in ERα stabilization, preventing itsproteasomal degradation. To demonstrate this, ERα transfected 293Tcells were treated with Akt inhibitor (Akt inhibitor #8, Calbiochem,Darnstadt, Germany) and 26S proteasome inhibitor MG132 (Calbio-chem) as shown in Fig. 3A. ERα was significantly decreased by Aktinhibitor (76% reduction, Fig. 3A bottom), and it was rescued by MG132treatment, suggesting that Akt1 led to an increase in ERα protein levelmainly by blocking proteasome-dependent degradation of ERα, notby up-regulation of ERα protein synthesis per se. When new proteinsynthesis was inhibited using cycloheximide in cycloheximide decayassays (Fig. 3B and C), co-expression of myr-Akt1 was shown to effec-tively enhance the stability of ERα.

Fig. 2. Akt1-induced up-regulation of ERα is maintained by direct phosphorylation on S167 and compensatory role of GSK3β. (A) Schematic representation of S167A mutant ERα.(B) Effect of Akt1 on ERαWTand ERαS167A. 293T cells were transfected with the constructs as indicated (top). The ERα bands frommore than three independent experiments werequantified and plotted (bottom). (C) Effect of GSK3β on ERα protein levels. 293Tcells were transfected as indicated and the samples were subjected towestern blot analyses. (D) Effectof LiCl on ERα protein level. 293T cells were transfected with ERα and treated with varying doses of LiCl (0–40 mM) for 24 h and harvested 48 h post-transfection for western blotanalyses. (E) Combined effect of Akt1 and GSK3β on ERα protein level. Indicated set of constructs were transfected to the cells. The cells were harvested 48 h post-transfection forwestern blot analyses (top). The ERα bands from more than three independent experiments were quantified and plotted (bottom).

1371S. Park et al. / Cellular Signalling 20 (2008) 1368–1374

3.4. Akt1 reduces the transcriptional activity of ERα

ERα is a transcription factor that binds to the target gene promo-ter, dimerizes and performs transcription upon activation by a ligandE2, as schematically represented in Fig. 4A. We were interested inthe transactivation function of ERα of which protein level has been

increased by Akt1. Dual luciferase assays using a luciferase constructhaving ERE indicated that the activity of ligand-dependent tran-scription was decreased by myr-Akt1 (Fig. 4B) although the pro-tein amount was increased (Fig. 1A). In contrast, co-transfection ofdnAkt1 induced increased ligand-dependent transcriptional activityof ERα.

Fig. 3. Up-regulation of ERα protein level by active Akt1 was maintained throughinhibition of proteasome-mediated ERα degradation. (A) Effect of MG132 on Aktinhibitor induced ERα down-regulation. 293T cells were transfected with ERα. AktInhibitor #8 was treated to the cells at 10 μM for 24 h, U0126 5 μM for 12 h, MG132 1 μMfor 24 h. The cells were harvested 48 h post-transfection for western blot analyses (top).The ERα bands from more than three independent experiments were quantified andplotted (bottom). (B) Cycloheximide decay assays performed with 20 μg/mL cyclohex-imide for indicated time periods in 48 h post-transfection. (C) The densities of ER αbands in (B) were quantified and expressed as percentage of each control band densities(the leftmost bands in each rows).

Fig. 4. Akt1 reduced the transcriptional activity of ERα without modulations in ERαsubcellular localization. (A) Schematic display of transcriptional mechanism of ERα.(B) Effect of Akt on ERα-dependent transcription in 293T cells. Estrogen responsiveelement-dependent luciferase assays were performed as described in Materials andmethods. Firefly luciferase activities were normalized to Renilla activities. Each barrepresents mean±S.D. from more than 3 sets of independent experiments. (C, D) Aktexpression did not affect subcellular localization of ERα. (C) Immunostaining wasperformedwith methanol-fixed 293Tcells. ERαwas visualized by immunostaining withERα antibody and nuclei were counterstained with Hoechst dye. (D) Subcellulardistribution of ectopic ERα in 293Tcells were determined by subcellular fractionation asdescribed in Materials and methods. (N, nuclear fraction; C, cytosolic fraction).

1372 S. Park et al. / Cellular Signalling 20 (2008) 1368–1374

Akt has been reported to modulate subcellular localization of itstarget protein [26,34]. To investigatewhether Akt1-mediated phosphor-ylation of ERα could lead to cytosolic mislocalization of ERα, weexamined the subcellular distribution of ERα in the presence of Akt1using immunofluorescence microscopy (Fig. 4C) and western blots ofcytosolic andnuclear subcellular fractions (Fig. 4D). The results indicatedthat Akt1 did not affect the nuclear localization of ERα, excluding thepossibility that ERα transcriptional activity was modulated by sub-cellular localization of the nuclear receptor. The similar patterns of ERαprotein up-regulation bymyr-Akt1 and ERα protein down-regulation bydnAkt1 was observed in nuclear fractions (Fig. 4D).

Since some reports indicated the relationships between ERα con-centration and its transcriptional activity [35,36], we examined thedosage-dependent effect of myr-Akt1 on ERα protein level and itstranscriptional activity (Fig. 5A and B). Cells were transfected withincreasing concentration of myr-Akt1 and the dosage-dependenteffect of myr-Akt1 was examined using dual luciferase assays and par-

allel western blot analyses with or without ligand treatment (10 nME2). The results indicated that myr-Akt1 could up-regulate ERα level ina dosage-dependent fashionwhile reducing its transcriptional activityin an apparent reciprocal manner at higher myr-Akt1 doses (above10 ng/well). To decide whether the declined transcriptional activity ofERα by myr-Akt1 could be attributable to epigenetic modulation ofERα-dependent transcription by myr-Akt1 or simply caused by up-regulation of ERα protein, the dosage-dependent effect of ectopic ERα

Fig. 5. Transcriptional activity of ERαwas regulated by its concentration. (A) Dosage-dependent effect of myr-Akt1 on transcriptional activity of ERα. 293T cells were transfected withincreasing amount of myr-Akt1 (0 ng – 1 ng – 5 ng⁎ – 10 ng – 50 ng – 100 ng – 500 ng⁎⁎) along with ERα and reporter constructs. Transcriptional activities of ERα were measuredusing dual luciferase assays in presence or absence of E2. (B) Dosage-dependent effect of myr-Akt1 on protein level of ERα. Cell lysates from A were subjected to western blots todetect the levels of ERα and Akt1. (C, D) Dosage-dependent effect of ERα on ERα transcriptional activity. Increasing amount of ERα construct (0 ng – 50 ng – 100 ng – 250 ng – 500 ng –1 μg – 2 μg) was transfected into 293T cells along with reporter constructs. Transcriptional activities of ERαwere measured using dual luciferase assays in presence or absence of E2.The amount of ERα proteinwas confirmed bywestern blots. (E) ChIP assays showing the association of ERα to pS2 promoter. Cells were transfected with ERα andmyr-Akt1 (5 ng⁎ and500 ng⁎⁎). ChIP assays were performed as described in Materials and methods.

1373S. Park et al. / Cellular Signalling 20 (2008) 1368–1374

on ERα-dependent transcription was investigated in the absence ofmyr-Akt1 co-transfection (Fig. 5C and D). Increasing ectopic ERαresulted in an initial increase in ERα-dependent transcription at lowerERα doses (Fig. 5C, 4th bar vs. 6th bar) but at higher ERα doses, the ERαtranscriptional activities were inversely declined as ERα concentrationincreased (Fig. 5C, 6th bar vs. 8th,10th,12th and 14th bars). These datamight suggest that transcriptional activity of ERα can also be modu-lated by its own concentration. The paradoxical inverse relationshipbetween ERα concentration and its transcriptional activity was foundat higher concentrations of ERα, suggesting that a small minimalamount of ERα protein is sufficient for maximum transcriptionalactivity of ERα. Indeed, ChIP assays indicated that when Akt was co-expressed at a higher dose (500 ng/well) the association of ERα to itstarget gene pS2 promoter was decreased as compared to the samplewith a lower dose of Akt (5 ng/well). Down-regulation of ERα proteinby E2 treatment (Fig. 5B and D) through ligand-dependent degrada-tion of ERα was also observed, which is in consistent with previousreports [12,13].

4. Discussion

In this report, we describe that Akt1 could increase ERα proteinstability via inhibition of proteasome-mediated ERα degradation. InFig. 3B, the initial ERα protein amounts were different in myr-Akt1,dnAkt1, CA-MEK1 co-expressing cells at starting point (designated as0 h in 48 h post-transfection), but when the intensities of the bands at0 h were normalized to 100%, there was a significant difference in the

slope of the decay curve of myr-Akt1 transfected cells from the othercells (Fig. 3B and C), indicating that Akt1 could increase the proteinlevel of ERα through inhibition of protein degradation mechanism.Themarginal difference in the slope of decaying curves suggested thatinhibition of the proteolytic degradation of ERα is the main mecha-nism responsible for myr-Akt1-mediated up-regulation of ERα. Sev-eral lines of evidence suggests that increased ERα protein level playscrucial roles in breast cancer tumorigenesis [37,38] via stimulation ofcell division and tumor growth [39]. Our results may suggest a novelmechanism bywhich activated Akt can contribute to tumorigenesis byup-regulating protein level of ERα.

Very recently, Grisouard et al. [31] reported that silencing of GSK3βusing small inhibitory RNA results in the reduction of ERα levels viainhibition of phosphorylation of ERα at S118 by GSK3β in ERα-positivebreast cancer cells. This study prompted us to analyze the possibleinvolvement of GSK3β in the regulation of ERα stability as GSK3β is adown-stream effector of Akt, which is negatively regulated by Akt [40],Our study using GSK3β expression plasmids revealed that dnGSK3βcould up-regulate ERα levels to augment the direct effect of Akt1 onERα levels through phosphorylation on S167 (Fig. 2D). Currently, we donot have full explanation for this discrepancy with the previous report[31]. These controversial phenomena may be originated from the dif-ferences in the cell lines used in the studies.

Our data in Fig. 4B suggested that myr-Akt1 could down-regulateligand-dependent transcriptional activity of ERα at higher Akt dose(500 ng/well), while increasing protein level of ERα. To explain theseparadoxical results, we have investigated the subcellular distribution

1374 S. Park et al. / Cellular Signalling 20 (2008) 1368–1374

of ERα in myr-Akt1 co-transfected cells (Fig. 4C and D). Though steroidreceptors including ERα were traditionally thought of as nuclear re-ceptors, functioning mainly as transcription factors, but emergingevidence have suggested that putative plasma membrane bound ERαcan initiate signal transduction [41,42]. Indeed, ERα has also beenshown to bind to the p85α regulatory subunit of PI3K leading to theactivation of Akt [24]. Our results indicated that majority of ectopicERα was localized mainly in the nucleus, excluding the possibilitiesthat non-genomic signaling of ERα or nuclear exclusion of ERα fromits target DNA sequence might be involved in down-regulation of ERαtranscriptional activity.

Data in Fig. 5 indicated that ectopic myr-Akt1 could down-regulateERα-dependent transcription by increasing protein concentration ofERα. Transient transfection of ERα expression construct in ERα negativeHeLa cells demonstrates that MG132-mediated stabilization and up-regulation of ERα protein can lead to attenuation of E2-responsiveluciferase expression, suggesting that proteasome-mediated degrada-tion is required for ERα-mediated transcription [10], The authors alsoshowed that inhibition of proteasome also interferedwith progesteronereceptor and thyroid hormone receptor-mediated transcription but notwith human glucocorticoid receptor-dependent transcription, indicat-ing the involvement of proteasome-mediated protein degradationpathways inhormone receptor-mediated transcription is not universallyconserved. Reid et al. demonstrated that proteasome-mediated degra-dation and ERα-mediated transactivation of downstream genes areinherently linked and cycling of transcriptionally active ERα onestrogen-responsive promoters is dependent on proteasome activity[43]. Taken togetherwith these previous reports, our datamight suggestthat Akt-mediated stabilization of ERα could block efficient turn-over ofERα, which could be an essential requirement for maintaining activetranscription complex on promoter regions of estrogen-responsivegenes. The accumulated ERα protein following proteasome inhibition, isreported to dissociate from its target promoter DNA sequences andbecome immobilizedwith nuclearmatrix [43]. Our ChIP assays in Fig. 5Ealso indicated that at a higher Akt dose (500 ng/well), which is highenough to induce maximum inhibition of ERα-dependent transcription(Fig. 5A) and maximum stabilization of ERα (Fig. 5B), association of ERαto pS2 gene promoter was decreased as compared to the sample fromcells transfected with a lower dose of Akt (5 ng/well) which showsmaximum increase in transcriptional activity (Fig. 5A).

There are conflicting literatures suggesting that PI3K/Akt couldincrease the activity of estrogen-independent AF-1 and estrogen-dependent AF-2 of ERα [16,23,44] and decrease in ERα protein level inERα-positive breast cancer cells [34]. In our experiments, co-expres-sion of myr-Akt1 at higher doses (above 10 ng/well) with ERα resultedin down-regulation of the transcriptional activity of ERα and up-regulation of ectopic ERα protein level in ERα-negative 293T cells(Fig. 1 and 4). Also in our hands, co-transfection of myr-Akt1 at lowerdose (below 10 ng/well) led to an increase in ERα-dependent tran-scription (Fig. 5A), suggesting that Akt can activate ERα function atlower doses without affecting ERα protein level.

Down-regulation of ERα protein by active Akt could be attribut-able to Akt-mediated inhibition of one of Forkhead transcriptionfactors, Foxo3a [34]. Guo et al. [34] also identified two major func-tional Forkhead transcription factor binding sites in human ERαpromoter B, which is the second major promoter in MCF-7 cells andmammary gland [45]. The authors concluded that down-regulation ofERα by Akt might be resulted from the inhibition of Forkhead by Akt.Since there are no known Forkhead transcription factor binding site(s)in CMV promoter, which was used in ectopic expression of ERα in ourexperiments, ectopic ERα could not be down-regulated by Akt, rather,via Akt-dependent phosphorylation on S167, ectopic ERα protein levelcould be up-regulated, resulting in blocking of efficient turn-over ofERα, which is an essential requirement for ERα-dependent transcription.

Acknowledgements

This work is supported by a grant from the National R&D Programfor Cancer Control, Ministry of Health and Welfare, Republic of Korea(0620400-1), FPR06C3-101 of 21st Century Frontier Functional Prote-omics Project from Korean Ministry of Science and Technology and bya Korea Research Foundation grant (KRF-2005-070-C00105).

References

[1] M. Callige, I. Kieffer, H. Richard-Foy, Mol. Cell Biol. 25 (11) (2005) 4349.[2] D.J. Mangelsdorf, C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B.

Blumberg, P. Kastner, M. Mark, P. Chambon, R.M. Evans, Cell 83 (6) (1995) 835.[3] D.P. McDonnell, J.D. Norris, Science 296 (5573) (2002) 1642.[4] N.J. McKenna, B.W. O'Malley, Ann. N.Y. Acad. Sci. 949 (2001) 3.[5] E. Enmark, J.A. Gustafsson, J. Intern. Med. 246 (2) (1999) 133.[6] J.M. Hall, J.F. Couse, K.S. Korach, J. Biol. Chem. 276 (40) (2001) 36869.[7] J.F. Couse, K.S. Korach, Endocr. Rev. 20 (3) (1999) 358.[8] E.T. Alarid, N. Bakopoulos, N. Solodin, Mol. Endocrinol. 13 (9) (1999) 1522.[9] A. El Khissiin, G. Leclercq, FEBS Lett. 448 (1) (1999) 160.[10] D.M. Lonard, Z. Nawaz, C.L. Smith, B.W. O'Malley, Mol. Cell 5 (6) (2000) 939.[11] X. Long, K.P. Nephew, J. Biol. Chem. 281 (14) (2006) 9607.[12] Z. Nawaz, D.M. Lonard, A.P. Dennis, C.L. Smith, B.W. O'Malley, Proc. Natl. Acad. Sci.

U. S. A. 96 (5) (1999) 1858.[13] M. Callige, H. Richard-Foy, Nucl. Recept. Signal 4 (2006) e004.[14] G. Bunone, P.A. Briand, R.J. Miksicek, D. Picard, Embo J. 15 (9) (1996) 2174.[15] P.B. Joel, J. Smith, T.W. Sturgill, T.L. Fisher, J. Blenis, D.A. Lannigan, Mol. Cell Biol. 18 (4)

(1998) 1978.[16] R.A. Campbell, P. Bhat-Nakshatri, N.M. Patel, D. Constantinidou, S. Ali, H. Nakshatri,

J. Biol. Chem. 276 (13) (2001) 9817.[17] J.R. Testa, A. Bellacosa, Proc. Natl. Acad. Sci. U. S. A. 98 (20) (2001) 10983.[18] S.R. Datta, A. Brunet, M.E. Greenberg, Genes Dev. 13 (22) (1999) 2905.[19] S.R. Datta, H. Dudek, X. Tao, S. Masters, H. Fu, Y. Gotoh, M.E. Greenberg, Cell 91 (2)

(1997) 231.[20] M.H. Cardone, N. Roy, H.R. Stennicke, G.S. Salvesen, T.F. Franke, E. Stanbridge, S.

Frisch, J.C. Reed, Science 282 (5392) (1998) 1318.[21] A. Brunet, A. Bonni, M.J. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J. Anderson, K.C. Arden,

J. Blenis, M.E. Greenberg, Cell 96 (6) (1999) 857.[22] D.A. Lannigan, Steroids 68 (1) (2003) 1.[23] A. Vilgelm, Z. Lian, H. Wang, S.L. Beauparlant, A. Klein-Szanto, L.H. Ellenson, A.

Di Cristofano, Cancer Res. 66 (7) (2006) 3375.[24] T. Simoncini, A. Hafezi-Moghadam, D.P. Brazil, K. Ley, W.W. Chin, J.K. Liao, Nature

407 (6803) (2000) 538.[25] Y.R. Lee, J. Park, H.N. Yu, J.S. Kim, H.J. Youn, S.H. Jung, Biochem. Biophys. Res.

Commun. 336 (4) (2005) 1221.[26] I. Shin, F.M. Yakes, F. Rojo, N.Y. Shin, A.V. Bakin, J. Baselga, C.L. Arteaga, Nat.Med. 8 (10)

(2002) 1145.[27] I. Shin, J. Edl, S. Biswas, P.C. Lin, R. Mernaugh, C.L. Arteaga, Cancer Res. 65 (7) (2005)

2815.[28] F.L. Graham, A.J. van der Eb, Virology 54 (2) (1973) 536.[29] L.C. Cantley, Science 296 (5573) (2002) 1655.[30] J. Faridi, J. Fawcett, L. Wang, R.A. Roth, Am. J. Physiol. Endocrinol. Metabol. 285 (5)

(2003) E964.[31] J. Grisouard, S. Medunjanin, A. Hermani, A. Shukla, D. Mayer, Mol. Endocrinol. 21 (10)

(2007) 2427.[32] J.R. Woodgett, Embo J. 9 (8) (1990) 2431.[33] D.A. Cross, D.R. Alessi, P. Cohen, M. Andjelkovich, B.A. Hemmings, Nature 378 (6559)

(1995) 785.[34] S. Guo, G.E. Sonenshein, Mol. Cell Biol. 24 (19) (2004) 8681.[35] A.M. Fowler, N.M. Solodin, C.C. Valley, E.T. Alarid, Mol Endocrinol 20 (2) (2006) 291.[36] M. Saceda, T.W. Grunt, R. Colomer,M.E. Lippman, R. Lupu,M.B.Martin, Endocrinology

137 (10) (1996) 4322.[37] G. Andry, S. Suciu, D. Pratola, R. Sylvester, G. Leclercq, P.M. da Costa, N. Legros, M.

Andry-t'Hooft, A. Verhest, W. Mattheiem, et al., Eur. J. Cancer Clin. Oncol. 25 (2)(1989) 319.

[38] A.M. Fowler, N. Solodin, M.T. Preisler-Mashek, P. Zhang, A.V. Lee, E.T. Alarid, Faseb J.18 (1) (2004) 81–93.

[39] D.C. Allred, P. Brown, D. Medina, Breast Cancer Res. 6 (6) (2004) 240.[40] J. Liang, J.M. Slingerland, Cell Cycle 2 (4) (2003) 339.[41] M. Razandi, A. Pedram, E.R. Levin, Mol Endocrinol 14 (9) (2000) 1434.[42] R.J. Pietras, C.M. Szego, Nature 265 (5589) (1977) 69.[43] G. Reid, M.R. Hubner, R. Metivier, H. Brand, S. Denger, D. Manu, J. Beaudouin, J.

Ellenberg, F. Gannon, Mol. Cell. 11 (3) (2003) 695.[44] I. Shin, C.L. Arteaga, IUBMB Life 58 (11) (2006) 664.[45] G. Reid, S. Denger, M. Kos, F. Gannon, Cell. Mol. Life Sci. 59 (5) (2002) 821.