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Journal List > Mol Biol Cell > v.15(7); Jul 2004

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Mol Biol Cell. 2004 July; 15(7): 3266–3284.
doi: 10.1091/mbc.E03-11-0823.
PMCID: PMC452582
Copyright © 2004, The American Society for Cell Biology
Estradiol Abrogates Apoptosis in Breast Cancer Cells through Inactivation of BAD: Ras-dependent Nongenomic Pathways Requiring Signaling through ERK and AktD in Box
Romaine Ingrid Fernando and Jay Wimalasena*
Department of Obstetrics and Gynecology, and the Comparative and Experimental Medicine Program, Graduate School of Medicine, University of Tennessee, Knoxville, Tennessee 37920
Keith Yamamoto, Monitoring Editor
* Corresponding author. E-mail address: jwimalas/at/utk.edu.
Received November 17, 2003; Revised April 16, 2004; Accepted April 18, 2004.
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Abstract
Estrogens such as 17-β estradiol (E2) play a critical role in sporadic breast cancer progression and decrease apoptosis in breast cancer cells. Our studies using estrogen receptor-positive MCF7 cells show that E2 abrogates apoptosis possibly through phosphorylation/inactivation of the proapoptotic protein BAD, which was rapidly phosphorylated at S112 and S136. Inhibition of BAD protein expression with specific antisense oligonucleotides reduced the effectiveness of tumor necrosis factor-α, H2O2, and serum starvation in causing apoptosis. Furthermore, the ability of E2 to prevent tumor necrosis factor-α-induced apoptosis was blocked by overexpression of the BAD S112A/S136A mutant but not the wild-type BAD. BAD S112A/S136A, which lacks phosphorylation sites for p90RSK1 and Akt, was not phosphorylated in response to E2 in vitro. E2 treatment rapidly activated phosphatidylinositol 3-kinase (PI-3K)/Akt and p90RSK1 to an extent similar to insulin-like growth factor-1 treatment. In agreement with p90RSK1 activation, E2 also rapidly activated extracellular signal-regulated kinase, and this activity was down-regulated by chemical and biological inhibition of PI-3K suggestive of cross talk between signaling pathways responding to E2. Dominant negative Ras blocked E2-induced BAD phosphorylation and the Raf-activator RasV12T35S induced BAD phosphorylation as well as enhanced E2-induced phosphorylation at S112. Chemical inhibition of PI-3K and mitogen-activated protein kinase kinase 1 inhibited E2-induced BAD phosphorylation at S112 and S136 and expression of dominant negative Ras-induced apoptosis in proliferating cells. Together, these data demonstrate a new nongenomic mechanism by which E2 prevents apoptosis.
INTRODUCTION
The growth of human and animal tumors is a balance between cellular proliferation and death. Cell death in tumors occurs through necrosis and apoptosis, and highly malignant tumors often have high rates of both apoptosis and necrosis (Gompel et al., 2000 blue right-pointing triangle). Thus, it is not surprising that mitogenic substances such as insulin-like growth factor (IGF-1) also serve as cellular survival factors and are able to decrease rates of apoptosis. Estrogens, in particular 17-β estradiol (E2), are well-characterized mitogens for mammary tissues and epithelial cells of the female reproductive tract. Whereas estrogens exert antiapoptotic influence in neurons (Zhang et al., 2001 blue right-pointing triangle), epithelial cells of the female reproductive tract (Leung and Wang, 1999 blue right-pointing triangle; Choi et al., 2001 blue right-pointing triangle), immune system, blood cells, and endothelial cells (Bynoe et al., 2000 blue right-pointing triangle; Haynes et al., 2000 blue right-pointing triangle), they also have significant apoptotic effects on certain classes of bone-derived cells (Hughes et al., 1996 blue right-pointing triangle; Kameda et al., 1997 blue right-pointing triangle) and some immune system cells (Zajchowski and Hoffman-Goetz, 2000 blue right-pointing triangle; Okasha et al., 2001 blue right-pointing triangle). Furthermore, in some estrogen receptor (ER)-negative cells, overexpression of ER often leads to apoptosis (Kushner et al., 1990 blue right-pointing triangle) perhaps through activation of p38MAPK (Srivastava et al., 1999 blue right-pointing triangle, and references therein). Whereas E2 promotes cell survival in ER-positive cells both in cell culture and in xenograft models, antiestrogens induce apoptosis in both ER-positive and ER-negative cells (Srivastava et al., 1999 blue right-pointing triangle; Chen et al., 2000 blue right-pointing triangle; Gandhi et al., 2000 blue right-pointing triangle). Whether the latter results can be explained by expression of ER at low levels or an isoform of ER different from ERα is presently unknown.
In the ER-positive MCF7 breast cancer cell line, antiapoptotic effects of E2 have been reported previously (Huang, 1997 blue right-pointing triangle; Perillo et al., 2000 blue right-pointing triangle; Ahamed et al., 2001 blue right-pointing triangle); however, the mechanisms of action are poorly defined. It is now established that E2 induces transcription of the BCL2 gene and increases expression of BCL2 protein, which exerts antiapoptotic action in many cell types (Huang, 1997 blue right-pointing triangle; Dong et al., 1999 blue right-pointing triangle; Leung and Wang, 1999 blue right-pointing triangle). In addition to genomic actions, E2 has been known to exert rapid, likely nongenomic actions both in the whole animal and in cultured cells (Szego, 1974 blue right-pointing triangle; Huang, 1997 blue right-pointing triangle; Razandi et al., 2000a,b). Previously, we had observed that estrogens require the function of the Ras, mitogen-activated protein kinase kinase (MEK)1 and extracellular signal-regulated kinase (ERK) pathway to induce G1/S phase transition (Ahamed et al., 2001 blue right-pointing triangle) and elicit down-regulation of the Cdk2 inhibitor p27kip1 (Foster, 2003 blue right-pointing triangle; Zhu et al., 2003 blue right-pointing triangle). Given that E2 is now known to activate kinase-regulated signal transduction pathways in MCF7 and other cells (Migliaccio et al., 1996 blue right-pointing triangle, and references therein; Singh et al., 2000 blue right-pointing triangle; Zhang and Shapiro, 2000 blue right-pointing triangle), we have examined potential nongenomic pathways involved in its antiapoptotic actions, in particular the phosphatidylinositol 3-kinase (PI-3K)/Akt/BAD pathway, which mediates BAD inactivation and favors cell survival. In this article, we demonstrate for the first time that E2 induces BAD phosphorylation through Ras/PI-3K/Akt as well as Ras/ERK/p90RSK1 pathways, and we also show that the function of the PI-3K/Akt pathway is required for E2 to block apoptosis induced by tumor necrosis factor (TNF-α), H2O2, and serum withdrawal. Our data further provide evidence that BAD function is required for induction of apoptosis in MCF7 cells by TNF-α, H2O2, and serum withdrawal and that overexpression of the dual phosphorylation site mutant of BAD, but not the wild type, abolished the antiapoptotic actions of E2.
MATERIALS AND METHODS
Cell Culture, Serum Starvation, Treatment with E2 and Other Agents
MCF7 cells (a gift from R.P. Shiu; Dubik and Shiu, 1992 blue right-pointing triangle) were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) supplemented with 5% fetal bovine serum, penicillin G, and streptomycin at 37°C in 5% CO2. Cells were kept in 1% serum for 24 h in DMEM (phenol red [PR]-free) medium and completely serum withdrawn for additional 24-48 h before the experiments. Before treatments, cells were washed once with warm phosphate-buffered saline and treated in medium free of serum and PR. LY294002 (5 μM in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium [MTT] assay, 20 μM in other assays) (Sigma-Aldrich) and ICI182,780 (1 μM), PD98059 (50 μM) were added 1 h before E2 (1 nM in MTT assay and 10 nM in other assays) or IGF-1 (10 ng/ml). ICI182,780 is a gift from Zeneca Pharmaceuticals (Wilmington, DE). Where appropriate, cells were treated with dimethyl sulfoxide or ethanol as controls.
Cell Survival Assay
At 20-30% confluence, cells were treated in triplicates in 24-well plates. At 24-h intervals, MTT assay was performed according to the manufacturer's (Sigma-Aldrich) protocol. Briefly, MTT (5 mg/ml) was added to equal 1/10 the culture volume and incubated for 3 h. Medium was removed, and the converted dye was solubilized in ice-cold isopropanol. Absorbency of dye was measured at 560 nm with background subtraction at 630 nm on a microplate reader (EL 340 Bio Kinetics Reader; Bio-Tek Instruments, Winooski, VT)
DNA Fragmentation Assay (Cell Death Detection ELISAPLUS)
Apoptosis was induced with H2O2 (2 mM) for 1.5 h, TNF-α (10 ng/ml) for 8 h, or by complete removal of growth factors for 72 h. Cells were pretreated with E2 or IGF-1 for 1 h before apoptotic stimuli. DNA fragmentation was quantified using enzyme-linked immunosorbent assay (ELISA) kit (Cell Death ELISA; Roche Diagnostics, Indianapolis, IN) with slight modification to the manufacturer's protocol. Briefly, 50,000 cells of each treatment were lysed in 100 μl of lysis buffer, and a fraction of the supernatant was subjected to reaction for 2 h with the immunocomplex of anti-DNA conjugated with peroxidase, which binds to nucleosomal DNA, and antihistone-biotin, which interacts with streptavidin-coated wells in a microtiter plate. At the end of the incubation, substrate was added, and color development was quantified at 405-nm wavelength.
Adenoviral Infections
PTEN (kindly donated by Dr. R Bookstein; Cheney et al., 1999 blue right-pointing triangle), DNAkt (kindly donated by Dr. K Walsh; Kureishi et al., 2000 blue right-pointing triangle), and LacZ (Q.Biogene, Carlsbad, CA) were transduced at 60, 30, and 30 multiplicity of infection (MOI), respectively. Optimum MOIs were determined by analyzing DNA profiles (by flow cytometry) in response to virus dosage. To study DNA fragmentation with TNF-α or H2O2, cells were maintained in DMEM (PR free) with 2.5% fetal bovine serum for 24 h after infection followed by induction of apoptosis. In 2A, cells were completely serum withdrawn for additional 48 h after infection.
Western Blot Analysis
Cells were washed once with ice-cold phosphate-buffered saline and lysed with buffer containing 50 mM Tris-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 nM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mM NaVO3, 1 mM NaF.
For the detection of poly(ADP-ribose) polymerase (PARP) cleavage, cells were lysed in ice-cold buffer containing 50 mM Tris-HCl, 6.5 M urea, 5% mercaptoethanol, 2% SDS, and protease inhibitors, sonicated for 20 s, and briefly centrifuged to remove cell debris (Xu et al., 2002 blue right-pointing triangle).
For cytochrome c detection, cytosolic fraction was prepared as described in Hirai and Wang (2001 blue right-pointing triangle) with some modifications. Cells were suspended in hypotonic buffer containing 3.3 mM HEPES, pH 7.5, 1.7 mM KCl, 0.25 mM MgCl2, 0.17 mM EGTA, and 0.17 mM EDTA, 6.6 μg/ml leupeptin, 0.3 mM phenylmethylsulfonyl fluoride, 16.7 mM NaF, 66.7 μM Na3VO4, and 8.3 mM sodium β-glycerophosphate. After incubation on ice for 30 min, cells were homogenized with a Dounce homogenizer and centrifuged at 1000 × g for 5min at 4°C to discard unbroken cells and nuclei. The resulting supernatant was centrifuged at 10,000 × g for 10 min at 4°C to obtain the heavy-membrane fraction (pellet) and the cytosol (supernatant).
Aliquots of cell extracts containing 50-100 μg of total proteins or immunoprecipitates of desired proteins were resolved on SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific antibodies to phospho-ERKs [pTEpY] (Promega, Madison, WI), phospho-BAD [S112 and S136] (Upstate Biotechnology, Lake Placid, NY), ERK2, PTEN, BAD, PARP, cytochrome c, Ras, and Akt (Santa Cruz Biotechnology, Santa Cruz, CA) followed by secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology). Required concentrations of antibodies for Western blot analyses were determined using manufacturers' protocols. Proteins were detected by ECL (Amersham Biosciences, Piscataway, NJ). The band intensities/fold activities were measured by Sigma Gel software (Jandel Scientific, Winooski, VT).
Immunocomplex Kinase Activity Assays
Antibodies against ERK2, Akt, GSK3β, p85 (PI-3K), and protein A/G plus beads were purchased from Santa Cruz Biotechnology. Anti p90RSK1antibody, soluble BAD, and IRS-1 were purchased from Upstate Biotechnology. Myelin basic protein (MBP) was from Invitrogen (Carlsbad, CA). One hundred to 200 μg of total protein was immunoprecipitated with antibody in excess and protein A/G plus beads at 4°C overnight. Precipitates were washed three times with TG buffer (250 mM NaCl, 20 nM Tris, 0.5% NP-40) and once with kinase buffer (50 nM HEPES, pH 7.4, 15 mM MgCl2). Kinase reactions were performed by incubating immunoprecipitates and the specific substrates (0.2-1 μg/sample) in kinase mixture (20 μM ATP, 5 μCi of [γ-32P]ATP, 1 mM dithiothreitol, 0.1 mM Na3VO4 in kinase buffer) for 30 min at room temperature. Reactions were stopped with 4× Laemmli's sample buffer and were resolved on SDS-PAGE. Band intensities of phosphorylated substrates in autoradiograms were measured by densitometry.
Phosphorylation Assay (Nonradioactive) with BAD Mutants
p-GEX-BADwt/mutant (kindly donated by Drs. P Cohen; Lizcano et al., 2000 blue right-pointing triangle) and AM Tolkovsky; Virdee et al., 2000 blue right-pointing triangle) were grown in Escherichia coli, and proteins were isolated using glutathione-agarose beads (Sigma-Aldrich) followed by elution in the presence of reduced glutathione. Ten micrograms of BAD protein was incubated with 75 μg of whole cell lysate in 50 μl of kinase buffer in the presence of 25 μM ATP for 30 min. At the end of the reaction, 15 μl of agarose-conjugated glutathione was added and shaken for 1 h at 40°C. Beads were collected, washed three times with TG buffer, and resuspended in 25 μl of 2× Laemmli's sample buffer.
Treatment with BAD Oligonucleotides
BAD antisense oligonucleotides (with phosphorothioate linkages) were synthesized and purified by Sigma-Genosys (Woodlands, TX). Cells were transfected twice with LipofectAMINE Plus reagent according to the manufacturer's (Invitrogen) protocol 48 h apart. Transfections were carried out in six-well plates with 1 μg of oligonulceotide per well. Twenty-four hours after the second transfection, cells were treated and collected for desired analyses. Antisense oligonucleotides used in this study encoded complimentary sequences to the coding regions including translation initiation (BAD antisense 1) and termination codon (BAD antisense 2) sites for the human BAD gene. Oligonucleotide sequences used were BAD antisense 1, 5′-TGGGATCTGTAACATGCT-3′; BAD antisense 1 (scrambled/control), 5′-GGTTAGGTCAATTACTCG-3′; and BAD antisense 2, 5′-CGAAGGTCACTGGGA-3′. Cells were treated with H2O2 or TNF-α (with or without E2) or completely serum withdrawn for 72 h to induce apoptosis. DNA content was analyzed by flow cytometry and sub-G1 population was considered as apoptotic (Ormerod et al., 1992 blue right-pointing triangle).
Transient Transfection, Microscopic Analysis of Nuclear Morphology, and Confocal Microscopy
MCF7 cells grown on coverslips were transiently transfected with FLAG-tagged BAD mutants and enhanced green fluorescent protein (EGFP, 15% of total DNA) by using LipofectAMINE Plus reagent according to the manufacturer's (Invitrogen) protocol. The BAD constructs used in this study were human BAD wild-type (Wt) and human BAD serine75 and serine99 (equivalent to mouse serine112 and serine136, respectively) mutated to alanine (Dm) and were kindly donated by Dr. H.G. Wang (Hirai and Wang, 2001 blue right-pointing triangle). Coverslips were fixed in 3% paraformaldehyde and mounted on to glass slides. Nuclei were stained with 3 μg/ml Hoechst 33342 (Sigma-Aldrich) and analyzed for the blue (DNA) and green (EGFP) fluorescence by using a fluorescence microscope (Leitz DMRB) or the confocal microscope. Cells expressing EGFP were considered as coexpressing BAD mutants. We have previously validated the coexpression of plasmid constructs in MCF7 cells (Foster, 2003 blue right-pointing triangle). Morphological changes in the nuclei were analyzed and condensed/shrunk nuclei were counted as apoptotic as opposed to normal round nuclei (Candolfi et al., 2002 blue right-pointing triangle).
FLAG-wtBAD and RasN17, RasV12T35S (kindly donated by Drs. G.L. Johnson and C.M. Counter (Hamad et al., 2002 blue right-pointing triangle, respectively) or empty vector were cotransfected at 1:1 ratio for coexpression studies. For macroscopic analyses, cells grown on coverslips were transfected with Ras or empty vector constructs together with EGFP (15% of total DNA) and processed and analyzed as described above.
Statistical Analysis
Statistical analysis was performed using Student's t test (Prism; GraphPad Software, San Diego, CA), and p ≤ 0.05 is considered significant.
RESULTS
Inhibition of Apoptosis in MCF7 Cells by E2 Is Dependent on the PI-3K/Akt Pathway
Previous work in our laboratory has shown that estrogens decreased cell death and apoptosis induced in MCF7 cells by mitogen withdrawal (Ahamed et al., 2001 blue right-pointing triangle). Recent work indicates that induction of cell growth by E2 in MCF7 cells may require activity of the PI-3K pathway (Lobenhofer et al., 2000 blue right-pointing triangle) and the Ras/ERK pathway (Castoria et al., 2001 blue right-pointing triangle). Furthermore, we reported that G1/S phase transition and Cdk2 activation induced by estrogens is inhibited by dominant-negative (DN) Ras mutants (Ahamed et al., 2001 blue right-pointing triangle; Foster, 2003 blue right-pointing triangle; Zhu et al., 2003 blue right-pointing triangle), and the MEK1 inhibitor PD98059 abrogated the increase in cyclin D1 accumulation resulting from estrogen treatment (Ahamed et al., 2001 blue right-pointing triangle).
Given this background, we sought to determine whether PI-3K plays an important role in estrogen action with respect to cell growth and apoptosis. In preliminary experiments (our unpublished data), we verified that E2 promoted cell survival/growth under low serum conditions and further established that the PI-3K inhibitor wortmannin inhibited this effect of E2. To confirm the requirement of PI-3K activity for the cell survival induced by E2, MCF7 cells were treated with E2 in the presence or absence of LY294002, a specific PI-3K inhibitor (Sanchez-Margalet et al., 1994 blue right-pointing triangle) in serum-free medium. In the absence of E2, there was significant cell loss reflected in MTT uptake over a 5-d period (p < 0.05). E2 completely blocked this decrease and maintained MTT uptake at or above day 1 level (Figure 1AFigure 1.). Treatment with LY294002 abolished the ability of E2 to increase cell numbers as indicated by MTT uptake (day 2) or maintain cell survival (day 4, 5) and also reduced viability in the control cultures at day 4 and 5. These results suggest that survival of MCF7 cells under conditions of serum withdrawal requires PI-3K activity and that the ability of E2 to protect cells from death as well as to induce growth is blocked by abrogation of PI-3K activity. MTT results simply demonstrate the amount of live cells present under given conditions and, in the case of E2, this is may present the sum of antiapoptotic and mitogenic effects.
Figure 1.
Figure 1.
Figure 1.
Inhibition of cell death in MCF7 cells by E2. (A) MCF7 cells were serum deprived after 20% confluence and treated in PR-free medium with indicated stimuli. LY294002 (5 μM) was added 30 min before E2 (1 nM) treatment. MTT uptake was measured at (more ...)
The above-mentioned results and prior observations suggest that E2 may decrease apoptosis in MCF7 cells. Because TNF-α induces apoptosis through activation of caspases 3, 6, 7, and 8 and/or JNK activation (Budihardjo et al., 1999 blue right-pointing triangle) in a variety of cell types, including MCF7 cells (Burow et al., 1999 blue right-pointing triangle; Guicciardi et al., 2000 blue right-pointing triangle; Tang et al., 2001 blue right-pointing triangle), we treated MCF7 cells with TNF-α in the presence of E2, 17-α estradiol (an estrogen of low potency), E2 plus an excess of the specific E2 antagonist ICI182,780. TNF-α increased apoptosis as measured by DNA fragmentation by four- to fivefold, and E2 was able to reduce this effect significantly, whereas 17α-estradiol had no protective effect. The presence of an excess of the E2 antagonist blocked the protective effect of E2 in TNF-α-treated cells (Figure 1BFigure 1.). PARP cleavage, a well-known late event in the apoptosis process (Duriez and Shah, 1997 blue right-pointing triangle), was induced by TNF-α, and this cleavage was significantly blocked by E2 pretreatment (Figure 1CFigure 1.)
The reactive oxygen species H2O2 is known to induce apoptosis in a variety of cell types, including MCF7 cells (Kim et al., 2000 blue right-pointing triangle), and the apoptotic effect of H2O2 in these cells is decreased by pretreatment with E2 or overexpression of BCL2 in a synergistic manner (Perillo et al., 2000 blue right-pointing triangle). Furthermore, E2 and overexpression of BCL2 exhibit additive inhibitory effects on cleavage of PARP (Duriez and Shah, 1997 blue right-pointing triangle) induced by H2O2. In confirmation of these data, we found that E2 pretreatment can block DNA fragmentation as well as PARP cleavage induced by 2 mM H2O2 (Figure 1, B and CFigure 1.). Pretreatment with 17-α estradiol had no significant protective effect against H2O2, suggesting that E2 effects are unlikely to be due to its potential free radical scavenger function (Behl et al., 1997 blue right-pointing triangle).
Apoptotic effects of certain stimuli, including those activating the extrinsic pathway, are manifested at least in part via their effects on mitochondrial membrane integrity (Downward, 1999 blue right-pointing triangle; Gross et al., 1999 blue right-pointing triangle). Release of cytochrome c from dysfunctional mitochondria contributes to the formation of the apoptosome, which ultimately activates the downstream caspase pathways (Budihardjo et al., 1999 blue right-pointing triangle). Because the results presented in Figure 1, A-CFigure 1., would predict that E2 may preserve mitochondrial integrity in the presence of apoptotic stimuli, we sought to determine whether E2 would preserve mitochondrial integrity by assessing cytoplasmic cytochrome c protein level (Figure 1DFigure 1.). In untreated or E2 only-treated cells, cytoplasmic cytochrome c was undetectable. As expected, H2O2, TNF-α, and serum withdrawal (see below) increased cytochrome c in the cytoplasm. Cotreatment with E2 decreased cytochrome c release induced by TNF-α to a similar extent as that observed in cells cotreated with serum and TNF-α. Together, these results suggest that E2 protects cells against apoptosis induced by TNF-α, H2O2, and serum withdrawal (our unpublished data), possibly by maintaining mitochondrial integrity.
Estradiol Effects on H2O2-, TNF-α-, and Serum Withdrawal-induced Apoptosis Are Abolished by Dominant Negative Akt (DNAkt) and Wild-Type PTEN
To establish the role of the PI-3K pathway in E2-induced antiapoptosis, we used adenoviral vectors to interfere with operation of this pathway. Cells were transduced with adenoviral wild-type PTEN/MMAC (Figure 2DFigure 2.) that effectively decreases activity of the PI-3K pathway by hydrolysis of PI-3K products (Maehama and Dixon, 1999 blue right-pointing triangle), and overexpression of PTEN has been used to decrease PI-3K products in other studies (Nakashima et al., 2000 blue right-pointing triangle). As depicted in Figure 2Figure 2., overexpression of PTEN completely blocked the ability of E2 or IGF-1 to prevent apoptosis induced by serum withdrawal (A), H2O2 (B), and TNF-α (C) compared with adenoviral LacZ (control)-infected cultures. Akt/PKB is a downstream target of PI-3K pathway that promotes cell survival in a number of cell types (Downward, 1998 blue right-pointing triangle; Datta et al., 1999 blue right-pointing triangle). Overexpression of DNAkt (Figure 2DFigure 2.) enhanced apoptosis induced by the agents and completely reversed the effects of either IGF-1 or E2 on apoptosis prevention (Figure 2, A-CFigure 2.). These data together with data derived using chemical inhibitors (Figure 1AFigure 1.) strongly suggest that PI-3K/ Akt pathway is essential for the antiapoptotic effects of E2 or IGF-1 in MCF7 cells. We repeatedly observed that DNA fragmentation (measured by the Cell Death ELISA) in viral-transduced cells was below the levels observed in parental cells (1.8-2.5 vs. 4-5 fold, compare Figures Figures1B1BFigure 1. and and2),2Figure 2.), which lead to an apparent decrease in the magnitude of apoptosis as measured by the Cell Death ELISA in Figure 2Figure 2..
Figure 2.
Figure 2.
Figure 2.
Inhibition of apoptosis in MCF7 cells by E2 is dependent on the PI-3K/Akt pathway. Viral vectors were used to overexpress PTEN, DNAkt, and Lac-Z (control) at predetermined MOIs. Forty-eight hours after the infection apoptosis was induced by complete serum (more ...)
Estradiol Induces BAD Phosphorylation
BAD is a proapoptotic BH3 domain-containing protein (Gross et al., 1999 blue right-pointing triangle; Adams and Cory, 2001 blue right-pointing triangle), which forms heterodimers with BCL2 or BCLxl (Condorelli et al., 2001 blue right-pointing triangle) resulting in cytochrome c release from mitochondria. Antiapoptotic agents such as IGF-1 induce BAD phosphorylation at specific serine residues, and phosphorylated BAD is sequestered away from its site of action in the mitochondria by binding to cytosolic 14-3-3 proteins (Datta et al., 1997 blue right-pointing triangle). Because BAD is phosphorylated on serine 136 (S136) by Akt (Datta et al., 1997 blue right-pointing triangle), we ascertained the ability of E2 to induce phosphorylation of BAD at specific residues by using BAD phosphorylation site mutants as in vitro substrates (Figure 3AFigure 3.). Clearly E2- or IGF-1-treated cell extracts were unable to induce phosphorylation of BAD at S112 in the serine-to-alanine mutant (S112A), whereas S136 phosphorylation was enhanced three- to fourfold. Similar specificity was observed when the S136A BAD mutant was used as the substrate (Figure 3AFigure 3., right). When the double phosphoryaltion site mutant (DM) was used as a substrate, only a small fraction of the phosphorylation that was observed with single mutants was detectable. Multiple immunoreactive bands have been previously observed when recombinant BAD was used as a substrate (Datta et al., 1997 blue right-pointing triangle).
Figure 3.
Figure 3.
Figure 3.
Estradiol induces BAD phosphorylation. (A) Glutathione-tagged BAD proteins (BAD S112A, BAD S136A, and BADS112/136A [DM]) were subjected to in vitro kinase assay for 30 min with extracts from MCF7 cells that were treated with E2 (10 nM) or IGF-1 (10 ng/ml) (more ...)
We also measured BAD phosphorylation in vivo by Western blot analysis. After treatment with E2 or IGF-1 for 15 min, both S112 and S136 phosphorylations were enhanced (>3-fold), and total BAD levels were unchanged in this time frame (Figure 3BFigure 3.).
Because our results demonstrated that E2 can induce BAD phosphorylation in vitro and in vivo and can prevent cell death in response to several stimuli, we studied the possible changes in phosphorylation status of BAD during apoptosis induction. E2 increased endogenous BAD phosphorylation at S112 and S136 above the basal level severalfold, whereas TNF-α and H2O2 decreased it below control level (Figure 3CFigure 3.). Cotreatment with E2 and H2O2 or TNF-α restored the BAD phosphorylation to above-control levels. Together, these data strongly suggest that E2 as well as IGF-1 exert antiapoptotic actions partly through phosphorylation and inactivation of the proapoptotic BAD protein and that phosphorylation of BAD is a consequence of activation of Akt and possibly ERK/p90RSK1 because S112 is a target site for p90RSK1 (Downward, 1999 blue right-pointing triangle).
Inhibition of BAD Is a Critical Event in Estradiol-induced Antiapoptosis in MCF7 Cells
Because the effects of apoptotic agents on decreasing BAD phosphorylation (inactivation) were largely reversed by E2, BAD inactivation may be a potential nongenomic effect of E2, which results in cell survival. To further understand the involvement of BAD in cell death and survival in MCF7 cells, we used antisense oligonucleotides to prevent the translation of BAD mRNA. The antisense sequences, BAD antisense 1 (BAD As 1) containing the complementary sequence to the translation initiation codon and BAD antisense 2 (BAD As 2) containing complementary sequence to the translation termination codon were equally effective in down-regulating BAD protein level (Figure 4AFigure 4.Figure 4.), whereas a scrambled oligonucleotide corresponding to BAD As1 (control oligo) was not. Quantification of DNA by flow-cytometry revealed a large reduction of the sub-G1 fraction-apoptotic cells (Ormerod et al., 1992 blue right-pointing triangle) in TNF-α-treated (Figure 4BFigure 4.Figure 4.) as well as serum-starved (our unpublished data) cells in BAD As1- and BAD As2-treated, but not in control oligonucleotide-treated cells. E2 did not have an additional effect alone or together with TNF-α, in antisense-treated cells compared with control oligonucleotide treated cells (our unpublished data). These observations suggest a critical role of BAD in apoptosis in MCF7 cells and agree with a previous study where BAD overexpression resulted in apoptosis of mammalian cells (Ottilie et al., 1997 blue right-pointing triangle; Virdee et al., 2000 blue right-pointing triangle). Change in apoptosis due to TNF-α was calculated by subtracting sub-G1 fraction of untreated from sub-G1 fraction of TNF-α-treated cells for each oligonucleotide-treated population. Data from three independent experiments are shown in Figure 4CFigure 4.Figure 4.. Introduction of BAD As1 or BAD As2 significantly reduced the magnitude of apoptosis in response to TNF-α (*p < 0.05), H2O2, and complete serum starvation (our unpublished data). We next measured DNA fragmentation in oligonucleotide-treated cells in the presence or absence of TNF-α. TNF-α induced four- to fivefold DNA fragmentation above the basal level (comparable to Figure 1BFigure 1.) in control oligonucleotide- or LipofectAMINE-treated cells. BAD As1 drastically reduced this effect of TNF-α (**p < 0.005) and maintained the DNA fragmentation near the basal level (Figure 4DFigure 4.Figure 4.). Similar results were observed using BAD As2 (our unpublished data). These data clearly suggest a critical involvement of BAD in cell survival and death in response to several agents in MCF7 breast cancer epithelial cells and agree with the reduction in basal BAD phosphorylation induced by TNF-α and H2O2 in the absence of E2 (Figure 3CFigure 3.).
Figure 4.Figure 4.
Figure 4.
Figure 4.
Figure 4.
BAD inhibition is a critical event in TNF-α-induced apoptosis and its prevention by E2 in MCF7 cells. (A) MCF7 cells were treated with BAD antisense (BAD As1 and BAD As2) and control oligonucleotides (see MATERIALS AND METHODS), and protein extracts (more ...)
To assess the effects on BAD in E2-induced cell survival and consequent changes in cellular morphology, we transiently expressed wild-type BAD (wtBAD) and dual phosphorylation site mutant of BAD (dmBAD) where S112 and S136 are mutated to unphosphorylatable alanine, together with an EGFP expression vector. After the desired treatments, coverslip cultures were stained with Hoechst 33342 and mounted on to glass slides. As shown in Figure 4EFigure 4.Figure 4., E2 reduced the TNF-α-induced apoptosis in wtBAD expressing cells by >50%, and importantly had no protective effect in dmBAD-expressing cells. Pretreatment of E2 had the same protective effect against TNF-α in cells expressing the control vector pcDNA3 or EGFP alone (our unpublished data). It must be emphasized that the overexpression of dmBAD did not alter the number of apoptotic nuclei compared with those in pcDNA3- or EGFP-transfected cultures. There is an absolute requirement for TNF-α to induce apoptosis in dmBAD as well as wtBAD expressing cells. The magnitude of apoptosis observed with cell counting (~8- to 9-fold in Figure 4EFigure 4.Figure 4.) is somewhat higher than that observed with DNA fragmentation ELISA assay (4- to 5-fold in Figures Figures1B1BFigure 1. and and4D)4DFigure 4.Figure 4.) in response to TNF-α. Possible explanation for such differences in magnitude may be the fact that only a fraction of all the cells (i.e., the transfected population) is counted in Figure 4EFigure 4.Figure 4., whereas in Figures Figures1B1BFigure 1. and and4D4DFigure 4.Figure 4. DNA fragmentation of the whole population is measured.
Similarly transfected and treated cells were examined by the confocal microscopy to further analyze the cellular morphological changes (Supplementary Figure 1). TNF-α-induced cellular shrinkage and atypical structure of nuclei in MCF7 cells as has been described previously for fibroblast (Luschen et al., 2000 blue right-pointing triangle) and neuronal cells (Candolfi et al., 2002 blue right-pointing triangle). It was difficult to observe the chromatin clumping (typical for apoptosis) in TNF-α-treated cells due to shrinkage of the nuclei as opposed to this feature readily apparent in paclitaxel-treated cells (our unpublished data). Under these conditions, expression of exogenous wtBAD or most importantly, dmBAD (white arrows in florescence images and respective differential interference contrast images) did not enhance basal apoptosis (1 and 4). Treatment with TNF-α in wt and dmBAD-transfected cells clearly induced apoptotic changes (2 and 5). Whereas E2 was able to impede morphological effects of TNF-α in wtBAD-expressing cells (3), it was unable to counteract the effects of TNF-α in dmBAD-expressing cells (6 and 7). Black arrows in 6 and 7 point to the untransfected cells that preserve the normal morphology. Under these conditions, expression of EGFP alone did not induce morphological changes (white arrows in the insert). These observations further support the proposed crucial role of BAD in MCF7 cells and the critical importance of the two specific phosphorylation sites in BAD for the antiapoptotic effects of E2. It is very clear that dmBAD by itself does not induce apoptosis in the absence of TNF-α. This suggests that in these mammary carcinoma cells, there are mechanisms to possibly sequester BAD away from mitochondria independent of S112 and S136 phosphorylation and binding to 14-3-3 proteins, thus making dmBAD ineffective as an apoptosis inducer by itself. Antiapoptotic effect of E2 was further evaluated in cells expressing increasing levels of BAD. MCF7 cells were transfected with 1/4×, 1/2×, 1×, and 2× (× = regular dose = 0.6 μg of DNA for 10 cm2) of each construct and were treated with TNF-α with or without E2. DNA fragmentation was assayed as in Figure 4DFigure 4.Figure 4.. As shown in Figure 4F, 1Figure 4.Figure 4., E2 reduced apoptosis by 45-60% in the wtBAD-expressing cells, irrespective of the dose, in agreement with Figure 4EFigure 4.Figure 4.. E2-mediated protection was dampened by the overexpression of dmBAD by 50% or more. A moderate dose dependency in response to dmBAD was observed in TNF-α as well as the E2 + TNF-α-treated cells (Figure 4F, 3Figure 4.Figure 4.). The values of apoptosis as measured by ELISA in pcDNA3, wtBAD, and dmBAD were unrelated to the dose of DNA. Therefore, when the DNA fragmentation values were averaged for the three types of transfections there was absolutely no difference among the constructs (O.D. values at 405 nm of 0.15, 0.16, and 0.15 for pcDNA3, wtBAD, and dmBAD, respectively). Increasing doses of wtBAD did not influence the basal apoptosis, TNF-α-induced apoptosis, or the protection offered by E2 as shown in Figure 4F, 2Figure 4.Figure 4.. Panel 4 demonstrates the fold induction of apoptosis in pcDNA3-expressing cells at regular dose of BAD. In wtBAD-transfected cells (Figure 4F, 1 and 2Figure 4.Figure 4.) E2 is able to decrease the apoptosis due to TNF-α to the same extent as in untransfected or pcDNA3-transfected cells (Figures (Figures1B1BFigure 1. and 4F, 4Figure 4.Figure 4.) by 50-60% when measured by the ELISA method. Therefore, untransfected cells are unlikely to influence results in the wtBAD-transfected cells. In contrast, the inability of E2 to prevent apoptosis in dmBAD mutant transfected cells is likely to be underrated as the ELISA measurement is an average of transfected and untransfected cells. In the latter, E2 will prevent apoptosis in the majority of the cells, thereby diluting the apoptotic effect of dmBAD-transfected cells. Together, the data in Figure 4, E and FFigure 4.Figure 4., and Supplementary Figure 1 strongly suggest that dmBAD is not an apoptosis inducer by itself in MCF7 cells. This reinforces our notion that in these cells, TNF-α (and perhaps other apoptotic stimuli) is able to translocate cytoplasmic BAD to its mitochondrial sites of action irrespective of phosphorylation at S112 and S136 residues.
Estradiol-induced BAD Phosphorylation Requires Ras-activated Signaling Pathways, and Ras Function Is Critical for Cell Survival of MCF7 Cells
Previous studies in our laboratory had demonstrated that E2 induced p27kip1 degradation and entry of quiescent MCF7 cells to the cell cycle requires Ras activity (Ahamed et al., 2001 blue right-pointing triangle, 2002 blue right-pointing triangle; Foster et al., 2001 blue right-pointing triangle). Other investigators have demonstrated that E2 rapidly activated Ras in MCF7 cells (Keshamouni et al., 2002 blue right-pointing triangle), and recent work in our laboratory (Foster, 2003 blue right-pointing triangle) demonstrated that Ras activity is required for nuclear export and degradation of p27kip1 in response to E2. The phosphorylation of BAD at S112 and S136 has been proposed to be mediated by ERK/p90RSK1 and PI-3K/Akt pathways, respectively, in addition to other mechanisms (Downward, 1999 blue right-pointing triangle, and references therein). Given the role of Ras discussed above and the evidence that the PI-3K and ERK pathways are activated by E2 (as discussed below), we used chemical inhibitors LY294002 and PD98059 to inhibit these pathways, respectively, and determined the effects of these treatments on BAD phosphorylation. As shown in Figure 5AFigure 5., E2 induced BAD phosphorylation on S136 as well as S112 was completely abrogated by LY294002, supporting the idea of cross talk between the MAPK/p90RSK1 and PI-3K/Akt pathways in enhancing the BAD phosphorylation (discussed under Figure 7Figure 7.). Similarly PD98059 effectively reduced the phosphorylation of BAD at S112 as determined by Western blots; however, the MEK inhibitor had considerably less effect on BAD S136 phosphorylation (compare E2 vs. PD + E2 on BAD phosphorylation on S136). None of the treatments changed total BAD levels in this time frame.
Figure 5.
Figure 5.
Figure 5.
Estradiol-induced BAD phosphorylation is mediated through Ras-activated signaling pathways. (A) MCF7 cells were pretreated with LY294002 (20 μM) or PD98059 (50 μM) for 1 h before E2 treatment for 15 min. BAD phosphorylation at S112 or (more ...)
Figure 7.
Figure 7.
Figure 7.
17-β-Estradiol induces activity of ERKs and p90RSK1. (A) Serum-starved MCF7 cells were treated with E2 (10 nM) for indicated times. ERK2 immunoprecipitates were subjected to in vitro kinase assay with MBP as a substrate. Band intensities were (more ...)
Because Ras is a well-known upstream regulator of the PI-3K/Akt and MEK/ERK/p90RSK1 pathways, we analyzed the effects of inhibition of endogenous Ras by using dominant negative Ras RasN17 (DNRas) or selectively activating the MEK/ERK pathway with RasV12T35S (Hamad et al., 2002 blue right-pointing triangle) in transient transfection experiments. FLAG-tagged BAD was cotransfected with control empty vector, RasN17, or RasV12T35S into MCF7 cells. Twenty-four hours after the transfection, cells were serum withdrawn for another 24 h and stimulated with E2 for 15 min. BAD phosphorylation was assessed by Western blotting. Clearly, the ability of E2 to increase both S112 and S136 phosphorylation (Figure 5B, 1Figure 5., lane2) of exogenous as well as endogenous BAD was inhibited by RasN17 (lane 4), which by itself did not regulate BAD phosphorylation (lane 3). The MEK/ERK-selective activator RasV12T35S by itself significantly activated BAD S112 phosphorylation and moderately activated exogenous BAD S136 phosphorylation (lane 5), and E2 was able to enhance this effect (lane 6). Together, the results in Figure 5, A and BFigure 5., strongly indicate that the ability of E2 to stimulate BAD phosphorylation and thereby prevent apoptosis is mediated through Ras-activated MEK/ERK and PI-3K/Akt pathways.
To directly analyze the apoptotic effects of RasN17 in these cells, we stained nuclei of Ras-transfected cells and studied them by using the confocal microscope. Nuclei corresponding to EGFP-expressing cells (cotransfected with Ras or control vector) were counted, and the graph (Figure 5CFigure 5.) shows the number of fragmented nuclei as a percentage of total number of cells. Transient overexpression of RasN17 induced seven- to eightfold apoptosis over a 48-h period, whereas the expression of RasV12T35S or empty vector had minimum effects on apoptosis observed in cells coexpressing EGFP. Representative morphological data are shown in Supplementary Figure 2 (1-5), which clearly demonstrates that expression of EGFP, empty vector, or RasV12T35S (1-3) did not alter nuclear morphology. However, RasN17-induced apoptotic nuclear morphology in the detached dead cells (5), whereas untransfected attached cells from the same cultures (4) had normal nuclei. These results demonstrate that Ras function is required to protect MCF7 cells from dying even in the presence of growth medium and that this effect may be mediated at least partly by phosphorylating and inactivating proapoptotic BAD. The observation that E2 induced BAD phosphorylation was abolished by inactivation of endogenous Ras (Figure 5BFigure 5.) further highlights the involvement of Ras-mediated pathways in E2 signaling and demonstrates that Ras plays a critical role in the antiapoptosis mediated by E2 in MCF7 breast cancer cells.
Estradiol Activates the PI-3K/Akt Pathway, Leading to Phosphorylation of Downstream Targets
Activation of PI-3K and Akt and Inactivation of GSK3β by E2 The above-mentioned data strongly argues that the ability of E2 to decrease apoptosis in MCF7 cells is at least partly mediated by the PI-3K/Akt pathway. Transient activation (measured by S473 phosphorylation) of Akt by E2 in MCF7 cells was reported previously (Castoria et al., 2001 blue right-pointing triangle; Razandi et al., 2002 blue right-pointing triangle) (see DISCUSSION) while this work was in progress (Fernando and Wimalasena, 2000 blue right-pointing triangle, 2002 blue right-pointing triangle). Our data suggest that the PI-3K/Akt pathway may need to be activated for a prolonged period to maintain BAD in an inactivated (phosphorylated) state. Therefore, we measured detailed kinetic behavior of the PI-3K response to E2. PI-3K activation was determined by phosphorylation of IRS-1 on S612 by in vitro kinase assay using immunoprecipitates of the p85 subunit of PI-3K. As shown in Figure 6AFigure 6., E2 was able to rapidly activate PI-3K approximately threefold, similar in magnitude to the activation elicited by IGF-1. A detailed time course of Akt catalytic activity measured by in vitro kinase assays (n = 3) using Akt immunoprecipitates and BAD as the substrate is shown in Figure 6BFigure 6.. Treatment with E2 does not change protein levels of Akt in this time period (our unpublished data). These data demonstrate that in contrast to the results from the S473 phosphorylation assay (Castoria et al., 2001 blue right-pointing triangle), catalytic activity of Akt is increased at least for 120 min. GSK3β is a downstream target of Akt (Cross et al., 1995 blue right-pointing triangle) and because of its potential role in the cell cycle, we determined GSK3β activity by in vitro kinase assay using immunoprecipitates of GSK3β. As shown in Figure 6CFigure 6., E2 rapidly and strongly inhibited GSK3β activity. As expected, another Akt activator IGF-1 also completely inhibited GSK3β activity. The effect of E2 was sustained for 60 min (our unpublished data), and such a time course is compatible with the increase in cyclin D1 protein observed 3-4 h after E2 addition to quiescent cells (Foster and Wimalasena, 1996 blue right-pointing triangle; Foster et al., 2001 blue right-pointing triangle).
Figure 6.
Figure 6.
Figure 6.
17-β-Estradiol activates the PI-3K/Akt pathway. Serum-deprived MCF7 cells were treated with E2 (10 nM) or IGF-1 (10 ng/ml) for indicated times. Immunoprecipitates of PI-3K (A), GSK3β (C), and p90RSK1 (E) were subjected to in vitro kinase (more ...)
Activation of ERKs by Estradiol. It is known that p90RSK1 is phosphorylated at S364 and T574 residues by ERKs, leading to its activation (Dalby et al., 1998 blue right-pointing triangle), and our results on BAD phosphorylation imply that E2 activates p90RSK1. Therefore, we directly determined that E2 rapidly activates p90RSK1 by in vitro kinase assay using immunoprecipitates of p90RSK1 (Figure 6, D and EFigure 6.), which shows that p90RSK1 is activated within 5 min by E2. A number of studies reported that E2 rapidly activates ERK1 and 2 in MCF7 cells (Improta-Brears et al., 1999 blue right-pointing triangle, and references therein) and in a number of other cell types (Singh et al., 1999 blue right-pointing triangle; Kousteni et al., 2001 blue right-pointing triangle). However, others failed to observe E2-mediated ERK activation in MCF7 cells (Dupont et al., 2000 blue right-pointing triangle; Lobenhofer et al., 2000 blue right-pointing triangle; Caristi et al., 2001 blue right-pointing triangle), and E2-induced ERK activity may not be a result of activation of the Ras/Raf signal transduction pathway but may follow rapid increase in intracellular Ca2+ (Improta-Brears et al., 1999 blue right-pointing triangle). Furthermore, we had previously demonstrated that increased cyclin D1 synthesis in response to estrogens is partly blocked by the MEK1 inhibitor PD98059 and that dominant negative ERK2 partially inhibited the induction of cell cycle transit by estrogens (Ahamed et al., 2001 blue right-pointing triangle). Therefore, we measured a detailed time course of ERK activation by E2. We found that E2 rapidly activated ERK2 with maximum activation of fivefold observed within 15 min (Figure 7AFigure 7.) and significant ERK2 activation was sustained for at least 120 min. Enhancement of ERK activity by E2 was partly blocked not only by pretreatment with the PI-3K inhibitor LY294002 but also by the specific estrogen antagonist ICI182,780 (Figure 7BFigure 7.). Given the above-mentioned results and recent work indicating that there is cross talk between the PI-3K and ERK pathways (Hawes et al., 1996 blue right-pointing triangle; Wennstrom and Downward, 1999 blue right-pointing triangle), we explored this interaction further by overexpression of adenoviral PTEN. Whereas E2 or IGF-1 activated ERKs in control Ad virus-infected cells, PTEN overexpression (~3× over the basal level; Figure 7C, 3Figure 7.) partly blocked ERK activation observed at 15 min of treatment with these two agonists (Figure 7C, 1Figure 7.). In confirmation of these data by using phospho-specific ERK antibodies, PTEN also partly inhibited ERK2 activity measured by MBP phosphorylation in an in vitro kinase assay (Figure 7C, 2Figure 7.). In extracts from similarly treated cells, BAD phosphorylation in vitro by Akt and p90RSK1 was also selectively abrogated by PTEN overexpression (Figure 7DFigure 7.), demonstrating that PTEN overexpression inhibited Akt and p90RSK1 activation. These results suggest that the PI-3K pathway may regulate the ERK response in addition to the Akt response in MCF7 cells and provide evidence for cross talk between these signal pathways in response to E2 or IGF-1 under our experimental conditions.
DISCUSSION
Regulation of Apoptosis by E2
E2 is known to support cell survival or induce cell death/apoptosis depending on the cell context (Choi et al., 2001 blue right-pointing triangle; Okasha et al., 2001 blue right-pointing triangle). Most of E2's actions on cell survival or death, as for many other steroid hormone actions, have been attributed to gene regulation. Emerging evidence for nongenomic actions of E2 (reviewed in Kelly and Levin, 2001 blue right-pointing triangle) prompted us to examine the signaling mechanisms of E2 with respect to regulation of cell survival in MCF7 breast cancer cells. We had previously shown that estrogens were able to decrease apoptosis induced by mitogen withdrawal in MCF7 cells (Ahamed et al., 2001 blue right-pointing triangle). Our present study shows that E2 significantly reduces apoptosis induced by TNF-α, H2O2, and serum withdrawal by using signal transduction pathways that lead to phosphorylation and inactivation of proapoptotic protein BAD. In Figure 8Figure 8. we summarize our results and present a model on how E2 is able to promote cell survival and abrogate apoptosis in MCF7 cells. However, E2 is unable to counteract the apoptosis-inducing effects of other agents such paclitaxel (our unpublished data); this observation is in contrast to recently published work (Razandi et al., 2002 blue right-pointing triangle).
Figure 8.
Figure 8.
Figure 8.
Schematic diagram showing the proposed survival mechanism induced by 17-β-estradiol. Estradiol activates ERK/p90RSK1 and PI-3K/Akt pathways possibly through Ras-dependent mechanisms. On activation of these signal pathways, proapoptotic BAD is (more ...)
Role of BAD in Apoptosis and Its Regulation by E2
E2 effects on BCL2 family members have been described previously: in cells where survival is promoted, E2 increased mRNA (Perillo et al., 2000 blue right-pointing triangle) and protein levels of antiapoptotic BCL2 (Dong et al., 1999 blue right-pointing triangle; Choi et al., 2001 blue right-pointing triangle) or down-regulated proapoptotic Bak protein (Leung et al., 1998 blue right-pointing triangle). In contrast to regulation of BCL2 and Bak at the transcriptional level, we find that E2 rapidly induces phosphorylation of BAD at two serine residues (112 and 136) without altering BAD protein levels (Figure 3BFigure 3.), suggestive of activation of nongenomic signal pathways. Given the fact that proapoptotic effects of BAD are decreased by phosphorylation and consequent sequestration by 14-3-3 proteins (Downward, 1999 blue right-pointing triangle), we sought to determine whether E2-induced phosphorylation of BAD is important for the antiapoptotic functions of E2.
BAD complexes with BCL2 and BCLxl, and leads to loss of mitochondrial integrity (Hirai and Wang, 2001 blue right-pointing triangle); therefore, release of cytochrome c into the cytoplasm is expected after BAD “activation.” The apoptotic stimuli increased the cytochrome c levels in the cytoplasm, and cotreatment with E2 reduced this effect concomitant with improved cell survival (Figure 1DFigure 1.). This suggests the restoration of mitochondrial integrity at least to some degree in E2-treated cells and may account for antiapoptotic effects of E2. In cells treated with TNF-α and H2O2, the extent of S112/136 BAD phosphorylation was lower than that observed in control cultures (Figure 3CFigure 3.), implicating the activation of BAD by these apoptotic stimuli. Estrogen treatment in this context restored phosphorylation to control levels. These experiments, along with those demonstrating that E2 prevents apoptosis in cells overexpressing wild-type but not the double phosphorylation site mutant (S112A/S136A) of BAD (Figure 4, E-FFigure 4.Figure 4.), suggest that BAD phosphorylation has a central role in apoptosis of MCF7 cells. Remarkably, the overexpression of BAD (S112A/S136A) did not cause apoptosis by itself, suggesting that in addition to phospho-serine-mediated cytosolic 14-3-3 binding there may be other mechanisms to sequester BAD away from mitochondria. These mechanisms effectively inactivating BAD are counteracted by apoptotic stimuli such as TNF-α. Binding of BAD to other cellular proteins such as glucokinase has been demonstrated in the rat liver (Danial et al., 2003 blue right-pointing triangle).
To confirm the importance of BAD in MCF7 cells, we used antisense oligonucleotides to suppress BAD protein translation (Figure 4AFigure 4.Figure 4.) and analyzed the effects of selected agents on MCF7 cell survival. Diminution of BAD in MCF7 cells significantly reduced the extent of apoptosis induced by TNF-α (Figure 4, B-DFigure 4.Figure 4.). This observation is in accordance with previously published studies where overexpression of BAD was shown to enhance cell death (Ottilie et al., 1997 blue right-pointing triangle; Virdee et al., 2000 blue right-pointing triangle). The fact that antisense BAD blocked ability of TNF-α to induce apoptosis (Figure 4, C and DFigure 4.Figure 4.) suggests that BAD phosphorylation/inactivation has a central role in apoptosis of MCF7 cells in response to stimuli such as TNF-α.
Agents such as TNF-α and H2O2 have been shown to have mixed effects on BAD protein levels and phosphorylation. TNF-α increased phosphorylation of BAD through PI-3K pathway in neutrophils (Cowburn et al., 2002 blue right-pointing triangle), and in HeLa cells (Pastorino et al., 1999 blue right-pointing triangle) where cell survival rather than the cell death was promoted. TNF-α also induced the production of more potent truncated form of BAD in a caspase 3-dependent manner, leading to apoptosis in Jurkat cells (Condorelli et al., 2001 blue right-pointing triangle). Similarly, H2O2 but not superoxide anion (O2-) has been shown to increase BAD protein levels in cardiomyocytes, leading to apoptosis (von Harsdorf et al., 1999 blue right-pointing triangle). Serum starvation may modulate activity of BAD and other apoptosis regulators, and such effects may be reversed by addition of growth factors or by manipulating signal transduction pathways (Yano et al., 1998 blue right-pointing triangle; Bai et al., 1999 blue right-pointing triangle). Recent evidence from BAD knockout mice suggests that BAD may have an important proapoptotic role in certain cell types such as the mammary tissue. Importance of BAD was especially underscored in apoptosis that occurs in the absence of growth factors (Ranger et al., 2003 blue right-pointing triangle) much like the importance of BAD inactivation in the response to E2, which is a well-known growth factor to breast cancer cells. However, it is also evident that BAD concentration or phosphorylation status could not be used to define survival or apoptosis in other cells such as PC12 cells (Maroto and Perez-Polo, 1997 blue right-pointing triangle).
Estradiol Signals through Two Pathways in Its Antiapoptotic Action
Roles of Ras/PI-3K/Akt and Ras/ERK/p90RSK Pathways in BAD Phosphorylation. The ability of DNAkt or PTEN to reverse the protective effects of E2 (Figure 2Figure 2.) suggests an essential role for signal transduction through PI-3K/Akt in E2 action in protecting cells from apoptosis. Akt is a known oncogene and is overexpressed in breast cancer cell lines compared with untransformed breast epithelial cells, as well as in breast cancer specimens where the majority of cells are ER positive (Sun et al., 2001 blue right-pointing triangle). Deranged PI-3K/Akt signaling due to PTEN deficiency or germline mutation has been proposed as a frequent cause for breast cancer (Mills et al., 2001 blue right-pointing triangle; Petrocelli and Slingerland, 2001 blue right-pointing triangle). Given the importance of PI-3K/Akt pathway to antiapoptotic action of E2, we examined aspects of this pathway, which might influence cell survival. Our results clearly indicate that PI-3K/Akt has a critical role in E2 action as a survival agent (Figure (Figure1A,1AFigure 1., ,2).2Figure 2.). While this work was in progress, Fernando and Wimalasena (2000 blue right-pointing triangle, 2002 blue right-pointing triangle), Castoria et al. (2001 blue right-pointing triangle), and Stoica et al. (2003a blue right-pointing triangle) demonstrated that E2 rapidly activates PI-3K/Akt in MCF7 cells, but others were unable to observe such activities of E2 (Ahmad et al., 1999 blue right-pointing triangle; Dupont et al., 2000 blue right-pointing triangle). However, in contrast to Castoria et al. (2001 blue right-pointing triangle), we found a rapid and sustained activation of Akt in response to E2 (Figure 6BFigure 6.). The reasons for these disparities, including conflicting results from one group (Ahmad et al., 1999 blue right-pointing triangle; Stoica et al., 2003a blue right-pointing triangle) are not clear but could relate to subtle culture conditions, treatment periods, or cell line variability. Prolonged activity of Akt is required to maintain BAD phosphorylation at S136 (Figure 3BFigure 3.; our unpublished data) as well as to maintain GSK3β in the inactive state (Figure 6CFigure 6.; our unpublished data). Active GSK3β may be required to stabilize cyclin D1 (Diehl et al., 1998 blue right-pointing triangle) and increased cyclin D1 protein is a characteristic response to E2 in MCF7 and other estrogen-responsive cells (Foster et al., 2001 blue right-pointing triangle, and references therein). E2 signaling also has been shown to be closely associated with the PI-3K/Akt pathway in vascular endothelial cells (Hisamoto et al., 2001 blue right-pointing triangle), and ERα is phosphorylated by activation of the PI-3K/Akt pathway in MCF7 cells in the absence of E2 (Campbell et al., 2001 blue right-pointing triangle; Stoica et al., 2003b blue right-pointing triangle).
In addition to Akt-mediated signaling, our data demonstrate that E2 also may signal through Ras/ERK/p90RSK pathway because endogenous and exogenous BAD were effectively phosphorylated at S112, a well-known ERK/p90RSK phosphorylation site (Downward, 1999 blue right-pointing triangle) in E2-treated cells (Figure 3, A and BFigure 3.). Our studies with activating and inactivating Ras mutants provides strong evidence for this line of argument and show that BAD S112 is a target of Ras-mediated MEK/ERK activity (Figure 5BFigure 5.). We could directly demonstrate that E2 is able to activate p90RSK1 (Figure 6, D and EFigure 6.), a downstream target of ERK1 and 2 (Zhao et al., 1996 blue right-pointing triangle; Bhatt and Ferrell, 1999 blue right-pointing triangle). Although the ability of E2 to activate ERK remains controversial (Lobenhofer et al., 2000 blue right-pointing triangle; Caristi et al., 2001 blue right-pointing triangle; Song et al., 2002 blue right-pointing triangle) due to unknown reasons, ERK activation seems to be important in the apoptotic response (Fang et al., 1999 blue right-pointing triangle; Shimamura et al., 2000 blue right-pointing triangle), perhaps through the regulation of BAD phosphorylation via p90RSK. The prolonged activation of ERK (Figure 7AFigure 7.) in contrast to transient activation reported by others (Migliaccio et al., 1996 blue right-pointing triangle) may be important to maintain the phosphorylation and inactivation of BAD, acting in concert with Akt. Prolonged ERK activation is also required for G1/S transition via enhanced cyclin D1 synthesis (Murphy et al., 2002 blue right-pointing triangle) and p27kip1 degradation (Foster, 2003 blue right-pointing triangle). Together, the data in Figure 5Figure 5. implicates a critical role for Ras/Raf/ERK in E2-promoted MCF7 cell survival. It is also possible that BAD can be phosphorylated by kinases other than Akt and p90RSK and that such phosphorylations may enhance or diminish the proapoptotic capacity of BAD (Harada et al., 1999 blue right-pointing triangle; Lizcano et al., 2000 blue right-pointing triangle; Donovan et al., 2002 blue right-pointing triangle; Konishi et al., 2002 blue right-pointing triangle).
Interaction and Regulation of the Estradiol-induced Signal Pathways. The two signaling pathways that we describe here may function as separate entities or interact to mediate E2 effects. Previous studies have shown that Ras/ERK and PI-3K/Akt exhibit regulatory cross talk (Wennstrom and Downward, 1999 blue right-pointing triangle; Shields et al., 2000 blue right-pointing triangle; York et al., 2000 blue right-pointing triangle). Regulation of ERK by E2 through the PI-3K/Akt pathway has not been previously reported to our knowledge. Using both biological and chemical inhibitors, we found that PI-3K may be required not only for E2 but also for IGF-1-induced ERK activation (Figure 7, B and CFigure 7.) as well as BAD phosphorylation at S112, a likely target of the ERK/p90RSK pathway (Figure 5AFigure 5.). Therefore, it is likely that E2 regulates ERK activation, in part, through PI-3K pathway, and our data suggest that E2 regulates a step upstream of PI-3K, possibly Ras function (Rodriguez-Viciana et al., 1994 blue right-pointing triangle). Overexpression of dominant negative Ras (RasN17) interfered with E2-induced BAD phosphorylation on both serine residues (Figure 5BFigure 5.) without affecting the basal BAD phosphorylation or protein levels supporting the notion that E2 regulates PI-3K through Ras (Khwaja et al., 1997 blue right-pointing triangle). However, we cannot exclude the possibility that E2 may regulate another kinase, which can phosphorylate BAD on S136. We previously demonstrated a role for Ras in estrogen-induced cell cycle progression (Ahamed et al., 2001 blue right-pointing triangle; Zhu et al., 2003 blue right-pointing triangle) and recently showed that the Ras/Raf/ERK pathway has a critical role in E2 induced p27kip1 degradation and relocalization in MCF7 cells (Foster, 2003 blue right-pointing triangle). Others (Migliaccio et al., 1996 blue right-pointing triangle; Filardo et al., 2002 blue right-pointing triangle) have reported rapid activation of Ras by E2. The ability of RasN17 to induce apoptosis in proliferating cells (Figure 5CFigure 5.) suggests that Ras plays a critical role in preventing apoptosis in MCF7 cells as observed in other cells (Wolfman et al., 2002 blue right-pointing triangle). Because 3′4′5′ or 3′4′PI phosphates regulate a variety of signal pathways such as Akt and PDK that could lead to modulation of Ras/Raf/ERK pathway (Rameh and Cantley, 1999 blue right-pointing triangle), nongenomic signaling by E2 may be very complex (Levin, 2003 blue right-pointing triangle). Furthermore, activation of PI-3K by E2 through a direct interaction of the p85 subunit with ERα was reported in human vascular endothelial cells (Simoncini et al., 2000 blue right-pointing triangle) and MCF7 cells (Sun et al., 2001 blue right-pointing triangle). Clearly, further studies are needed to delineate signaling mechanisms and their interactions in the perspective of estradiol's antiapoptotic and cell cycle effects.
Interestingly, many of the responses to E2 observed in this study also were elicited by IGF-1, as has been demonstrated in the context of MCF7 cell proliferation (Stewart et al., 1990 blue right-pointing triangle; Dupont et al., 2000 blue right-pointing triangle). E2 may indirectly activate Akt genomic actions (increased IGF-1 receptor expression, IGF-1 synthesis/secretion) (Ahmad et al., 1999 blue right-pointing triangle; Lee et al., 1999 blue right-pointing triangle); however, the responses we have described here are elicited by E2 within a 2- to 15-min time frame, which is not tenable with genomic actions of E2. On the other hand, we cannot formally exclude the possibility that E2 may activate immediate release of IGF-1 (or other growth factors) from an inactive storage form(s), as recently documented (Filardo et al., 2000 blue right-pointing triangle) for epidermal growth factor. The nongenomic signaling of E2 is postulated to be mediated by membrane-associated ER (Kelly and Levin, 2001 blue right-pointing triangle), and this hypothesis is further supported by the recent demonstrations of membrane located ERα in MCF7 and other cell types (Marquez and Pietras, 2001 blue right-pointing triangle; Powell et al., 2001 blue right-pointing triangle). According to these studies, the fraction of plasma membrane-bound ERα ranged from 3 to 20% of the total in MCF7 cells. Agreeing with this notion, our data show that E2 induced rapid ERK phosphorylation, and the protective effects against apoptotic agents was substantially blocked by the antiestrogen ICI182,780 (Figures (Figures1B1BFigure 1. and and7B7BFigure 7.).
SUMMARY
This study demonstrates that E2 as well as IGF-1 inhibit apoptosis in MCF7 cells by enhanced phosphorylation of BAD and indicates that BAD inactivation results from activation of Ras/ERK/p90RSK and Ras/PI-3K/Akt pathways by E2. Our results also indicate that BAD plays an essential role in apoptosis induced by H2O2, TNF-α, and serum withdrawal and also demonstrate that even the double phosphorylation site mutant of BAD (S112/136A) is inactive as an inducer of apoptosis in the absence of TNF-α. Our results suggest that the ability of E2 to prevent apoptosis in MCF7 cells requires BAD inactivation, which occurs in response to Ras activation by E2. Thus, we have demonstrated a novel nongenomic pathway by which E2 promotes survival of breast cancer cells in culture. Furthermore, this is the first report to our knowledge demonstrating the role of the Ras/PI-3K/Akt/BAD and Ras/Raf/ERK/p90RSK/BAD pathways as potential nongenomic mediators of the antiapoptotic actions of E2 or any other steroid hormone. The crucial contributions of aberrant Ras as well as those of Akt function to human cancer development is widely accepted (Datta et al., 1999 blue right-pointing triangle; Shields et al., 2000 blue right-pointing triangle), and inactive mutations or down-regulation of tumor suppressor PTEN have been linked to accelerated tumor growth in breast and other cancers (Maehama and Dixon, 1999 blue right-pointing triangle). Therefore, our study provides a mechanism whereby the functions of these oncogenes and tumor suppressors may converge and dictate the fate of the breast cancer cell.
Supplementary Material
Supplemental Figures
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Acknowledgments
We thank Drs. Don Torry, Roger Carroll, H.R. Wang, and Ming Zou for critical review of the manuscript; Steve Foster and Don Henley for advice and support throughout the study; Andy Shannon for preparing plasmids; Dr. Jonathan Wall and Craig Wooliver for the assistance in fluorescence microscopy; Dr. John Dunlap for the assistance in confocal microscopy; Richard Andrews for flow cytometric analysis; and Eva Bukovska and Will Laxtyon for editorial support. This study was supported by National Cancer Institute grant 84048 to J.W. R.I.F. gratefully acknowledges the graduate student stipend from the University of Tennessee Graduate School of Medicine in Knoxville.
Notes
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03-11-0823. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03-11-0823.
Abbreviations used: Dm, double mutant; DN, dominant negative; Wt, wild type.
D in BoxOnline version of this article contains supporting material. Online version is available at www.molbiolcell.org.
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