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Molecular Endocrinology, doi:10.1210/me.2002-0330
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Molecular Endocrinology 17 (5): 818-830
Copyright © 2003 by The Endocrine Society

Estradiol Rapidly Activates Akt via the ErbB2 Signaling Pathway

Gerald E. Stoica, Thomas F. Franke, Anton Wellstein, Frank Czubayko, Heinz-Joachim List, Ronald Reiter, Elisha Morgan, Mary Beth Martin and Adriana Stoica

Department of Oncology (G.E.S., A.W., H.-J.L., R.R.,M.B.M., A.S.), Lombardi Cancer Center, Georgetown University, Washington, DC 20007; Department of Pharmacology (T.F.F.), Columbia University, New York, New York 10032; Department of Pharmacology and Toxicology (F.C.), Philipps University, School of Medicine, 35033 Marburg, Germany; and School of Nursing and Health Studies (E.M., A.S.), Georgetown University, Washington, DC 20007

Address all correspondence and requests for reprints to: Adriana Stoica, Ph.D., E411 Research Building, 3970 Reservoir Road NW, Washington, DC 20007. E-mail: stoicaa{at}georgetown.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-{alpha} (ER{alpha}) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ER{alpha} into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ER{alpha}. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ER{alpha}, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTRADIOL EXERTS MOST of its effects by direct binding to the estrogen receptor (ER), which homodimerizes and interacts with estrogen response elements to stimulate transcription of target genes (genomic effects). The ER is a phosphoprotein and belongs to a nuclear receptor superfamily of ligand-regulatable transcription factors (1). In the absence of hormone, the ER is associated with a host of proteins that prevent it from interacting with the cellular transcription apparatus. Upon binding estradiol, the receptor undergoes an activating conformational change, facilitating its association with target genes and permitting it to regulate gene transcription (2).

ER-positive breast cancer cells produce growth factors that may act in an autocrine and/or paracrine manner to influence the proliferation and responsiveness of breast cancer (3). Cell responses to growth factors are mediated by cell-surface receptor tyrosine kinases (RTKs) that possess an intrinsic protein kinase activity. Ligand binding induces activation of tyrosine kinase and subsequent recruitment of target proteins, which initiate a complex signaling cascade of phosphorylation-dephosphorylation reactions (4, 5, 6). These signaling cascades propagate signals to the nucleus to elicit changes in gene expression (7).

The ErbB family of RTKs includes four receptors [epidermal growth factor receptor (EGFR)/ErbB1, ErbB2/HER-2/Neu, ErbB3/HER3, and ErbB4/HER4] (8) and several ligands (six EGFR ligands and two families of heregulins). Each of these ligands has a different preference to stabilize distinct receptor dimers, and each ligand-induced receptor dimer signals through a unique set of pathways by recruiting a different set of effector proteins (8). Unlike homodimers, the activities of which are relatively weak, heterodimers are more potent. Heterodimers between ErbB3 and ErbB2 are the most mitogenic (9, 10, 11, 12), leading to proliferation, growth, and transformation. The activation of ErbB RTKs eventually results in the activation of signaling molecules, the most important of which are PI 3-K and Ras (13). These two signaling pathways are often activated simultaneously in breast cancer cells and, depending on cell type, duration, or strength of the stimulus, one or the other may be preferred (14). For example, the p85 subunit of PI 3-K associates only with ErbB3 (15, 16, 17, 18).

Evidence has accumulated for the presence of rapid, posttranslational (nongenomic) estrogen actions similar to those evoked by growth factors (reviewed in Refs. 19, 20, 21). Physiologically important nonnuclear estrogen-signaling pathways occur in human vascular endothelial cells, rat primary cortical neurons, and human breast cancer cells (MCF-7) (22, 23, 24, 25, 26). These effects are mediated by ERs. Cell surface and nuclear ERs coexist in many target cells; however, the great majority of receptors are nuclear (80–90%; Refs. 22, 27, 28). Upon ligand binding, these ERs generate both rapid and long lasting responses (19). Ligand affinity, molecular weights, and immunological properties of the membrane and nuclear receptors are identical (20). It has been demonstrated recently that cell membrane and nuclear ERs originate from a single transcript (29). Confocal microscopy studies determined rapid formation of membrane ruffles, pseudopodia, and ER{alpha} membrane translocation in MCF-7 cells upon estradiol treatment (30). Furthermore, estradiol can act as a ligand for ErbB2 or for the type I IGF receptor/insulin receptor substrate-1 (IRS-1; Refs. 23 and 31).

The serine/threonine protein kinase Akt is downstream of many growth factor signaling cascades (32, 33), including EGF (26, 34), IGF-I (26, 35), and heregulin (36, 37, 38). ER{alpha}, but not ERß, can bind, in a ligand-dependent manner, to the p85{alpha} regulatory subunit of PI 3-K in human vascular endothelial cells and in fibroblasts transfected with ER{alpha}. Stimulation with physiological concentrations of estradiol increased ER{alpha}-associated PI 3-K activity, leading to activation of Akt and endothelial nitric oxide synthase (22). These data suggest that PI 3-K is recruited and activated by a small subset of ligand-bound membrane-associated ERs. The PI 3-K signaling cascade is also involved in the neuroprotective mechanism stimulated by estradiol in rat primary cortical neurons (24). Inhibition of PI 3-K activity in MCF-7 cells prevented estrogen-induced mitogenesis (25) and increased the activation of both activation functions, AF-1 and AF-2, of ER{alpha} (39). However, Akt increased the activity of only AF-1 (26, 39).

We have demonstrated previously that the two mitogenic growth factors EGF and IGF-I can regulate ER{alpha} gene expression and activity via Akt in the hormone-dependent breast cancer cell line, MCF-7 (26). In this paper, we now show that estradiol can also rapidly activate Akt. Selective inhibitor and anti-ErbB2 ribozyme experiments demonstrate that the effect of estradiol on Akt kinase activity is mediated by the ErbB2 RTK.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Estradiol on Akt Kinase Activity
Previous results from this laboratory demonstrate that the PI 3-K/Akt pathway mediates the effects of EGF and IGF-I on the amount and activity of ER{alpha} in the breast cancer cell line, MCF-7 (26, 40, 41). To determine whether estradiol can also activate this pathway, its ability to induce Akt activity was determined. MCF-7 cells were serum starved for 24 h and treated with 10-9 M estradiol for 10 min with or without preincubation with 10-7 M of the PI 3-K inhibitor, wortmannin, or 5 x 10-7 M of the antiestrogen ICI 182,780 for 20 min. The phosphorylation of immunoprecipitated Akt is presented in Fig. 1AGo (no exogenous substrate), and the phosphorylation of H2B by Akt is presented in Fig. 1BGo. Akt is catalytically inactive in serum-starved MCF-7 cells. Estradiol, similar to EGF and IGF-I, induces Akt kinase activity as demonstrated by an increase in phosphorylation. The phosphorylated kinase migrated at approximately 60 kDa as a tightly spaced triplet, consistent with the proposed multiple phosphorylation sites on the enzyme (26). For quantification, H2B was employed as a substrate, and similar results were obtained. In the absence of treatment, the amount of phosphorylated H2B was low. Treatment with 10-9 M estradiol resulted in a 9-fold activation of Akt (compared with a 14-fold activation by EGF and a 16-fold increase by IGF-I). When estradiol and either EGF or IGF-I were added together to the cells, an additive response was obtained. The effects of estradiol, EGF, and IGF-I were blocked by the PI 3-K inhibitor wortmannin (Fig. 1BGo), suggesting that the activation of Akt was PI 3-K dependent. In addition, the effect of estradiol on Akt activity was blocked by the antiestrogen ICI 182,780 (Fig. 1BGo), suggesting the involvement of ER.



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  		Figure 1. Effects of Estradiol and Growth Factors on Akt Kinase Activity

MCF-7 cells were serum-starved for 24 h, preincubated for 20 min with the PI 3-K inhibitor wortmannin W (10-7 M) or ICI 182,780 (5 x 10-7 M), and treated for 10 min with estradiol (10-9 M), EGF (100 ng/ml), or IGF-I (40 ng/ml). Akt was immunoprecipitated with an anti-Akt antibody from cell lysates, and an in vitro kinase assay was performed in the presence or absence of the histone protein H2B. A, In vitro kinase assay of Akt in the absence of H2B. Representative immunoblot. B, In vitro kinase assay of Akt in the presence of H2B. H2B was quantified by autoradiography using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and results are expressed as percent of control cells (mean value ± SD, n = 3).

 
The time course of the activation of Akt by estradiol is presented in Fig. 2AGo. Similar to the growth factors EGF and IGF-I (26), estradiol rapidly induced Akt activity, with a maximum increase of 9-fold by 20 min, which remained increased for 3 h (data not shown). The concentration dependence of Akt activation by estradiol is presented in Fig. 2BGo. Physiological concentrations of estradiol (10-12–10-8 M) induced Akt activity with a maximum increase at 10-9–10-8 M (9-fold increase). Taken together, these data suggest that estradiol induces Akt kinase activity in MCF-7 cells, an effect that requires PI 3-K and ER.



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  		Figure 2. Time and Concentration Dependence of Akt Activation by Estradiol

MCF-7 cells were serum starved for 24 h and treated with estradiol (10-9 M) for the times indicated (A) and for 10 min and the concentrations indicated (B). Akt was immunoprecipitated with an anti-Akt antibody from cell lysates, and an in vitro kinase assay was performed in the presence of the histone protein H2B. A, Time course of the effect of estradiol on Akt activity as measured by H2B phosphorylation (mean value ± SD, n = 3). B, Concentration dependence of 17ß-estradiol on Akt activity (mean value ± SD, n = 3).

 
To determine whether the effect of estradiol on Akt activation is stereospecific, the 17{alpha} isoform of estradiol was employed. Treatment of MCF-7 cells with the {alpha}-isoform did not result in Akt activation (Fig. 3AGo). To test whether the response to estradiol is specific for estrogens, the effects of estradiol, diethylstilbestrol (DES), and estrone were compared with the effect of the progestin promegestone (R5020) and the glucocorticoid dexamethasone (10-9 M). Although DES and estrone induced Akt activity, only a 3- to 4-fold induction, respectively, was observed as compared with a 9-fold induction by 17ß-estradiol. R5020 and dexamethasone were inactive.



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  		Figure 3. Specificity of Akt Activation by Estradiol

A, Effect of 17ß-estradiol, 17{alpha}-estradiol, E2-BSA, DES, estrone, R5020, and dexamethasone on Akt activity. MCF-7 cells were serum starved for 24 h and treated with 10-9 M of 17ß-estradiol, 17{alpha}-estradiol, DES, estrone, charcoal-stripped E2-BSA, R5020, or dexamethasone for 10 min. Akt was immunoprecipitated with an anti-Akt antibody from cell lysates, and an in vitro kinase assay was performed in the presence of the histone protein H2B. B, Effect of 17ß-estradiol and E2-BSA (10-9 M) on ER{alpha} activity. MCF-7 cells were treated with either free 17ß-estradiol or E2-BSA (10-9 M) for 6 h. PR mRNA and pS2 mRNA were determined by an RNase protection assay as described in Materials and Methods. Results in panels A and B are expressed as percent of control cells and represent the mean value of three independent experiments ± SD.

 
To confirm that the effect of estradiol is mediated by ER{alpha}, located at the extracellular membrane, the effect of 17ß-estradiol coupled to BSA (E2-BSA, 10-9 M) on Akt activity was measured. This conjugate is generally considered membrane impermeant and more water soluble than free estradiol (27, 29, 42, 43). However, it is not stable in solution, especially in the presence of cells, and can release measurable amounts of steroid (44). Therefore, aliquots of the estradiol-BSA conjugate were preabsorbed with dextran-coated charcoal (29, 43). Exposure of cells to conjugated estradiol resulted in an 11-fold increase in Akt kinase activity, suggesting that the effect of estradiol is mediated by a membrane-associated receptor. To demonstrate that estradiol was not released from E2-BSA, MCF-7 cells were treated with the conjugate, and the genomic effect on ER-{alpha} was measured. No significant effect on progesterone receptor (PR) mRNA or pS2 mRNA was found (Fig. 3BGo), a finding in agreement with earlier reports (29, 43, 45, 46). Also, BSA alone (10-9 M) had no effect on PR mRNA or pS2 mRNA (data not shown).

Effects of Estradiol on the Phosphorylation of Akt on Serine S473
Activation of Akt is associated with phosphorylation of serine S473. To determine whether serine S473 is phosphorylated after activation by estradiol, Western blot analyses were performed using an antibody that specifically recognizes Akt, phosphorylated on S473 (P-Akt). Serum-starved MCF-7 cells were treated with 10-9 M estradiol, 100 ng/ml EGF, or 40 ng/ml IGF-I in the presence or absence of 10-7 M wortmannin, 10-5 M LY294,002, 10-7 M genistein, or 20 ng/ml of rapamycin, the ribosomal protein kinase p70S6K inhibitor. The cells were lysed, the amount of total and phosphorylated Akt was measured on immunoblots, and the phosphorylation was normalized for the amount of total Akt. The results in Fig. 4Go, A and B, demonstrate that in the absence of treatment, the amount of phosphorylation on serine S473 was minimal. Treatment with estradiol or growth factors induced a 9-, 12-, and 16-fold increase in the phosphorylation of Akt on serine S473, respectively. The effect of estradiol was inhibited by wortmannin and LY 294,002 but not by rapamycin, suggesting that Akt is a downstream target of PI 3-K and that the p70S6K pathway is not essential for the effect of estradiol on Akt (Fig. 4Go, A and B).



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  		Figure 4. Effect of Estradiol on the Phosphorylation of S473

A and B, Estradiol effects in MCF-7 cells. C and E, Estradiol effects in MCF-7/ADR Cells. D and E, Estradiol effects in MCF-7/ADR cells transiently transfected with ER{alpha}. Cells were serum starved for 24 h and treated with 10-9 M estradiol, 100 ng/ml EGF, and 40 ng/ml IGF-I in the presence or absence of the PI 3-K inhibitors wortmannin W (10-7 M) or LY294,002 (10-5 M), the antiestrogens ICI 182,780 or 4-hydroxytamoxifen (Tam; 5 x 10-7 M), rapamycin (rapa; 20 ng/ml), or genistein (gen; 10-7 M; for 30 min). Western blots were probed using anti-phospho-Akt (P-Akt; Ser 473) or control anti-Akt antibody. A, C, and D are representative immunoblots from three independent experiments. B and E, The ratio between phosphorylated and total Akt was determined by densitometry, and results are represented as percent of control cells. The experiments were repeated three times ± SD.

 
To determine that the activation of Akt by estradiol requires ER{alpha}, two antiestrogens (ICI 182,780 and 4-hydroxytamoxifen) were used and cotransfection experiments of wild-type ER{alpha} into in an ER-negative variant of MCF-7, MCF-7/ADR, were performed. The activation of Akt by estradiol was blocked by both antiestrogens (Fig. 4Go, A and B). In the ER-negative MCF-7/ADR cells, EGF and IGF-I activated Akt, but estradiol was not capable of inducing Akt activity (Fig. 4CGo). However, transient transfection of wild-type ER{alpha} into these cells could restore the ability of estradiol to activate Akt activity (Fig. 4DGo), suggesting that estradiol activation of Akt requires ER{alpha}.

Role of ErbB2 in the Activation of Akt by Estradiol
To determine the mechanism of activation of Akt by estradiol, we used a genetic and a pharmacological approach. In the genetic approach, we used MCF-7 cells stably transfected with anti-ErbB2-targeted ribozyme expressed under the control of a tetracycline-regulated promoter system. As expected, in the cells that do not express the ribozyme, estradiol activates Akt activity while in cells that expressed ErbB2 ribozyme, estradiol and EGF did not activate Akt (Fig. 5AGo). In support of the genetic approach, Akt activation by estradiol was partially inhibited by genistein, a protein kinase inhibitor, suggesting an interaction of estradiol with ErbB2 (Fig. 4BGo). Because genistein has also a role as an ER agonist, our results suggest that the kinase inhibition activity predominates over the ER agonist activity, further reinforcing the requirement for both the protein kinase and estrogen signaling pathways. To identify the specific kinase, the effect of estradiol on Akt activity was tested in the presence of selective ErbB inhibitors (Fig. 5Go, B and C). AG825, a selective ErbB2 inhibitor, blocked the estradiol activation of Akt; however, the effect of estradiol was not inhibited by the selective EGFR inhibitor, AG30, suggesting that ErbB2, but not EGFR, is needed for activation of Akt by estradiol. The effect of EGF, however, was blocked by the selective EGFR inhibitor, AG30, suggesting that EGFR is needed for the EGF effect. To determine which Akt isoform is expressed in MCF-7 cells, Western blot analyses were performed using anti-AKT1, AKT2, and AKT3 antibodies (Fig. 5AGo and data not shown). Because only AKT1 was expressed in these cells, we can conclude that estradiol activates AKT1 (Fig. 5AGo).



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  		Figure 5. Role of ErbB2 in the Activation of Akt by Estradiol

A and C, MCF-7 cells were stably transfected with an anti-ErbB2-targeted ribozyme expressed under the control of a tetracycline-regulated promoter system and treated with 10-9 M estradiol or 100 ng/ml EGF for 10 min. B and D, MCF-7 cells were treated with 10-9 M estradiol or 100 ng/ml EGF for 10 minutes, in the presence or absence of the selective ErbB2 inhibitor A6825 or the selective EGFR inhibitor AG30 (20 min preincubation). Western blots of cell extracts were probed using anti-phospho-Akt (P-Akt) (Ser 473), anti-AKT1, or control anti-Akt antibody. A and B, Representative immunoblots of three independent experiments. C and D, The ratio between phosphorylated and total Akt was determined by densitometry and results are represented as percent of control cells. The experiments were repeated three times ± SD.

 
Role of Akt Mutants in the Estradiol-Induced Activation of Akt
Previously, we have stably transfected MCF-7 cells with a dominant negative Akt (K179M-Akt) and a constitutively active Akt mutant (myr-Akt; Ref. 38) and a mutant carrying an arginine-to-cysteine mutation at amino acid 25 (R25C-Akt) in the pleckstrin homology (PH) domain, a characteristic feature of the Akt family members. A comparable mutation (R28C) in the PH domain of Bruton’s tyrosine kinase (Btk) results in X-linked immunodeficiency in CBA/N mice (47). The PH mutation used in this study does not affect its kinase function, but NIH 3T3 cells cotransfected with this Akt mutant failed to respond to platelet-derived growth factor (32). Western blot analysis using an anti-phospho-Akt (S473) antibody showed that estradiol did not activate Akt in the MCF-7 cell pool stably transfected with either the kinase-defective or the R25C-Akt mutant. As expected, basal Akt activity was observed in the MCF-7 cell pool stably transfected with the constitutively active Akt, but treatment with estradiol did not result in further increase in Akt activity (Fig. 6Go). These results suggest that the estradiol-induced activation of Akt is dependent not only on the kinase domain, but also on R25 in the PH domain.



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  		Figure 6. Role of Akt Mutants in the Estradiol-Induced Activation of Akt

MCF-7 cells were stably transfected with the Akt mutants as described in Materials and Methods. Pooled clones were selected, serum starved, and treated with 10-9 M estradiol for 10 min. Western blot analysis was performed using an anti-phospho-Akt (S473) or control Akt antibody. A, Representative immunoblots of three independent experiments. MCF-7, Parental cells; myr-Akt, constitutively active mutant; K179M-Akt, dominant negative kinase mutant; R25C-Akt, PH domain mutant. B, The ratio between phosphorylated and total Akt was determined by densitometry, and results are represented as percent of control cells. The experiments were repeated three times ± SD.

 
Role of Akt Isoforms on the Effect of Estradiol
It was shown previously that estradiol can also stimulate Akt activity in ER-negative cell lines (54); however, the mechanism of estradiol action and the Akt isoform(s) involved were not identified. We show in this study that 10-9 M estradiol and 100 ng/ml EGF can both activate Akt in the ER-negative cell line, MDA-MB231 (Fig. 7BGo). In contrast to MCF-7 cells, the effects of estradiol and EGF can not be blocked by the antiestrogen ICI 182,780 (Fig. 7Go, A and B), suggesting two different mechanisms in ER-positive vs. ER-negative cell lines.



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  		Figure 7. Role of Akt Isoforms on the Effect of Estradiol

A, Effects of estradiol and EGF in MCF-7 cells. B, Effects of estradiol and EGF in MDA-MB231 cells. Cells were serum starved for 24 h and treated for 10 min with 10-9 M estradiol or 100 ng/ml EGF in the presence or absence of the antiestrogen ICI 182,780 (5 x 10-7 M; for 30 min). Western blots were probed using anti-phospho-Akt (P-Akt) (Ser 473) or control anti-Akt antibody. Panels A and B are representative immunoblots from three independent experiments. C, Role of Akt isoforms on the effect of estradiol. MCF-7 and MDA-MB231 cells were serum starved for 24 h and treated with 10-9 M estradiol for 10 min. Cells were lysed, immunoprecipitated with anti-Akt1, -Akt2, or -Akt3 antibodies, and kinase assays were performed as described in Materials and Methods. Results were expressed as relative Akt activity, normalized to the background activity of nonspecific mouse IgG, and are expressed as percent of control cells ± SD (n = 3).

 
To determine whether selective activation of Akt isoforms plays a role in the effect of estradiol, an in vitro kinase assay was performed using the Akt1 immunoprecipitation kinase assay kit and anti-Akt1-, Akt2-, and Akt3-specific antibodies in serum-starved and estradiol-treated MCF-7 and MDA-MB231 cells. The Akt/SGK-specific peptide was employed as substrate (Fig. 7CGo). Treatment of MCF-7 cells with 10-9 M estradiol resulted in a 2-fold increase in Akt1 activity. In contrast, MDA-MB231 cells treated with 10-9 M estradiol resulted in a 2-fold activation of Akt3. No activation of Akt2 by estradiol was observed in either cell line. However, when cells were treated with 100 ng/ml EGF, all Akt isoforms were activated in both cell lines (3-fold increase in Akt1, 2-fold increase in Akt2, and 30-fold increase in Akt3 in MCF-7 cells; and a 20-fold increase in Akt1, and a 34-fold increase in Akt3 in MDA-MB231 cells; data not shown). Taken together, these results suggest that Akt1 is activated by estradiol in MCF-7 cells and membrane ER{alpha} is needed for this effect, whereas Akt3 is the isoform activated in MDA-MB231 cells and ER{alpha} is not involved in the effect of estradiol.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The actions of estrogens are required for development and maintenance of reproductive tissue and, in some instances, are essential for the growth and survival of tumors that develop from these tissues. To date, it is unclear whether these diverse estrogen-mediated biological effects are entirely manifested via the known ERs (ER{alpha} and ERß). In addition to promoting ER-dependent gene transcription, estrogens rapidly trigger a variety of second messenger signaling events, including production of cAMP (48), mobilization of intracellular calcium (28), and activation of the MAPKs, Erk1 and Erk2 (45, 49, 50, 51). In this report we show that estradiol can also rapidly activate the PI 3-K/Akt pathway in MCF-7 breast cancer cells. We demonstrate that this estrogenic effect is a specific nongenomic effect since it satisfies all the criteria that define nongenomic effects, namely physiologically relevant concentration dependence, steroid specificity, stereospecificity, rapid effect, and membrane effect. Similarly, estradiol alone or conjugated with BSA, rapidly induced phosphorylation and activation of endothelial nitric oxide synthase through the PI 3-K/Akt pathway in human endothelial cells (52, 53) and ER{alpha} membrane translocation in MCF-7 cells, leading to MAPK activation by ER{alpha}-Shc association (30).

Using a genetic approach (MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme), as well as a pharmacological method (selective ErbB inhibitors), we demonstrate for the first time that the effect of estradiol on Akt activity requires ErbB2 and not EGFR. Moreover, the effects of estradiol on Akt activity can also be blocked by the antiestrogens tamoxifen and ICI 182,780 and cannot be reproduced in the ER-negative MCF-7/ADR cells. Transient transfection of ER{alpha} into these cells, however, restored Akt activation by estradiol, suggesting the involvement of ER{alpha}. In other ER-negative breast cell lines, such as HS785t or MDA MB 231 (Fig. 7BGo) or MDA MB435 and 293 (54), estradiol can also stimulate Akt activity. In the central nervous system, in specific cell types that do not express ER{alpha} or ERß (hypothalamic, striatal, and hippocampal neurons) and/or in transgenic mice that have these receptors deleted (ERKO mice), there have also been shown many rapid membrane effects of estradiol that cannot be attributed to the binding of the steroid ligand to nuclear or cytoplasmic ERs (55, 56). This apparent contradiction may be due to the existence of different Akt isoforms, expressed in different breast cancer cell lines. Whereas Akt1 is overexpressed in MCF-7 cells (57), Akt3 is overexpressed and overactive in some ER-negative breast tumors and cell lines (58). We demonstrate herein that Akt1 is the isoform that is activated by estradiol in MCF-7 cells (Figs. 7CGo and 5AGo), and ER{alpha} is needed for its effect. In the ER-negative cell lines, estradiol activation of Akt3 is ER{alpha} independent (Fig. 7CGo and Ref. 54) because the effect was not blocked by the antiestrogen, suggesting another molecular mechanism than in ER-positive breast cancer cells (e.g. via PI 3-K/Src; Ref. 54).

Thus, cumulative evidence argues for the existence of another ER or an isoform of ER that is associated with the membrane. However, how ER interpolates with the membrane and couples to intracellular signaling cascades is not known. In one model, ER spans the membrane, enabling estradiol to bind to the extracellular domain. Because no obvious sequences exist that would code for hydrophobic, membrane-spanning regions within the ERs, one speculation is that a posttranslational lipid modification of ER could occur in the endoplasmic reticulum that could facilitate the movement of the receptor into the membrane (27). A second and probably more compelling model is that, at least for a certain time, the ER exists entirely within the plasma membrane bilayer (59, 60) and can be found within the low-density, caveolin-coated plasmalemmal vesicles, called calveoli (61). Growth factor receptors also localize to the caveoli, where downstream signaling, including Ras, Src, and PI 3-K, may be facilitated (62). Caveolin appears to organize the association of signaling molecules within the caveoli (62) where second messengers move to the membrane from cytoplasm to be activated (63). Therefore, our results demonstrate, for the first time, a new molecular mechanism through which estradiol can rapidly activate Akt via a growth factor receptor/second messenger pathway. In ER-positive breast cancer cells, estradiol mimics the effects of ErbB ligands. Estradiol was reported previously to mimic ligand activity of the ErbB2 product in NIH 3T3 cells cotransfected with pSV2ErbB2 expression plasmid (31). A physical and functional interaction of estrogens and the ErbB2 protein was demonstrated. Estrogens and a putative ligand(s) of ErbB2 might recognize a common region of ErbB2 and compete for binding to the receptor due to the significant homology (~36%) of the ErbB2 extracellular domain with that of the ligand-binding domain of the ER (residues 449–568; Ref. 31). In our model (Fig. 8Go), estradiol, bound to ER{alpha}, interacts with an ErbB heterodimer containing ErbB2, leading to tyrosine phosphorylation of the ErbB receptors. Because we have demonstrated that EGFR is not needed for this rapid estrogen effect (Fig. 5Go, B and C), and MCF-7 cells overexpress ErbB3, but not ErbB4 (64), we expect that estradiol may activate the ErbB2 ErbB3 heterodimer. Activated ErbB3 may recruit and activate PI 3-K via phosphorylation of YXXM motifs in the ErbB3 carboxy-terminal domain (16, 17, 18, 37). Activated PI 3-K, in turn, leads to Akt activation. A cross-talk between ErbB2 and the androgen receptor pathways has also been demonstrated (65). ErbB2, in the absence of androgen, was shown to promote androgen-independent survival and growth of prostate cancer cells through the Akt pathway, and Akt specifically bound to the androgen receptor, phosphorylating serines S213 and S791 (66). Therefore, the ErbB2/Akt/steroid receptor cross-talk appears to be a more general mechanism in hormone-dependent cancer settings.



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  		Figure 8. A New Molecular Mechanism of the Rapid Activation of Akt by Estradiol

Estradiol, bound to membrane ER{alpha}, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.

 
The MAPK pathway is also thought to play an important role in mediating the effects of estrogens on activation and expression of ER{alpha}. Activation of MAPK by estradiol in MCF-7 cells was shown to potentiate the transactivation function of ER{alpha} by phosphorylating serines in the AF-1 domain of the receptor (4, 26, 67, 68). It requires the G protein-coupled receptor, GPR30, and occurs independently of known ERs via transactivation of the EGF receptor through release of heparin-binding EGF (51). Also, a linkage of the rapid estrogen action to MAPK activation by ER{alpha}-Shc association and Shc pathway activation was established (30, 51). More recently, estradiol was also shown to bind to and rapidly (within minutes) induce phosphorylation of the IGF receptor IGFR-1R and ERKs 1 and 2 in COS-7 cells cotransfected with ER{alpha} but not with ERß and in L6 cells (69). The ability of all these pathways to affect ER{alpha} function suggests that ER{alpha} is a point of convergence of the Akt and MAPK pathway, and it is not clear to date whether these pathways are alternate or whether they cross-talk, resulting in receptor activation. Although the precise pathways that mediate the effects of estrogen and growth factors on ER{alpha} expression and activity remain to be defined, our data suggest that the mechanism of regulation is complex, involving multiple signaling pathways.

The similarities between the nongenomic actions of estradiol and the growth factor-signaling pathway suggest that these two distinct pathways converge in such a manner to permit cross-talk. Estradiol may cause rapid PI 3-K-dependent activation of Akt in the ER-positive breast cancer cell line, MCF-7, in a membrane-associated ER{alpha}-dependent fashion. The elucidation of the mechanism(s) by which estradiol and growth factors induce Akt and influence ER may have a critical role in breast cancer prognosis and treatment in ER-positive patients with normal levels of ErbB2. In these patients, Akt overexpression may lead to resistance to tamoxifen treatment. Therefore, Akt may constitute a new prognostic marker as well as a new target for an alternative therapeutic intervention to the classical endocrine therapy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
Monolayer cultures of MCF-7 cells were grown in improved MEM (IMEM), supplemented with 5% fetal calf serum. At 80% confluence, the medium was replaced with phenol red-free IMEM containing 5% charcoal-treated calf serum (70). The calf serum was pretreated with sulfatase and dextran-coated charcoal to remove endogenous steroids (71). After 2 d, the medium was changed into serum-free, phenol red-free IMEM supplemented with fibronectin, glutamine, HEPES, trace elements, and transferrin, after which 10-9 M estradiol, 100 ng/ml EGF, or 40 ng/ml IGF-I were added. Cells were harvested at the times indicated.

EGF was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY); IGF-I was obtained from Biosource International, (Camarillo, CA); 17ß-estradiol, 17{alpha}-estradiol, E2-BSA, the glucocorticoid dexamethasone, 4-hydroxytamoxifen, and wortmannin were purchased from Sigma (St. Louis, MO). Rapamycin, genistein, the selective ErbB receptor inhibitors AG 30 (for EGFR) and AG 825 (for ErbB2), and LY 294,002 were purchased from Calbiochem (San Diego, CA). The progestin promegestone (R5020) was obtained from NEN Life Science Products (Boston, MA).

Plasmids
The clone 36B4 was constructed by subcloning a 220-bp fragment of 36B4 into the PstI restriction site of the pGem polylinker (71). The clones for pS2 (73), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (72), and progesterone receptor (PR; Ref. 74) were used as previously described.

The expression vector for Akt (HA-Akt; Ref. 32), the kinase-defective Akt [HA-Akt (K179M)], the constitutively active Akt (myrAkt-HA; Ref. 75), and the Akt mutant carrying an arginine-to-cysteine mutation at amino acid 25 (R25C-Akt; Ref. 32) were generated as HindIII-BamHI inserts in pCMV-6. The expression vector for ER{alpha} (HEG0) was described elsewhere (76).

Measurement of PR mRNA and pS2 mRNA
Total cellular RNA was extracted from MCF-7 cells by the RNazol method. The amounts of PR, pS2, 36B4, and GAPDH were determined by an ribonuclease (RNase) protection assay (71). Briefly, homogeneously 32P-labeled antisense cRNA was synthesized in vitro from pS2, 36B4, and pGAPDH using T7 polymerase and from PR using SP6 polymerase. Sixty micrograms of total RNA were hybridized for 12–16 h to the radiolabeled cRNA. After a 30-min digestion at 25 C with RNase A, 32P-labeled cRNA probes protected by total RNA were separated by electrophoresis on 6% polyacrylamide gels. The bands were visualized by autoradiography and quantified using a phosphoimager. The amounts of pS2 mRNA and PR mRNA were normalized to the internal control 36B4 and GAPDH, respectively.

Immunoprecipitation and in Vitro Akt Kinase Assay
To assay for Akt protein kinase activity, serum-starved, estradiol-, or growth factor-treated MCF-7 cells were lysed in Nonidet P-40 (NP-40) lysis buffer [1% NP-40, 10% glycerol, 137 mM NaCl, 20 mM Tris-HCl (pH 7.4)] containing 2 µg/ml aprotinin, 2 µg/ml leupeptin, 10-3 M pefabloc, 2 x 10-2 M NaF, 10-3 M sodium phosphate, and 10-3 M Na3VO4. Equal amounts of lysates (300 µg) were precleared by centrifugation and preabsorbed with protein A-protein G (1:1) agarose slurry. Immunoprecipitation was carried out for 16–18 h using anti-Akt antibody (1:500 dilution; Transduction Laboratories, Inc., Lexington, KY). Immunoprecipitates were washed three times with lysis buffer, once with water, and once with the Akt kinase buffer [20 nM HEPES-NaOH, 10 mM MgCl2DNM–, 10 mM MnCl2 (pH 7.4)]. Kinase assays were carried out in Akt kinase buffer containing 10 µCi [{gamma}-32P] ATP (3000 Ci/mmol), 5 x 10-6 M cold ATP, and 10-3 M dithiothreitol. Histone H2B (Roche Molecular Biochemicals, Indianapolis, IN) was added as exogenous substrates at a final concentration of 0.05 mg/ml. After 20 min at room temperature, kinase assays were stopped by the addition of loading buffer and separated in 12.5% sodium dodecyl sulfate-polyacrylamide gels. Detection was performed by autoradiography and phosphoimaging.

The assay for Akt1, Akt2, and Akt3 protein kinase activity was conducted in serum-starved and/or estradiol-treated MCF-7 and MDA-MB231 cells using an Akt1 immunoprecipitation kinase assay kit and anti-Akt1, -Akt2, and -Akt3 antibodies (Upstate Biotechnology, Inc., Lake Placid, NY) as described by the manufacturer (www.Upstatebiotech.com). Immunoprecipitations were performed with 4 µg anti-Akt1, -Akt2, or -Akt3 antibodies, respectively, and 2 mg of cell lysates per sample. As a control, 4 µg of a nonspecific mouse IgG antibody (Upstate Biotechnology, Inc.) were employed. {gamma}-32P-ATP (specific activity, 6000 Ci/mmol, 10 µCi/assay) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). The radioactivity-containing phosphocellulose paper squares were counted in a liquid scintillation counter.

Western Blot Analysis
MCF-7 cells [parental, stably transfected with a dominant negative Akt mutant (26), an arginine-to-cysteine mutant at the amino acid 25 in the PH domain of Akt (R25C), or with an anti-ErbB2-targeted ribozyme (77)] were preincubated for 20 min with the inhibitors of PI 3-K, wortmannin (10-7 M), and LY 294,002 (10-5 M), the inhibitor of p70S6K, rapamycin (20 ng/ml), genistein (10-7 M), a protein tyrosine kinase inhibitor, or the selective inhibitors for ErbB AG 30 and AG 825 (10-7 M) and treated with estradiol (10-9 M), EGF (100 ng/ml), or IGF-I (40 ng/ml) for 10 min. Cells were lysed in NP-40 lysis buffer; the lysates were heated to 95-100 C for 5 min, and equal amounts of protein (100 µg) were loaded onto sodium dodecyl sulfate-polyacrylamide gels. Gels were electrotransferred to nitrocellulose membranes, washed in PBS five times at room temperature. Membranes were kept in blocking buffer overnight at 4 C and incubated with the primary antibody [either anti-phospho-Akt antibody (Ser 473) or anti-Akt antibody (New England Biolabs, Inc., Beverly, MA)] for 1 h at room temperature. After three additional washes in PBS, membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:2000) in blocking buffer for 1 h at room temperature. Detection was performed by chemiluminescence, using Super Signal chemiluminescent substrate (Pierce Chemical Co., Rockford, IL).

Transfections
Stable transfection of MCF-7 cells (passage 47) with a constitutively active Akt (myr-Akt), a dominant negative Akt (K179M-Akt), and an arginine-to-cysteine Akt mutant at amino acid 25 in the PH domain (R25C-Akt) was performed with lipofectamine Plus (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s instructions and is described elsewhere (26). Transfected cells were selected in IMEM supplemented with 10% fetal calf serum and 500 µg/ml G418 for about 1 month. Single colonies as well as pools of transfected colonies were picked up, serum starved, and treated with 10-9 M estradiol, and enzymatic activity of Akt was tested.

Transient transfection of ER{alpha} (HEG0) into MCF-7/ADR cells was also performed with lipofectamine Plus (Life Technologies, Inc.). Briefly, 106 cells were plated into 100-mm dishes and transfected with 5 µg of HEG0. After 16–18 h, cells were washed and serum-free medium was added. One day later, transfected cells were used for Western blot analysis for Akt activity and expression.


   ACKNOWLEDGMENTS
 
We thank Dr. P. Chambon for the ER{alpha} expression vector and Drs. S. Byers, D. Gamett, and W. Li for critical reading of the manuscript.


   FOOTNOTES
 
This work was supported by grants from the Milheim Foundation and Georgetown University (Dean of Research) (to A.S.) and, in part, by Career Development Award DAMD17-00-1-0214 (to T.F.F.).

Abbreviations: AF, Activation function; DES, diethylstilbestrol; E2-BSA, 17ß-estradiol coupled to BSA; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IMEM, improved MEM; NP-40, Nonidet P-40; PH, pleckstrin homology; PI 3-K, phosphatidylinositol 3-kinase; PR, progesterone receptor; RNase, ribonuclease; RTK, receptor tyrosine kinase.

Received for publication September 19, 2002. Accepted for publication January 16, 2003.


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