Linkage of Rapid Estrogen Action to MAPK Activation by ER{alpha}-Shc Association and Shc Pathway Activation

Robert X.-D. Song, Robert A. McPherson, Liana Adam, Yongde Bao, Margaret Shupnik, Rakesh Kumar and Richard J. Santen

Departments of Internal Medicine (R.X.-D.S., R.A.M., M.S., R.J.S.) and Biomolecular Research Facility (Y.B.), University of Virginia School of Medicine, Charlottesville, Virginia 22908; and Department of Molecular & Cellular Oncology (L.A., R.K.), University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Robert X. Song, Division of Hematology and Oncology, University of Virginia Health Science Center, Charlottesville, Virginia 22901. E-mail: rs5wf{at}virginia.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E2 rapidly activates MAPK in breast cancer cells, and the mechanism for this effect has not been fully identified. Since growth factor-induced MAPK activation involves signaling via the adapter protein Shc (Src-homology and collagen homology) and its association with membrane receptors, we hypothesized that breast cancer cells utilize similar signaling mechanisms in response to E2. In the present study, we demonstrated that E2 rapidly induced Shc phosphorylation and Shc-Grb2 (growth factor receptor binding protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells. Overexpression of dominant negative Shc blocked the effect of E2 on MAPK, indicating a critical role of Shc in E2 action. Using selective inhibitors, we also demonstrated that ER{alpha} and Src are upstream regulators of Shc. A rapid physical association between ER{alpha} and Shc upon E2 stimulation further evidenced the role of ER{alpha} on Shc activation. Mutagenesis studies showed that the phosphotyrosine binding and SH2 domains of Shc are required to interact with the activation function 1, but not activation function 2, domain of ER{alpha}. Using a glutathione-S-transferanse-Shc pull-down assay, we demonstrated that this ER{alpha}-Shc association was direct. Biological consequences of this pathway were further investigated at the genomic and nongenomic levels. E2 stimulated MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy studies showed that E2 rapidly induced formation of membrane ruffles, pseudopodia, and ER{alpha} membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ER{alpha} can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E2 IS a steroid hormone that plays an important role in the genesis of human breast cancer and in the growth of established tumors (1). Classically, E2 elicits genomic effects on transcription via ER{alpha} and ERß, which are mainly located in the nucleus. As ligand-dependent transcriptional factors, the hormone-bound ERs interact with estrogen response elements to stimulate certain genes in E2-responsive tissues and to regulate gene transactivation (2). The genomic actions of E2 are characterized by delayed onset of action and sensitivity to inhibitors of transcription and translation. Many of the E2-induced transcriptional mechanisms involve ER{alpha}, which can be functionally regulated by posttranslational phosphorylation of serine residues at the 104, 106, 118, 167, and 236 positions (3, 4, 5, 6). In addition, tyrosine-537 of ER{alpha} has been reported to be phosphorylated in MCF-7 cells and in ER{alpha}-expressing Sf9 insect cells (7). The biological function of ER{alpha} is also regulated by association with a subset of nuclear proteins, such as coactivators, corepressors, and integrator proteins (8, 9, 10, 11).

More recently, investigators have recognized rapid nongenomic actions of E2 on several cellular processes, such as activation of Ras (12), Raf-1 (13), PKC (14), PKA (15), Maxi-K channels (16), increments in intracellular calcium levels (17), and an increase of nitric oxide (17). Elicited responses depend upon the cell types studied and the conditions used. In contrast to its transcriptional effects, the precise nongenomic signaling pathways of E2, especially the involvement of ER{alpha} in this process, are not well understood and have not been extensively studied (18, 19, 20).

Several recent reports demonstrated that E2 rapidly activates MAPK in a number of model systems (13, 21, 22), but the mechanisms responsible for this are still controversial (23, 24, 25, 26). Many growth factor receptors on the cell membrane, such as the receptors for epidermal growth factor (27), nerve growth factor (28), platelet-derived growth factor (27), and IGF (29), activate MAPK through a Shc-mediated pathway. We hypothesized that E2 might co-opt a pathway using Shc-Grb2-Sos (Src homology-growth factor receptor binding protein 2-son of sevenless) to activate MAPK. The adapter protein Shc has no intrinsic kinase domain and transduces signals dependent on the association with membrane receptors. Three domains mediating protein-protein interactions have been reported on Shc. Two of these, the phosphotyrosine binding (PTB) domain in the amino-terminal region and the Src homology 2 (SH2) domain in the carboxy-terminal region are separated by a region rich in proline and glycine residues, called the collagen homology (CH) domain (30). Shc binds rapidly to the specific phosphotyrosine residues of receptors through its PTB or SH2 domain and becomes phosphorylated itself on tyrosine residues of the CH domain (27). The phosphorylated tyrosine residues on the CH domain provide the docking sites for the binding of the SH2 domain of Grb2 and hence recruit Sos, a guanine nucleotide exchange protein. This adapter complex formation allows Ras activation via the Sos protein, leading to the activation of the MAPK pathway (27, 31, 32). Since ER{alpha} has been reported to be associated with the membrane of MCF-7 cells (20), we hypothesized that this receptor might be involved in mediating E2 action on MAPK activation by physical interaction with Shc in a manner analogous to that in growth factor pathways.

To test our hypothesis, we used MCF-7 and its variant LTED cells. LTED cells were developed from parental MCF-7 cells growing long-term in estrogen-depleted medium (33). This maneuver causes a 5- to 10-fold up-regulation of ER{alpha} expression (33). In the present study, we demonstrated that E2 rapidly stimulated both MAPK and Shc phosphorylation and induced the association of Shc with ER{alpha}. Using selective inhibitors and a dominant mutant Shc, we further demonstrated that Shc is upstream of MAPK and that the rapid actions of E2 are ER{alpha}-dependent and involve ER{alpha}/Shc association. Furthermore, a direct physical association of ER{alpha} with Shc was evidenced by protein pull-down assay. Our results show that the PTB and SH2 domains of Shc interacted with the AF-1 region of ER{alpha}. Tyrosine-537 of ER{alpha} was not involved in this interaction. Measurement of Elk-1 activation and morphological changes by confocal microscopy provided further evidence of the rapid, nongenomic role of E2 in these cells. Together, our data suggest that the nongenomic effects of E2 on Shc and MAPK activation are mediated by the classical ER{alpha} receptor, that E2 can induce an association of ER{alpha} with Shc, and that Shc appears to be a crucial step for the activation of MAPK.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Effects of Selective Inhibitors and Dominant-Negative Shc on E2-Induced MAPK Phosphorylation
Prior studies reported that E2 rapidly activated MAPK in MCF-7 cells (34). To investigate the upstream signals mediating E2 action on MAPK, we first confirmed that E2 increased MAPK activation in our MCF-7 cells by measuring MAPK phosphorylation. Figure 1AGo shows that E2 increased both p42 and p44 MAPK phosphorylation in a time-dependent manner with the peak at 15-min treatment. We then questioned whether the effects of E2 on MAPK involved ER{alpha}, MEK (MAPK kinase), and Src tyrosine kinase. As shown in Fig. 1BGo, the antiestrogen ICI, the MEK inhibitor PD98059, and a pan Src inhibitor PP2 all blocked E2-induced MAPK phosphorylation, implying that ER{alpha}, MEK, and Src family members are required for this step. PP2 alone abolished basal MAPK phosphorylation, suggesting the role of Src in maintaining basal MAPK function (Fig. 1BGo).



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Figure 1. MAPK Phosphorylation in MCF-7 Cells

A, E2-induced MAPK phosphorylation. MCF-7 cells were treated with vehicle or E2 at 10-10 M for the times indicated. The phosphorylation status of MAPK (p42 and p44) from whole-cell lysates was assessed using an antibody recognizing dual phosphorylated MAPK (top panel). Levels of total MAPK protein were determined on the same blot to control for loading variations (bottom panel). The positions of phosphorylated and total MAPK proteins are indicated at the right. B, Effects of pathway-selective inhibitors on E2-induced MAPK phosphorylation. MCF-7 cells were pretreated with 23 µM PD98059, 10-9 M ICI 182 780 (ICI), and 5 nM PP2 for 30 min. Cells were then treated with vehicle or 10-10 M E2 for 15 min. The phosphorylated (top panel) and total MAPK (bottom panel) were determined. C, Overexpression of mutant Shc blocked E2-induced MAPK phosphorylation. MCF-7 cells were either not transfected or transfected with the mutant Shc (ShcFFF) or control vectors (pEBG) for 2 d. Then cells were treated with vehicle or 10-10 M E2 for 15 min. The phosphorylated (top panel) and total MAPK (middle panel) were detected. Both GST-ShcFFF and endogenous Shc expression are shown (bottom panel). The above experiments have been repeated three times, and one of the representative experiments is shown.

 
Activation of MAPK through classical growth factor signaling pathways involves the phosphorylation and activation of the adapter protein Shc. To test whether Shc is necessary for E2-induced MAPK phosphorylation, we transiently transfected MCF-7 cells with a dominant-negative Shc mutant. The mutant is a glutathione-S-transferase (GST) fusion protein with phenylalanines substituted for tyrosines at the 239/240 and 317 positions of the Shc CH domain (ShcFFF), rendering it defective in signaling to MAPK (35, 36). Expression of ShcFFF decreased E2-induced MAPK phosphorylation compared with untransfected and empty vector (pEBG)-transfected cells (Fig. 1CGo). Together, these results indicated that ER{alpha}, Src, and Shc are all upstream components regulating MAPK activation.

E2 Rapidly Induces Shc Pathway Activation
To further confirm the involvement of Shc in the E2 rapid signaling pathway, we evaluated this step in both MCF-7 and LTED cells that express a 10-fold elevation of ER{alpha} levels. As shown in the top panel of Fig. 2AGo, E2 at 10-10 M increased p52 kDa Shc phosphorylation in a time-dependent manner with the maximum response at 3 min. When LTED cells were compared with MCF-7 cells, Shc was phosphorylated to a greater extent in LTED cells (left in Fig. 2AGo) than parental MCF-7 cells (right in Fig. 2AGo) under basal conditions and increased proportionately in response to E2. To validate the results, the polyvinylidene difluoride (PVDF) membranes were reprobed with anti-Shc antibodies. The results show a similar amount of Shc proteins of both p46 and p52 kDa in MCF-7 and LTED cells (Fig. 2AGo, bottom panels). E2-induced Shc phosphorylation was also dose-dependent with the optimal dose at 10-10 M (data not shown).



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Figure 2. E2-Induced Shc Pathway Activation in MCF-7 and Its Variant Cells

A, Effects of E2 on Shc phosphorylation. Both LTED and MCF-7 cells were treated with vehicle or 10-10 M E2 for the times indicated and then extracted as described in Materials and Methods. Shc phosphorylation was measured by immunoprecipitation (IP) of Shc protein and detection of tyrosine phosphorylation (top panel) on immunoblot (IB). To normalize Shc protein loading, the membranes were stripped and reprobed with polyclonal anti-Shc antibodies (bottom panel). Nonspecific monoclonal or polyclonal IgG were employed in all immunoprecipitation (IP) steps of the following experiments as negative controls. B, Effects of E2 on Shc-mediated adapter protein complex formation. LTED cells were treated with vehicle or E2 at 10-10 M for 3 min. The association of Grb2/Shc and Grb2/Sos was measured by immunoprecipitation of Grb2 and detection of Shc (left top panel) or Sos (middle top panel) on Western blots. The same membrane was reprobed with antibodies against Grb2 to detect Grb2 protein loading (left and middle bottom panels). The Grb2/Sos association was further confirmed by reciprocal antibody method (right panel). C, Effects of the selective inhibitors on E2-induced Shc phosphorylation in MCF-7 cells. MCF-7 cells were pretreated with 5 nM PP2, 23 µM PD98059, and 10-9 M ICI for 30 min. Cells then were challenged with vehicle or 10-10 M E2 for 3 min. The phosphorylation of Shc was determined as described in Materials and Methods (top panel). The membrane was reprobed with anti-Shc antibodies to show Shc protein loading (bottom panel). Above experiments were done three times and a representative experiment is shown.

 
Classical pathways of Shc activation by growth factors involve Shc association with the adapter proteins, Grb2 and Sos. Accordingly, we examined the effects of E2 on Shc-Grb2-Sos complex formation. LTED cells were used in this study because of their enhanced Shc phosphorylation in response to E2. As shown in Fig. 2BGo, Grb2/Shc (left panel) and Grb2/Sos (middle and right panels) complexes were faintly apparent in the vehicle-treated group, but greatly increased by 10-10 M E2 treatment at 3 min. Together, these results suggest that E2 is capable of activating the Shc-mediated signaling pathway in MCF-7 cells, which is regulated by the level of ER{alpha} expression.

Effects of Selective Signaling Pathway Inhibitors on E2-Induced Shc Phosphorylation
Because Shc is responsible for the activation of MAPK by E2, we wished to determine components responsible for Shc phosphorylation. Additionally, we wished to demonstrate a role for ER{alpha} in this process and to exclude MAPK as the cause of Shc phosphorylation. Shc has been reported to be a substrate of c-Src in HEK-293 cells (37). Accordingly, we examined the effects of PP2, ICI, and PD98059 on E2-induced Shc phosphorylation. In the presence of the inhibitors, MCF-7 cells were stimulated with vehicle or 10-10 M E2 for 3 min, and the status of Shc phosphorylation was examined. Figure 2CGo shows that both PP2 and ICI effectively inhibited E2-induced Shc phosphorylation, indicating that Src family kinases and ER{alpha} are required for Shc activation. As expected, PD98059 did not influence Shc phosphorylation status, suggesting that MAPK functions downstream of Shc. No effects of these inhibitors were apparent in the absence of E2 stimulation. These results indicate that both ER{alpha} and Src are upstream components of Shc functionality, and their involvement is required for Shc phosphorylation.

E2-Stimulated ER{alpha} Association with Shc
We postulated that ER{alpha} might mediate effects of E2 on Shc by directly or indirectly associating with the Shc adapter protein. This concept was based on the fact that activated Shc transduces signals by associating with membrane receptors and our evidence that ICI blocked E2-induced Shc phosphorylation in MCF-7 cells. To test this hypothesis, we immunoprecipitated Shc protein and detected ER{alpha} on Western blots. As shown in Fig. 3Go, E2 rapidly induced the association of ER{alpha} with Shc in both LTED and MCF-7 cells (top panel). The induction was seen as early as 1 min of E2 treatment and reached a plateau from 3 to 10 min. Compared with MCF-7 cells, ER{alpha}/Shc complexes were present basally in LTED cells, which express 5- to 10-fold higher levels of ER{alpha}, and greatly increased in response to E2 treatment. Consistency of protein loading was confirmed by reprobing the membrane with anti-Shc antibody (bottom panel). Thus, the association between ER{alpha} and Shc was observed not only in MCF-7 cells, but it was greatly enhanced in ER{alpha} up-regulated LTED cells. The results were also confirmed by the immunoprecipitation of ER{alpha} and the detection of Shc on immunoblot (data not shown). Together, our data suggest that as an upstream signal of Shc, the level of ER{alpha} expression affects the level of Shc phosphorylation and ER{alpha}/Shc association.



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Figure 3. E2 Rapidly Induced the Physical Association Between ER{alpha} and Shc in LTED and MCF-7 Cells

Cells were treated with vehicle or 10-10 M E2 for the times indicated. Subsequently, the association of ER{alpha} with Shc proteins was determined by the immunoprecipitation of Shc and detection of coimmunoprecipitated ER{alpha} protein on immunoblot (top panel). The same membrane was stripped and reprobed with anti-Shc antibodies to normalize the Shc protein loading (bottom panel). The positions of ER{alpha} and Shc proteins are indicated at the right. The experiment has been done three times, and one of the representative experiments is shown.

 
Study of Molecular Basis of ER{alpha}/Shc Interactions
To study the molecular basis of the interacting domains on ER{alpha} and Shc, various GST-tagged Shc and ER{alpha} mutants were employed (Fig. 4AGo). Furthermore, to minimize factors specific to breast cancer cells, COS-1 cells that do not have detectable endogenous ER{alpha} were used and electroporated with different ER{alpha} and Shc expressing vectors (Fig. 4AGo). First, we examined the Shc-interacting domain(s) on ER{alpha} by electroporating wild-type Shc (ShcWT) with either wild-type ER{alpha} expressing vector (HEGO) or a mutant ER{alpha} expressing vector in which phenylalanine was substituted for tyrosine in the 537-position (Y537F). As shown in Fig. 4BGo, the Y537F mutation did not alter the Shc/ER{alpha} complex formation compared with wild-type ER{alpha}, suggesting that Y537 of ER{alpha} is not the site interacting with Shc. Coexpression of either the truncated activation function 1 (AF-1) or activation function 2 (AF-2) domains of ER{alpha} with ShcWT in COS-1 cells confirmed that it is the AF-1, but not AF-2 (contains Y537), domain of ER{alpha} that interacts with Shc (Fig. 4, C and D). E2 increased the association of AF-1 with Shc (Fig. 4CGo). The mechanism for this is not known and was unexpected, since AF-1 does not contain a binding site for E2. We postulate that E2 might exert effects on AF-1 or Shc binding proteins independently of ER{alpha}, leading to an increase of AF-1/Shc association.



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Figure 4. Study of Shc/ER{alpha} Interaction

A, Schematic ER{alpha} and GST-tagged Shc constructs for eukaryotic expression. In left panel, HEGO is wild-type ER{alpha}-expressing vector. Y537F is a vector expressing mutant ER{alpha} in which the tyrosine-537 was mutated into phenylalanine. The AF-1 vector encodes amino acids 1–367 of ER{alpha} and AF-2 encodes 179–595. In the right panel, the GST-Shc fusion protein-expressing vectors are shown. The oval region in the N terminus of Shc corresponds to the GST fusion protein. The white, gray, and diagonally lined rectangles represent the PTB, CH, and SH2 domains of Shc, respectively. B, Examination of tyrosine-537 of ER{alpha} interaction with Shc. Wild-type Shc vector (ShcWT) was cotransfected with either HEGO or Y537F ER{alpha}-expressing vectors into COS-1 cells. Cells were then treated with vehicle or 10-10 M E2 for 5 min, and the ER{alpha}/Shc association was assessed. C and D, The AF-1 domain of ER{alpha} is required to interact with Shc. COS-1 cells were cotransfected with GST-tagged wild-type Shc (ShcWT) and different ER{alpha} constructs as indicated for 3 d. Cells were then treated with vehicle or 10-10 M E2 for 5 min. The ER{alpha}-Shc association (top panels) was measured by immunoprecipitation of GST and detection of ER{alpha} on immunoblot using either anti-C (panel C) or anti-N-terminal (panel D) ER{alpha} antibodies. The membrane was reprobed with anti-GST antibodies to normalize the protein loading (bottom panels). To confirm the positions of AF-1 and AF-2, the ER-transfected COS-1 cell extracts were loaded on the same gels, and the expression of immunoprotein bands of HEGO, AF-1, and AF-2 is shown on the right sides of the top panels. E, Mapping the ER{alpha}-interacting domains on Shc. Wild-type ER{alpha} (HEGO) was transiently transfected into COS-1 cells with different GST-tagged Shc expression vectors as indicated. On d 3, cells were extracted, and the interactions between ER{alpha} and wild-type Shc (top left panel) or mutated Shc proteins (top right panel) were measured as described in Materials and Methods. The same membranes were stripped and reprobed with anti-ER{alpha} antibody to normalize the protein loading (bottom panels). Quantitation of the GST-Shc immunoprotein bands normalized by ER{alpha} protein loading is shown as fold induction. All figures are representative blots of the study that was performed three times. F, GST pull-down assay of ER{alpha}-Shc interaction. GST-tagged fusion proteins expressing Shc-selective domains were purified by the beads of Glutathione Sepharose 4B. Beads (15 µl) conjugated with or without GST, GST-SH2, or GST-PTB+CH domains of Shc were incubated with 5 ng recombinant full-length human ER{alpha} protein overnight. To control the specificity, the full-length human ER{alpha} was also incubated with anti-vitamin D receptor, anti-ER{alpha}, or nonspecific immunoglobulin (IgG) as indicated. After washing the beads three times, the eluted proteins were resolved on 10% SDS-PAGE, transferred onto a PVDF membrane, and detected with anti-ER{alpha} antibody (top panel). The gel was stained with Coomassie blue to show the positions of the Shc fusion proteins (bottom panel). One of four experiments is shown.

 
Next we further mapped the ER{alpha}-interacting site(s) on Shc. When ShcWT was coexpressed with ER{alpha} (HEGO) in COS-1 cells, we demonstrated an association of GST-Shc protein with ER{alpha} under basal conditions and further enhancement of this association with 5 min of E2 treatment (Fig. 4EGo, top left panel). We then used selective domain-expressing Shc mutants to examine which domains of Shc were necessary for this association. Both the PTB (ShcPTB)- and the SH2 (ShcSH2) domain-expressing proteins formed complexes with ER{alpha}, which further increased in intensity upon E2 stimulation (Fig. 4EGo, top right panel). Proteins that only expressed the GST or CH domain of Shc failed to interact with ER{alpha}. After normalizing the GST-Shc immunoprotein bands (Fig. 4EGo, top panels) for ER{alpha} protein loading (Fig. 4EGo, bottom panels), we show that the Shc/ER{alpha} association induced by E2 is 2- to 3-fold higher than the control in cells expressing PTB or SH2 and is similar to that in cells containing ShcWT.

Shc can be a substrate of c-Src that associates with ER{alpha} in MCF-7 cells after E2 treatment (38). It would be of interest to know whether c-Src is involved in this ER{alpha}-Shc complex in our breast cancer system. Using several different anti-c-Src (B-12, N-16, and SRC2, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-ER{alpha} antibodies (D-12 and MC-20, Santa Cruz Biotechnology, Inc.), we did not detect c-Src in ER{alpha}-Shc complexes of either MCF-7 or LTED cells (data not shown). To further address the molecular basis of Shc-ER{alpha} interaction, we tested whether this interaction is direct or mediated by third-party proteins using GST-pull-down assays. Both GST-tagged SH2 and PTH+CH domain fusion proteins of Shc were produced at the size of 37 kDa and 68 kDa (Fig. 4FGo, bottom panel). The 62-kDa recombinant full-length ER{alpha} was pulled down by both GST-tagged Shc proteins (Fig. 4FGo, top panel). As negative control, the beads and GST protein alone did not pull down the ER{alpha} protein. The specificity was confirmed by immunoprecipitation and detection of ER{alpha} with anti-ER{alpha} antibodies, but not by the nonspecific or anti-vitamin D receptor antibodies. Together, our results demonstrate that the interaction between ER{alpha} and Shc is direct and does not require the presence of any other proteins.

Biological Effects of E2-Induced Nongenomic Pathway Activation
To further provide evidence that the ER{alpha}-Shc-MAPK pathway is biologically relevant, we used two approaches. First, we evaluated the role of MAPK on the activation of Elk-1, which is a transcriptional factor phosphorylated and activated by MAPK (39). Second, we examined the rapid effects of E2 on morphological changes of MCF-7 cells.

To study Elk-1 activation by E2, a well characterized reporter system utilizing both GAL4-Elk-1 and GAL4-luciferase vectors (40) was cotransfected into MCF-7 cells. Figure 5Go shows that E2 increased Elk-1 activation in a dose-dependent manner at 6 h as shown by luciferase assay. The E2 doses higher than 10-12 M significantly stimulated Elk-1 activation, indicating the involvement of MAPK in E2 action.



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Figure 5. E2-Stimulated MAPK Substrate Elk-1 Activation

A transient transfection method was used to study MAPK-mediated Elk-1 activation. MCF-7 cells were cotransfected with GAL4-Elk-1 and GAL4-E1B-Luciferase vectors. Twenty-four hours after transfection, cells were stimulated with vehicle or different doses of E2 for 6 h. Luciferase activity for each lysate was determined. The data are presented as mean ± SD of three experiments. *, P < 0.05 compared with untreated control.

 
To further provide direct biological evidence of E2 stimulation of Shc-MAPK pathways, effects of E2 on cell morphology were examined. We reasoned that E2 might elicit effects similar to those induced by exogenous growth factors since it used a pathway normally activated by growth factors, namely the Shc-MAPK pathway. Our coauthors recently used the confocal microscopic method to demonstrate that heregulin induces several rapid cellular effects, such as formation of membrane ruffles, filopodia, pseudopodia, actin polymerization, and loss of focal adhesion kinase plaques (41). Using identical methods, we studied rapid cell morphological changes and their substructures using LTED cells due to their strong responses with respect to ER{alpha}/Shc association and Shc phosphorylation upon E2 treatment.

Under basal conditions (Fig. 6AGo), LTED cells exhibited a low level of membrane ruffles (red), indicating minimal actin polymerization sites. ER{alpha} (green) appeared to be predominantly in the nucleus with some also present in the cytoplasm. Beginning at 10 min (data not shown) and increasing by 20 min, E2 induced dramatic morphological effects, including the formation of additional membrane ruffles and cell shape alterations (Fig. 6BGo). Even more dramatic was the formation of pseudopodia shown in Fig. 6CGo as an arm-like extension of the cell membrane with a fist-like structure at its terminus. To demonstrate that ER{alpha} mediated these morphological responses, the effects of E2 were abrogated by coadministration of the pure antiestrogen ICI along with E2 (Fig. 6DGo). ICI alone resulted in no morphological changes (Fig. 6EGo).



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Figure 6. Confocal Analysis of E2-Induced Morphological Changes and ER{alpha} Subcellular Localization in LTED Cells

Three-color-merged images are shown in panels A–E. Red fluorescent color indicates filamentous actin; green represents ER{alpha}; and blue indicates vinculin. Panel A illustrates cells grown under control conditions without E2. The arrow is directed toward a membrane ruffle that consists of concentrated bundles of filamentous actin. The regions indicated by the arrow in this and other panels (i.e. A–E) are further illustrated in the higher power views of specific areas shown as insets in panel F. Panel B illustrates cells treated with 10-10 M E2 for 20 min and shows the formation of additional membrane ruffles; panel C shows the formation of a pseudopodia under the same conditions. Panel D demonstrates that ICI at 10-9 M reduced the E2-induced ruffle formation, and panel E shows that ICI exerted no effect by itself. The insets in F represent the higher power views of specific areas of the membrane regions shown by arrows in panels A–E. On the left panels of the insets are shown the three-color ER{alpha}/filamentous actin/vinculin merged views; on the right, the two-color ER{alpha}/vinculin merged views are shown.

 
Use of confocal microscopy and immunofluorescence provided a dynamic means of assessing ER{alpha} location and alterations in response to E2. Accordingly, we focused on the region contiguous to the cell membrane. The insets in Fig. 6FGo show enlarged areas indicated by arrows in panels A–E. Under basal conditions, minimal green staining could be observed along the cell membrane of the cells (Fig. 6AGo, inset a, right panel). In marked contrast, E2 appeared to translocate ER{alpha} into the region along the membrane ruffles as indicated by the strong appearance of green staining (Fig. 6BGo, inset b,right panel). As shown by merging the red (actin) and green (ER{alpha}) views, the ER{alpha} appeared as yellow (Fig. 6BGo, inset b, left panel), indicating colocalization with actin in the membrane ruffles. Striking also was the translocation of the ER{alpha} into the fist-like region of the pseudopodia as shown by both the green staining (Fig. 6CGo, inset c, right panel) and yellow merged views (Fig. 6CGo, inset c, left panel). ICI blocked E2-induced ER{alpha} membrane translocation with little effect by it alone (Fig. 6Go, D and E; inset d, right and left panels; and inset e, right and left panels). The immunofluorescence staining for vinculin (blue) represents the focal adhesion contacts of the cells that are not highly visible.

The morphological changes demonstrated in Fig. 6Go represent examples of consistent changes observed in many cells. To establish that these changes were statistically significant, we used an integrated measure of membrane changes called dynamic membrane formation (DM). This integrated parameter includes both membrane ruffles and pseudopodia. As shown in Fig. 7Go, the E2-induced increase in DM was statistically significant and could be blocked by ICI. Collectively, these results demonstrated that E2 rapidly induced specific changes on cell morphology in addition to its effects on membrane-mediated Shc-MAPK activation.



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Figure 7. Summary of Dynamic Membrane Formation in LTED Cells

The changes in ruffles and pseudopodias are combined under the term, dynamic membrane (DM), formation. Quantitative results from 12 individual cells are summarized. *, P < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our studies demonstrated that MCF-7 breast cancer cells utilize a classical growth factor signaling pathway to mediate the nongenomic effects of E2. This pathway involves the direct association of ER{alpha} with Shc, the phosphorylation of Shc, and the formation of Shc-Grb2-Sos complexes. Shc is critical for E2-induced MAPK activation. Using a mutagenesis approach, we demonstrated that the SH2 and PTB domains of Shc are the sites interacting with ER{alpha} and that the AF-1 region of ER{alpha} is required to interact with Shc.

Evidence of the biological relevance of this pathway is inferred from the demonstration that Elk-1, one of the mediators of MAPK, was increased by E2. More importantly, we obtained direct biological evidence of the nongenomic effects of E2 by demonstrating morphological changes with confocal microscopy. These studies showed that E2 rapidly induced formation of membrane ruffles and pseudopodia, but also translocation of ER{alpha} to the membrane and into perimembrane regions of pseudopodia. Finally our results demonstrated that E2, mediated by ER{alpha}, activated a classical growth factor-signaling pathway involving Shc adapter proteins.

The adapter protein Shc mediates the effects of many growth factors to activate MAPK. However, several recent studies demonstrated different mechanisms whereby E2 can induce MAPK activation. McDonnell and associates (34) showed that calcium is required for E2-induced MAPK activation in MCF-7 cells, and this event is ER{alpha} dependent. E2-induced MAPK activation was also reported to be independent of ER{alpha} or ERß but required the existence of epidermal growth factor receptor (EGFR) and GRP30, a G protein-coupled receptor homolog in an ER{alpha}-negative breast cancer cell line MDA-MB-231 (25). EGFR expression in MCF-7 cells is very low, and Parsons and colleagues (42) have reported that EGFR protein is not detectable in MCF-7 cells either by immunoprecipitation or by immunoblotting methods. In our system, we do not detect EGFR in either MCF-7 or its variant cells (data not shown).

In the present study, we demonstrated that ER{alpha}, as a key mediator, is required for E2 action on Shc/ER{alpha} association and Shc and MAPK phosphorylation. Approximately 0.5% of total ER{alpha} was estimated to be associated with Shc after E2 stimulation as shown by an ER{alpha}-Shc coimmunoprecipitation study (data not shown). The activation of MAPK with E2 is less than that by growth factors, as reported by other investigators (34). Our unpublished data also show that MAPK phosphorylation induced by 10 ng/ml TGF{alpha} was 35-fold higher than untreated controls, compared with 2.5-fold induction induced by 10-10 M E2. ER{alpha}-dependent MAPK activation has been reported by many laboratories using MCF-7 and other cell lines treated with E2 (39, 43). The differences regarding ER{alpha} involvement in MAPK activation under varying conditions are unknown. Nevertheless, it is not surprising that E2 can activate multiple rapid signaling pathways in different cell lines due to the fact that it can exert nongenomic effects on nitrous oxide, PKC, PKA, and MAPK. Detailed examination of E2 induction through each of these mechanisms will be required before a full understanding of the biological relevance of these various pathways emerges.

A single time point of Shc phosphorylation induced by E2 was first reported in MCF-7 cells (13). We extended these observations by conducting detailed time studies and showed the effects as early as 1 min after E2 treatment. ER{alpha} is required for Shc phosphorylation, and the association of ER{alpha} with Shc appears to be important for Shc activation. It has been suggested that PTB and SH2 are two distinct binding domains, which may link to phosphorylated tyrosine residues in a different manner (44). Both PTB and SH2 domains of Shc are required to associate with EGFR (45), but Neuregulin-activated ErbB2 and ErbB3 bind only to PTB domain of Shc (46). The functional roles of the PTB or SH2 sites in their interaction with ER{alpha} are now under investigation.

The PTB and SH2 domains of Shc interact with phosphorylated tyrosine residue(s) of other proteins. The exact tyrosine residue of the ER{alpha} responsible for binding to Shc is unknown. Since tyrosine-537 of human ER{alpha} may serve as a constitutively phosphorylated site (7), we considered it a possible docking site for Shc. However, our data show that the AF-1 domain of ER{alpha}, but not the AF-2 or tyrosine-537 region, interacts with Shc, indicating that there might be other tyrosine residues phosphorylated on the N-terminal ER{alpha}. We postulated that 1 of 10 potential tyrosine residues in the AF-1 region (amino acids 1–179) might be involved in the association with Shc, such as tyrosine 43, 52, 54, 60, 73, 80, 130/131, 139, and 150. Further investigation of these sites is now being conducted in our laboratory.

ER{alpha} has been reported to associate functionally with many cell membrane proteins, such as IRS-1, the p85 subunit of PI3K, and caveolin (47, 48, 49). c-Src has been reported to physically associate with ER{alpha} of bovine uterus, but there is no direct evidence that ER{alpha}-associated c-Src is responsible for Shc phosphorylation (13). The present study demonstrated that the Src inhibitor PP2 blocked Shc phosphorylation induced by E2. This evidence suggests that a Src protein family member is functionally involved in E2-induced Shc phosphorylation, even though Src is not detectable in the ER{alpha}-Shc complex. It is possible that other Src family members are involved in phosphorylating Shc or that the Src-ER{alpha} complex is unstable during immunoprecipitation.

The breast cancer models used in our studies involve parental MCF-7 and its variant LTED cells. When compared with its parental cells, LTED cells show an increased growth rate in E2-deprived media and respond to very low concentrations of E2 when grown in nude mice (50). ER{alpha}, but not ERß, is detectable in our MCF-7 and its variant cells (33, 50). The presence of increased amounts of ER{alpha} appears to amplify the components examined in the ER{alpha}-Shc signaling pathway. Whenever effects, such as ER{alpha}/Shc association and Shc phosphorylation, were compared in LTED and MCF-7 cells, the responses were qualitatively similar but quantitatively greater in the variants. For this reason, we used this variant for demonstrating certain effects such as Grb2/Sos interactions and morphological changes by confocal microscopy.

Our studies demonstrated rapid, nongenomic effects of E2 by two separate methods, biochemical assessment of the Shc-MAPK pathway and morphological effects on cell shape, membrane ruffles, pseudopodia, and ER{alpha} translocation. Both effects could be blocked by the antiestrogen ICI, indicating that Shc-MAPK activation and morphological changes were all initiated by ER{alpha}. Even though we do not yet have direct evidence that the observed E2 effects on cell structure are mediated by activation of the Shc and MAPK pathway, the role of Shc and MAPK on cell adhesion and motility has been demonstrated in several cell lines (51, 52). The interaction of Shc with membrane integrin in MCF-7 cells was reported to regulate the locomotion and cell adhesion of MCF-7 cells (53). We postulate that the E2-induced membrane signaling pathway activation, especially ER{alpha}-involved activation of Shc, might be linked to cell morphology changes in our cell models.

The suggestion that ER{alpha} can reside in or near the cell membrane has been controversial in the past (54). Recent transfection studies provided additional support that ER{alpha} can be present in the cell membrane (55). Razandi et al. (55) transfected ER{alpha} into Chinese hamster ovary (CHO) cells and demonstrated that 3% of expressed ER{alpha} was associated with cell membranes. Immunochemical studies with several anti-ER{alpha} antibodies on fixed cells including MCF-7 cells also suggested the presence of membrane ER{alpha} (20). Our observation using confocal microscopy showed that E2 increased the amounts of ER{alpha} in the region of the cell membrane. Our findings provide the first evidence that E2 can functionally influence the amount of ER{alpha} translocated into the perimembranous area. At the present time, we do not have direct morphological evidence that Shc is associated with this membrane-associated ER{alpha}, but we plan to examine this issue in the future.

In conclusion, we demonstrated that the signaling pathway mediating the rapid action of E2 involved the interaction of Shc with ER{alpha}, Shc activation, and the phosphorylation of MAPK. The PTB and SH2 domains of Shc as well as AF-1 domain of ER{alpha} are the sites mediating the interaction. Using selective pathway inhibitors and a dominant negative Shc mutant, we confirmed that ER{alpha}-mediated Shc activation resulted in MAPK phosphorylation in MCF-7 cells. As evidence of functionality, E2 rapidly stimulated cell morphological changes and induced activation of the MAPK substrate, Elk-1. Additional studies are now required to provide further proof of the role of each of the signaling molecules in the biological events observed.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents and Plasmids
Tissue culture supplies were obtained from Fisher Scientific (Pittsburgh, PA). Improved MEM (IMEM) with or without phenol red (zinc option, Richter’s modification) and FBS were products of Life Technologies, Inc. (Gaithersburg, MD). E2 was obtained from Steraloids (Wilton, NH). ICI was from AstraZeneca, Inc. (Wilmington, DE). E2 and ICI were dissolved in ethanol with a final concentration of ethanol in medium of less than 0.01%. PP2 and PD98059 were from Calbiochem (La Jolla, CA) and dissolved in dimethylsulfoxide. Full-length ER{alpha} recombinant protein was purchased from Affinity BioReagents, Inc. (Golden, CO). Protease inhibitors, leupeptin, pepstatin, and aprotinin, were purchased from Sigma (St. Louis, MO).

Mammalian expression vectors encoding human 52-kDa forms or partial regions of Shc with GST fusion proteins were gifts from Dr. Ravichandran Kodi (University of Virginia, Charlottesville, VA) and have been described previously (46). The amino acid sequences encoded by the different Shc constructs are: full-length (1–471), PTB (17–203), CH (233–377), and SH2 (377–471). The full-length human ER{alpha} expression vector (HEGO) was a gift from Dr. Rosalie Uht (University of Virginia) and has been previously reported (56). The ER{alpha} expression vectors of AF-1, AF-2, and the Y537F mutant in which tyrosine 537 was mutated into phenylalanine were constructed in our laboratory by PCR and site-directed mutagenesis (QuickChange, Stratagene, La Jolla, CA) as previously described (57). The fusion gene GAL4-Elk-1 and its reporter gene GAL4-E1B-Luc were generously provided by Dr. Michael J. Weber (University of Virginia) and have been described previously (40).

Cell Culture
The MCF-7 cells and COS-1 cells were maintained in 5% FBS-IMEM and 10% FBS-DMEM, respectively. The LTED cells were developed from MCF-7 cells by growing long term (i.e. 6 months to 2 yr) in phenol red-free IMEM supplemented with 5% Dextran-coated charcoal-stripped FBS and have been extensively characterized with respect to growth characteristics, responsiveness to E2, and ER content (33, 50). As determined by ligand binding assays as well as mRNA and Western blots, these cells contain 5- to 10-fold higher levels of endogenous ER{alpha} than the parental MCF-7 cells. The content of ERß is negligible to absent as demonstrated by Western blot and PCR (data not shown). All cell lines were routinely cultured in a humidified 95% air-5% CO2 incubator at 37 C.

Cell Stimulation and Lysate Preparation
For rapid E2-induced signaling pathway studies, cells cultured in IMEM containing 1% dextran-coated charcoal-stripped FBS were treated at 37 C with vehicle or E2. Time and doses are indicated in the figure legends. If inhibitors were used, cells were pretreated with the inhibitors for 30 min and then challenged with E2. Cells were washed once with ice-cold PBS containing 1 mM Na2VO4 and extracted with binding buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 5% glycerol, 1% Triton X-100, 25 mM NaF, 2 mM Na2VO4, and 10 µg/ml of each aprotinin, leupeptin, and pepstatin). Cell lysates were centrifuged at 14,000 x g for 10 min at 4 C to pellet insoluble material. The supernatant of cell extract was analyzed for protein concentration by a DC protein assay kit based on the Lowry method (Bio-Rad Laboratories, Inc.).

Immunoprecipitation and Immunoblotting
Immunoprecipitation and immunoblotting were carried out as described previously (58). Briefly, 1 mg of cell lysate from each treatment was immunoprecipitated using one of the following antibodies: 1 µg monoclonal anti-Shc antibody (PG-797; Santa Cruz Biotechnology, Inc.), 1 µg monoclonal anti-ER{alpha} antibody (D-12, Santa Cruz Biotechnology, Inc.), 1.2 µg polyclonal anti-Grb2 antibody (C-23, Santa Cruz Biotechnology, Inc.), 1 µg polyclonal anti-GST antibody, and 1 µg monoclonal anti-Sos antibody (Transduction Laboratories, Inc., Lexington, KY). Incubations proceeded for 4 h at room temperature or overnight at 4 C in the presence of 35 µl of 50% slurry protein-G or protein-A Sepharose beads (Life Technologies, Inc.). The beads were washed three times in cold binding buffer. The proteins eluted from the beads were analyzed on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. The PVDF membranes were probed with one of the following primary antibodies: horseradish peroxidase-conjugated monoclonal antiphosphotyrosine antibody (PY20, Transduction Laboratories, Inc.), polyclonal anti-ER{alpha} antibody (HC-20, Santa Cruz Biotechnology, Inc.), polyclonal anti-Shc (Transduction Laboratories, Inc.), polyclonal anti-GST antibodies (Z-5, Santa Cruz Biotechnology, Inc.), monoclonal anti-Grb2 (Transduction Laboratories, Inc.), monoclonal anti-GST, monoclonal anti-ER{alpha} N-terminal antibody (COIM2133, Fisher Scientific), polyclonal anti-C-terminal ER{alpha} antibodies (MC-20, Santa Cruz Biotechnology, Inc.), and polyclonal anti-Sos antibodies (Transduction Laboratories). After washing the PVDF membrane, the immunoblots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h [donkey against rabbit IgG from Pierce Chemical Co. (Rockford, IL) or sheep against mouse IgG from Amersham Pharmacia Biotech (Piscataway, NJ)] and further developed using the chemiluminescence detection system (Pierce Chemical Co.). The reciprocal studies for all protein-protein interactions were conducted and the same results were confirmed. All experiments were done at least three times.

GST Pull-Down Assay
Prokaryotic vectors expressing both PTB+CH and SH2 domains of Shc were gifts from Dr. Ravichandran Kodi (University of Virginia), expressed in Escherichia coli BL21 and purified with a GST-glutathione affinity system (Amersham Pharmacia Biotech). GST, GST-ShcPTB+CH, and GST-ShcSH2 proteins were induced, solubilized, and bound to Glutathione beads. To test the protein interaction, 15 µl of the suspension were incubated in binding buffer with 5 ng of human full-length ER{alpha} expressed and purified from Baculovirus. After incubation, the beads were washed three times with the buffer. Bound proteins were eluted with 40 µl of 2x SDS-PAGE buffer, electrophoretically separated in 10% SDS gel, and transferred on the PVDF membrane. The membrane was probed with monoclonal anti-ER{alpha} antibody, and the immunoprotein bands were visualized as described above.

Electroporation and Transfection
COS-1 cells were electroporated exactly as described by Roy et al. (59) with 5 µg each of Shc constructs and 5 µg of each ER{alpha}-expressing vectors. Cells were then incubated in DMEM supplemented with 10% FBS for 48 h. The medium was changed again with serum-free and phenol-free DMEM for 1 d. Four hours before E2 treatment, the medium was changed again with fresh serum-free and phenol-free medium. Cells were then treated with E2 for 5 min and extracted with binding buffer. At this step, the samples were either stored at -80 C or prepared for protein-protein association assay. To study MAPK-mediated Elk-1 activation, MCF-7 cells were transiently transfected with both 2 ng of GAL4-Elk-1 and 0.1 µg of GAL4-E1B-Luc reporter vectors encoding GAL-Elk fusion protein and GAL-luciferase, respectively. Transfection was performed with Effectene transfection reagent (QIAGEN, Valencia, CA) according to company specifications; 24 h after transfection, cells were treated with different doses of E2 for 6 h. Luciferase activity was measured for each lysates, and samples were normalized by ß-galactosidase cotransfected with above vectors. All transfections were conducted at least three times and the data are presented as mean ± SD.

Confocal Analysis of E2-Induced Cell Morphology Changes
Cells were fixed in paraformaldehyde followed by chilled acetone and blocked in normal goat serum followed by incubation with monoclonal antibody against vinculin (Sigma) and with polyclonal antibodies against ER{alpha} (Santa Cruz Biotechnology, Inc.). For triple costaining of filamentous actin, ER{alpha} and vinculin and 0.1 µM Alexa 546-conjugated phalloidin were included during incubation with the Alexa 633-goat antimouse and Alexa 488 goat antirabbit secondary antibodies. Slides were mounted, and analyzed by a LSM inverted microscope (Carl Zeiss, Thornwood, NY) (60). Each image represents Z sections at the same cellular level and magnification. Confocal analysis was performed using a Carl Zeiss laser scanning confocal microscope and established methods, involving processing of the same section for each detector and comparing pixel by pixel. Localization of ER{alpha} with ruffles is demonstrated by the development of yellow color due to red and green overlapped pixels. The effect of E2 on the changes of filamentous actin in terms of dynamic membrane was measured based on the formation of ruffles and pseudopodia from 12 cells in each treatment. Extensive application of identical methods for analysis of effects of heregulin has been recently published by Adam et al. (41).


    ACKNOWLEDGMENTS
 
We thank Dr. Ravichandran Kodi for supplying the Shc-expressing constructs and Dr. Michael J. Weber for providing us with the Elk-1 vector. We also thank Dr. Rosalie Uht for her gift of ER{alpha}-expressing vector.


    FOOTNOTES
 
This work was supported by NIH Grants CA-65622 (to R.J.S.), CA-80066 (to R.K.), and DK-57082 (to M.A.S.).

Abbreviations: AF-1 and AF-2 domains, Activation function 1 and 2 domains; CH domain, collagen homology domain; EGFR, epidermal growth factor receptor; Grb2, growth factor receptor binding protein 2; GST, glutathione-S-transferase; HEGO, human ER{alpha} expression vector; ICI, ICI 182 780; IMEM, improved MEM; LTED cells, MCF-7 cells growing long-term in estrogen-depleted medium; MEK, MAPK kinase; PTB domain, phosphotyrosine binding domain; PVDF, polyvinylidene difluoride; Shc, Src-homology and collagen homology; ShcWT, wild-type Shc; Sos, Son of Sevenless; Src, Src family of tyrosine kinases; c-Src, p60 of Src.

Received for publication February 20, 2001. Accepted for publication September 6, 2001.


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