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Proximal Events in Signaling by Plasma Membrane Estrogen Receptors*

Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for G (cid:1) q and G (cid:1) i blocked E2-induced, EGFR-dependent ERK activation, and G (cid:2)(cid:3) also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER

Steroid hormones such as estrogen are essential to the development and reproductive functions of prokaryotic and eukaryotic organisms. Traditionally, steroid hormone action was exclusively attributed to the binding of nuclear receptors and the subsequent transactivation of target genes that led to cell biological effects (1). More recently, it has become clear that steroids rapidly act on cells, in seconds to minutes, effects that are classified as "nongenomic" (reviewed in Ref. 2). For estrogen, this has been attributed in most cells to binding a population of receptors that exists within caveolar rafts and other domains in the plasma membrane (3)(4)(5). It is at the plasma membrane that estradiol (E2) 1 -liganded estrogen receptors (ER) physically associate with the scaffold protein, caveolin-1 (5), and a variety of proximal signaling molecules, including G proteins (6,7), Src and Ras (8,9), and B-Raf (10). This results in the activation of cascades of signal transduction, mainly evolving from G protein activation. Comparable with many other G protein coupled receptors (GPCR), G protein activation by ER (6,7) leads to the stimulation of phospholipase C (11), protein kinase C (12), ERK (9), and phosphatidylinositol 3-kinase and nitric-oxide synthase (13). These positive signaling effects are cell context-specific, and in some cells, estrogen inhibits cytokine-related signal transduction to cell differentiation, proliferation, migration, or cell death (14 -17).
What is the nature of the membrane ER, and how does it enact signal transduction? Current evidence favors the idea that the membrane and nuclear ER are the same protein.
Antibodies directed against many epitopes of the classical ER␣ receptor identify membrane ER by immunocytochemistry (18). Expression of antisense DNA to the "nuclear" ER also abrogates the detectable expression of membrane ER in cells containing endogenous receptor (19). In CHO cells, expression of a single cDNA for either ER␣ or ER␤ produces both membrane and nuclear receptor populations and results in E2 activation of signal transduction from the membrane (6). In many cell types, endogenous membrane ER have been identified (15,18,20) and appear to reflect the localization of receptors that also have the capacity to translocate to the nucleus. The structural aspects of the membrane ER that allow it to activate signaling molecules are not well defined. Assuming that the sequence of the nuclear receptor is the same as the membrane ER, there is no catalytic or kinase sequence inherent to the structure. Recent evidence favors the idea that the E domain of the membrane ER is essential (and perhaps sufficient) for activation of the ERK cascade (5), leading to cell survival (17). Additionally, the AF-1 domain of ER␣ has been identified to interact with the adapter protein, Shc, in whole cell homogenates (21). Thus, the membrane ER acts similarly to many other GPCR that also lack catalytic or kinase domains yet signal to important events in cell biology.
As a GPCR, the membrane ER associates with and activates several G proteins. In transfected CHO cells, membrane ER␣ or ER␤ co-precipitates with and activates G␣ s and G␣ q proteins (6). This leads to the expected downstream signaling to cAMP and inositol 1,4,5-trisphosphate generation, signaling that has been shown in cells expressing endogenous ER (22,23). In EC, endogenous membrane ER physically associates with G␣ i and activate endothelial nitric-oxide synthase; this probably takes place within caveolae (7). Additionally, it has been proposed in breast cancer cells that E2/ER transactivates the epidermal growth factor receptor (EGFR), leading to the downstream signaling to ERK activation (24,25). This occurs through the activation of G␤␥, the liberation of heparin-binding EGF (HB-EGF), which results in the binding and activation of the EGFR, and the subsequent stimulation of the ERK signaling cascade. In some of these respects, the membrane ER acts similarly to a wide range of GPCR (26). However, it was further proposed in breast cancer cells that E2 in some undefined way activates the orphan GPCR, GPR30, to stimulate signaling, and this interaction does not require ER (25). These latter data are not in concert with many studies from other laboratories, indicating that E2 requires an ER for signaling from the membrane in various cell types (5,6,8,20,27,28).
The utilization of EGFR by E2/ER to signal results from a linked series of events involving multiple upstream molecules, only some of which have been defined. For instance, we do not know the range of G proteins that can be activated to cross-talk to EGFR activation, and it is not clear what signals immediately downstream of G proteins are important. Src participates in the transactivation of EGFR in response to other GPCR ligands and is probably upstream of HB-EGF shedding (29), but its exact role and requirement for ER signaling is unclear. Furthermore, although matrix metalloproteinase (MMP) activation is required for HB-EGF liberation (and subsequent EGFR activation), the identity of the required MMP(s) is mainly undefined, especially as regards ER signaling. These issues are addressed in the studies described here. Finally, much of the interaction between GPCRs and EGFR has examined ERK activation. Thus, we sought additional signaling molecules in several cell types and the structural requirements within ER that utilize this interactive mechanism following endogenous ER ligation by E2.

EXPERIMENTAL PROCEDURES
Materials-Antibodies and substrate for kinase activation/activity were from Santa Cruz Biotechnology (Santa Cruz, CA). PD 98059 was a generous gift from Dr. Alan Saltiel (Parke-Davis). LipofectAMINE was from Invitrogen. Primary cultures of bovine aortic EC were prepared and used as previously described (30). In transfection studies, EC were generally used in passages 4 and 5, based upon the previous observation that this greatly increases the transfection efficiency of these cells. Breast cancer cell lines were obtained from ATCC. The cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 or RPMI 1640 with serum until 48 h prior to experimentation, when they were placed in serum-free conditions and in medium without phenol red. Gelatin was from Sigma, and kinase substrates were from Upstate Biotechnology, Inc. (Lake Placid, NY) or Santa Cruz Biotechnology. PP2, Src family kinase inhibitor, and GM6001, a matrix metalloproteinase (MMP) inhibitor, were from Calbiochem (San Diego, CA).
Kinase Activity Assays-For ERK or p38␤ activity assays, the cells were synchronized for 24 h in serum-and growth factor-free medium. The cells were then exposed to E2 for 8 (ERK) or 15 (p38) minutes, with or without additional substances, as previously described (30,33). The cells were lysed, and lysate was immunoprecipitated with protein A-Sepharose conjugated to antiserum for p38 or ERK. Immunoprecipitated kinases were washed and then added to the proteins ATF-2 (for p38) or myelin basic protein (for ERK) for in vitro kinase assays. This was followed by SDS-PAGE separation and autoradiography/laser densitometry. In addition, the E2-induced phosphorylation of AKT kinases at 10 min was determined to assess activation. Cultured cell lysates were pelleted and dissolved in SDS sample buffer, boiled, separated, and then transferred to nitrocellulose. Phosphorylated kinase proteins were detected using phospho-specific monoclonal antibodies (Santa Cruz) and the ECL Western blot kit (Amersham Biosciences). Equal samples from the cells were also immunoprecipitated, and immunoblots of the precipitated kinase protein from each experimental condition were determined to show equal gel loading. All of the experiments were repeated two or three times.
Transient Transfections-MCF-7, HCC-1569, ZR-75-1, or bovine aortic endothelial cells (passages 4 and 5) were grown to 40 -50% confluence and then transiently transfected with 1.5 g (each well of 6-well plates) or 10 g of fusion plasmid DNA (100-mm dishes). Plasmids included wild type mouse ER␣ (31) (kindly provided by Dr. Ken Korach) PRK5-HER, a dominant negative EGF receptor construct (kindly provided by Dr. A. Ullrich (32), a dominant negative Src construct, pRC-csrc-K298M (kindly provided by Drs. Louis Luttrell and Robert Lefkowitz (26), a dominant negative, truncated ␤-adrenergic receptor kinase plasmid (BARK1-CT pRK5) from Dr. Walter Koch (34), and truncated G␣ subunit plasmids, serving as specific dominant negative constructs for G s , G q , G i , G 12 , and G 13 (35). Transfection was carried out using LipofectAMINE (Invitrogen). The cells were incubated with liposome-DNA complexes at 37°C for 5 h, followed by overnight recovery in culture medium containing 10% fetal bovine serum, 24 h of synchronization in serum-free medium, and then treatment with E2 with or without other substances.
Gelatin Zymography Substrate Cleavage and Antisense Studies for MMP Activity-MMP activity, as secreted into the medium of cultured MCF-7 cells, was analyzed by substrate gel electrophoresis (zymography). The cells were synchronized in serum-free medium for 24 h and then incubated in medium with or without 10 nM estrogen for 2 min at 37°C in a CO 2 incubator. The cell medium was removed, concentrated 20-fold by ultrafiltration, and mixed with native gel sample buffer (Bio-Rad), and the proteins were separated by electrophoresis on an 8% gel co-polymerized with 1 mg/ml gelatin (Sigma). Active MMP-2 and MMP-9 (Calbiochem) was loaded into additional lanes on the gel. After electrophoresis, the gels were washed in 2.5% Triton X-100 at room temperature for 1 h and incubated for16 h at 37°C (in 0.05 M Tris, pH 7.5, 5 mM CaCl 2 , 0.02% NaN 3 ). The gel was stained with 0.5% Coomassie Blue and destained in 10% acetic acid, 10% propanol. The study was repeated twice. Gelatinolytic activity appears as a clear band on a blue background. For the fluorescent substrate assay, MCF-7 cells were synchronized for 24 h and then incubated without or with 10 nM estrogen for 2 min. The incubation medium was concentrated 10-fold, and 1 ml of assay buffer (100 mM Tris, pH 7.5, 100 nM NaCl 2 ) containing 5 M of the Mca-Pro-Leu-Dpa-Ala-Arg-NH 2 substrate for MMP-2/MMP-9 was added and then incubated at 37°C for 3 h. Excitation at 328 nm and emission at 393 nm were determined in a fluorimeter. To implicate MMP-2 and MMP-9 in the shedding of HB-EGF, the cells were incubated with antisense (ASO) or scrambled antisense (MSO) with the same base composition for each of the two MMPs. The oligonucleotides were: MMP-2, ASO, CCGGGCCATTAGCGCCTCCAT, and MSO, TCACCGCGGTACGCATGCCCT; and MMP-9, ASO, CAGGGGCTGC-CAGAGGCTCAT, and MSO, GCGAGCTAGGACTGTGCAGCC. The oligonucleotides were added with LipofectAMINE for 5 h, and the cells were recovered overnight and synchronized in the absence of serum for 12 h. Transfection efficiency exceeded 60%, based upon co-expression of PEGFPc2. Western blot studies were carried out to confirm the efficacy of the ASO but not the MSO to inhibit specific protein production. Studies of E2-induced signaling were then carried out in cells expressing the various oligonucleotides.
Western Blot for HB-EGF and EGFR Phosphorylation-Subconfluent, transfected, or nontransfected cultured bovine aortic endothelial cells were serum-deprived for 24 h and then incubated under various conditions for 10 min with inhibitors followed by 10 min of treatment with stimulants. This included several 17-␤-E2 concentrations, ICI 182780 (1 M), and 100 nM GM6001, a broad MMP inhibitor. The cells were lysed, and antibodies to HB-EGF or EGFR (tyrosine 1138) (1:50 dilution) were conjugated to Sepharose beads and then added to the cell lysate for 2 h at 4°C. After pelleting and washing, the samples were electrophoretically separated on a 7% SDS gel, transferred to nitrocellulose, and immunoblotted. Detection utilized the ECL kit (Amersham Biosciences).

FIG. 1. E2 activates ERK via ER and EGFR.
A, 17-␤-E2 activates ERK only when ER is present. HCC-1569 cells (ER negative) were incubated with 10 nM 17-␤-E2 or were transfected to express wild type mouse ER␣ (mERa) and then incubated with 17-␤-E2 or 17-␣-E2, and ERK activity (against myelin basic protein) was determined after 8 min in an in vitro tube assay as described under "Experimental Procedures." Immunoblots of total ERK protein are shown below the activity. The bar graph represents three combined experiments. *, p Ͻ 0.05 for control versus E2 or EGF; ϩ, p Ͻ 0.05 for E2 versus E2 with ICI182780 (ER antagonist). B, inhibition of EGFR tyrosine kinase function with tyrphostin AG1478 prevents E2-induced ERK activation in MCF-7 cells (left panel), in ZR-75-1 cells (center panel), or in endothelial cells (right panel). The cells were incubated as described above with 17-␤-E2 with or without a specific EGFR tyrosine kinase inhibitor, and ERK activity was determined. Each bar graph represents three combined experiments. *, p Ͻ 0.05 for control versus E2 or EGF; ϩ, p Ͻ 0.05 for E2 versus E2 with ICI182780 (ER antagonist) or E2 or EGF versus either E2 or EGF with AG1478 (tyrphostin). C, expression of a dominant negative EGFR (EGFR (DN)) prevents E2-induced ERK activation in MCF-7 cells. The cells were transfected to transiently express PRK5-HER dominant negative EGFR, recovered overnight in 10% serum, and 24 h after cell recovery, E2 activation of ERK was determined after 8 min of incubation. *, p Ͻ 0.05 for control versus E2; ϩ, p Ͻ 0.05 for E2 versus PRK5-HER transfected cells incubated with E2.

Activation of ERK by E2 Requires an ER and the Activation of EGFR by HB-EGF-
We first established that E2 required both the presence of an ER and the activation of EGFR to signal to ERK. HCC-1569 cells lack ER, and the cells did not respond to E2 with ERK activation (Fig. 1A, lanes 1 and 2). When ER␣ was expressed in these cells, 17-␤-E2 (lane 4), but not 17-␣-E2 (lane 8), was capable of activating ERK, and this was substantially blocked by the ER antagonist, ICI182780 (lane 5). As a positive control, these cells express the EGFR and appropriately respond to EGF (lane 7). The requirement of ER is similar to our previous findings in CHO-K1 cells (6). We then asked whether E2 activation of ERK depends upon EGFR tyrosine kinase activity. We examined this in MCF-7 and ZR-75-1 breast cancer cells and EC (all with ER). Tyrphostin AG1478, specifically directed against the EGFR tyrosine kinase function, prevented EGFR-induced ERK activation in both MCF-7 and ZR-75-1 cells (Fig. 1B, left and center panels). Importantly, tyrphostin AG1478 also substantially prevented the ability of E2 to activate ERK in the three cell types (Fig. 1B, all panels, lanes 2 versus lanes 6). To corroborate this finding, we expressed a dominant negative EGFR (31) in MCF-7 cells, and E2 was much less effective in stimulating this MAP kinase, compared with cells expressing the empty vector (control) (Fig. 1C).
What ligand for EGFR is involved in the transactivation of this receptor by E2? Although there are many members of the EGF family that can bind the EGFR, HB-EGF has often been implicated in the setting of GPCR signaling via this receptor (36). To examine this, we first determined whether E2 could stimulate the secretion of HB-EGF, determined by Western blot. As seen in Fig. 2A, E2 dose-responsively induced a significant enhancement of HB-EGF shedding/secretion from the MCF-7 cells after 3 min of incubation. This was prevented by ICI182780 and by GM6001, an MMP inhibitor. To determine that HB-EGF was the important ligand for EGFR signaling to ERK, we incubated the MCF-7 cells with 10 nM E2, in the presence or absence of antibody to HB-EGF. In the setting of this added antibody, E2 could not significantly activate ERK (Fig. 2B). In contrast, antibody to TGF␣-1, another ligand for the EGFR, had no effect on E2-induced ERK, and the antibodies by themselves did not affect basal ERK activity. Similarly, antibody to HB-EGF (but not to TGF␣-1) prevented E2-induced phosphorylation of the EGFR (Fig. 2C). Identical findings were determined from EC incubated with E2 (data not shown). These results support the interactions of secreted HB-EGF with EGFR, leading to ERK activation in breast cancer and vascular cells. The data also support ER-mediated, MMP-dependent release of HB-EGF.
Matrix Metalloproteinases 2 and 9 Are Activated and Are Necessary for Signaling by E2-Current evidence supports the idea that GPCRs activate MMP activity, thereby liberating HB-EGF from the cell matrix, leading to the transactivation of the EGFR (36,37). Therefore, MMP activation represents the step immediately upstream of HB-EGF liberation. In many cell paradigms, including E2 action, the precise MMP(s) activated by GPCR signaling are unknown. We therefore showed that E2 activates MMP activity by demonstrating that the incubation medium from MCF-7 cells treated with E2 for 2 min induces the cleavage of substrate specific for MMP-2 and MMP-9 (Fig.  3A). In contrast, substrate specific for MMP-13 or MMP-3 was not cleaved by the E2-treated cell medium (data not shown), even though breast cancer cells produce these proteolytic enzymes. We then sought to further identify the MMPs by carrying out gelatin zymography. E2 treatment of the cultured MCF-7 cells for 2 min led to the increased secretion and activation of MMP-2 and -9 (Fig. 3B, first and second lanes). To corroborate the identify of the digested gelatin band activities, active MMPs (Calbiochem) were also run in parallel on a separate gel (data not shown). Functionally, activation of MMP activity was necessary for E2-induced ERK. This was shown in that the MMP inhibitor completely reversed the ability of E2 to activate ERK in both MCF-7 and ZR-75-1 cells (Fig. 3C, left and right panels). This compound did not affect EGF-induced ERK activation, supporting the idea that MMP-related events occur upstream to EGFR activation in this pathway.
Although E2 activates these two MMPs, it is not clear that they are responsible for E2-induced HB-EGF shedding. We therefore used ASO or MSO, with the latter comprised of the same base composition as the ASO for MMP-2 and MMP-9, and expressed them in MCF-7 cells. First, we validated the constructs by showing that the ASO (but not the MSO) for MMP-2 or MMP-9 inhibited the respective protein production in a dose-related manner (Fig. 4A, left panel). Similarly, we validated the function of the MMP-2 or MMP-9 to specifically inhibit only the intended protein target (Fig. 4A, right panel). Using these ASO and MSO, we next determined whether MMP-2 and MMP-9 each contributed to HB-EGF shedding and ERK activation (Fig. 4, B and C). Each ASO significantly downregulated E2-induced HB-EGF liberation, and expressing the ASO to both MMPs completely blocked this E2 action. The ASO to MMP-2 almost completely prevented the ability of E2 to activate ERK in MCF-7 cells, whereas the ASO to MMP-9 was also substantially able to prevent this signaling; neither MSO had any effect, and the results were similar to those in EC. E2/ER stimulation of MMP-2 and MMP-9 may therefore underlie several important actions in breast cancer, including signaling through ERK to cell proliferation and survival (5,9). Metalloproteinase activation also contributes to the disengagement of cells from matrix, a necessary initial step preceding invasion and migration behaviors (38). MMP-2 and MMP-9 are well recognized to contribute to these events in various contexts (38,39).
Specific G Proteins Are Involved in E2-induced Transactivation of EGFR-It has previously been established that E2 can activate G␣ s and G␣ q , as well as G␣ i in several cell models (6,7). Therefore, one or more G proteins activated by E2 could ultimately result in EGFR signaling to ERK. To examine this issue, we expressed mini-genes for G␣ subunits of G s , G i , G 12 , G 13 , and G q , constructs that have been shown to act as dominant negatives for specific endogenous G protein subunit activation (35). As seen in Fig. 5A, ERK activation in response to E2 in cells expressing the control plasmid, G␣ ir (lane 3), was substantially prevented after expressing the inhibitory minigenes for G␣ i and G␣ q (lanes 4 and 5). However, dominant negative constructs for the ␣ subunits of G s , G 12 , and G 13 had insignificant effects on this signaling. We also expressed a C-terminal truncated ␤-adrenergic receptor kinase, pRK5-BARK1-(495-689), that inhibits G␤␥ signaling (33). Expression of this construct significantly but incompletely prevented the ability of E2 to activate ERK and HB-EGF liberation (Fig.  5B). Upon expressing ER␣ in HCC-1569 cells, E2 could now activate ERK in a G␣ i , G␣ q , and G␤␥-dependent fashion (Fig.  5C). Therefore, both G␣ and G␤␥ subunits contribute to the ability of E2/ER to activate the signaling pathway that ultimately results in EGFR transactivation.
Calcium, PLC, and PKC Activities Mediate E2-induced MMP Activation-The signaling through the identified G proteins potentially leads to the activation of MMP activity and the subsequent downstream signaling through EGFR. We examined which signal pathways immediately downstream of G protein activation that we identified here could mediate MMP activation. E2-induced MMP activity was significantly inhib-ited by EDTA, an extracellular calcium chelator, but was not affected by BAPTA-AM ( Fig. 2A). This indicates that calcium entry through surface channels, but not the mobilization of intracellular calcium, contributes to E2-induced MMP activation. It has previously been shown that E2 activates several calcium channels that lead to an influx of calcium into the cell (40,41), and this can result from G␣ q or G␤␥ activation.
We also found that soluble inhibitors of PLC and PKC (calphostin C and U-73122, respectively) significantly prevented E2 activation of MMP activity (Fig. 3A). This is consistent with our identification here of G␣ q and G␤␥ as mediating E2-induced ERK activation, because PLC and PKC up-regulation results from the activation of these G protein subunits. We previously showed that E2 can activate G␣ q , PLC, and inositol 1,4,5-trisphosphate generation via membrane ER (6), and E2 has been described to stimulate PKC activity in several cell types (reviewed in Ref. 42). PKC-dependent signaling in growth plate chondrocytes mediates E2-induced regulation of these cells, and originates from membrane action of the steroid (43). These findings link the most proximal signaling events to later events (MMP activation and HB-EGF shedding), mediating EGFR transactivation.
Role of Src in Shedding of HB-EGF-It has been documented that E2-liganded ER complex with and activate the Src tyrosine kinase, and this is necessary for E2 stimulation of ERK (8,17). Src could potentially play a role both upstream and downstream of EGFR activation. We therefore determined where Src activation is required for the proximal signaling induced by E2, leading to EGFR transactivation. As shown in Fig. 3B, E2-induced MMP-2 and MMP-9 activation and secretion at 2 min (first lane versus second lane). This was substantially prevented by the Src family kinase inhibitor, PP2 (third lane), or by expressing a specific dominant negative Src construct, pRC-csrc-K298M (26) (fifth and sixth lanes compared with first and second lanes). Thus, these results define a novel role for Src in E2-induced signaling from the membrane, and we suggest that this molecule may play a similar role in other GPCRinduced activation of EGFR through this mechanism.
ER Is Required for Proximal Signaling Events-We earlier showed that E2 requires an ER to activate ERK (Fig. 1). To further support the idea of the necessity of ER presence for E2 action, we expressed ER␣ in HCC-1569 cells and determined the proximal signaling events implicated. We first demonstrated that expression of the dominant negative G␣ i and G␣ q mini-genes substantially blocked E2-induced ERK, compared with kinase activity in the presence of the control (inactive) construct, G␣ ir (Fig. 6A). Similarly, expression of C-terminal truncated ␤-adrenergic receptor kinase (BARK1) also downregulated E2/ER-induced ERK, whereas the truncated minigene for G␣ 13 was without effect (similar to control). We also examined HB-EGF secretion and found that E2 stimulated the secretion of this receptor ligand only when ER was expressed (Fig. 6B, lanes 1 and 2 versus lanes 3 and 4). ICI182780 and MMP inhibition significantly prevented the stimulation of HB-EGF secretion. Finally, we found that in the presence (but not mined by Western blot. The bar graph is three experiments combined. *, p Ͻ 0.05 for control versus E2; ϩ, p Ͻ 0.05 for E2. B, antibody to HB-EGF but not TGF␣ blocks E2-induced ERK activation. MCF-7 were incubated with 10 nM E2 with or without 10 g/ml antibody to HB-EGF or TGF␣, and ERK activity was determined after 8 min. The antibodies alone had no effect on ERK activity. C, HB-EGF but not TGF␣ antibody blocks the E2-induced transactivation/phosphorylation of EGFR. The cells were incubated with 10 nM E2 with or without antibodies for 8 min, and lysate was subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antibody to tyrosine 1173 of the EGF receptor. *, p Ͻ 0.05 for control versus E2 or E2 plus TGF␣ antibody; ϩ, p Ͻ 0.05 for E2 versus E2 with HB-EGF antibody.  3. E2 activates matrix metalloproteinase 2 and 9 secretion and activity. A, cells were incubated with or without E2 with or without BAPTA (intracellular calcium inhibitor), EDTA (chelates extracellular calcium), a specific PLC inhibitor, U73122, or a specific PKC inhibitor, calphostin C for 2 min. Cleavage of substrate for MMP-2/MMP-9 by the medium from MCF-7 cells incubated with 10 nM E2 for 2 min was determined by spectroflurometry. The data are from triplicate determinations in a representative experiment, repeated twice. B, MCF-7 cells were in the absence) of ER␣, E2 stimulated the activation of MMP-2 and MMP-9 (Fig. 6C). This was partially dependent upon extracellular calcium, PLC, and PKC signaling. These data strongly support the idea that the classical ER␣ is required for ing, ERK and cAMP generation are the two pathways that have been identified to require EGFR activation (24,25), but this has only been established in breast cancer cells. To further define the role of EGFR in E2-induced signaling from membrane ER, we examined breast cancer cells and EC, cells that express endogenous ER (9,20). In MCF-7 cells, we found that E2 activated protein kinase B (AKT) (Fig. 7A). This was substantially prevented by the soluble inhibitor of MMP activity and by tyrphostin AG1478, implicating the EGFR. These two compounds had no effects by themselves (data not shown). In EC, we previously showed that E2 activates the p38␤ member of the MAP kinase family, and this was essential for E2 to act as a cell survival factor during hypoxia, to preserve EC morphology after metabolic insult, and to stimulate EC migration and primitive capillary formation (33). Here, we show that MMP inhibition or EGFR tyrosine kinase inhibition significantly prevents E2 signaling to the activation of p38␤ (Fig. 7B). Thus, additional signal transduction pathways are rapidly triggered by E2 in several cell types, and these pathways require EGFR transactivation via the linked events we show here.
E Domain Activation of Signaling-What structural aspect of the membrane ER is necessary for activation of the signal cascade that results in EGFR activation and ERK up-regulation? This is an important issue, and assuming that the membrane and nuclear proteins are the same (6), there is no typical catalytic or kinase domain sequence present in ER␣ or ER␤. It has recently been shown in CHO cells lacking endogenous ER that targeting of the E domain of ER␣ to the plasma membrane is sufficient to allow strong activation of ERK by E2 (5). This same, localized construct rescues HeLa cells from apoptotic cell death in response to etoposide (17), and in both situations, targeting of the E domain to the nucleus had no effect. We therefore asked whether targeting the E domain of ER␣ to the plasma membrane was sufficient to activate the signal pathway that we define here. This was accomplished in the HCC-1569 breast cancer cells that do not express ER. Targeting the E domain to the plasma membrane resulted in MMP activation (Fig. 8A) and EGFR activation (Fig. 8B), leading to ERK upregulation (5). In the absence of the expressed E domain, E2 was unable to stimulate any of these events. Targeting the E domain to the nucleus also did not result in activation of this pathway. Therefore, the E domain appears to be sufficient for the complex interactions at the plasma membrane that allow assembly and activation of the signalsome in response to E2.

DISCUSSION
The ability of E2 to signal from membrane ER is increasingly appreciated as being important to the effects of this sex steroid. E2 triggers calcium increases in seconds and rapidly activates PKC and adenylate cyclase. Downstream activation of several kinases then leads to cell biological effects in a variety of cell types (44). Ischemia reperfusion injury of muscle in rats is limited by E2, and this occurs through the physical association of ER with phosphatidylinositol 3-kinase, the subsequent upregulation of kinase activity, and the generation of NO (13). E2 acts as a survival factor for neurons (27), breast cancer (16,45), and osteoblasts (17) while suppressing osteoclast differentiation (14). The sex steroid also acts as a survival and angiogenesis-promoting factor for EC (33). These effects are related to the modulation of ERK, JNK, and p38 MAP kinases, regulated through membrane ER. Recently, it was shown that the administration of antibodies to ER␣ in nude mice blocked the growth of human breast cancer xenografts. This probably resulted from the antibodies inhibiting membrane ER signaling to ERK and phosphatidylinositol 3-kinase (46). Therefore, it is important to understand how E2 acts through membrane ER to trigger signal transduction. Now established for a variety of GPCRs, membrane ER localize substantially to caveolae (3)(4)(5). Here, they can physically complex with or activate signal molecules, including G proteins, receptor tyrosine kinases (insulin-like growth factor-1 receptor and EGFR), nonreceptor tyrosine kinases (Src family), and a variety of adapter and threonine/serine kinase proteins. This probably occurs on a scaffold platform provided by caveolin-1 (47,48) and in part related to tyrosine 14 phosphorylation of this protein (49). Interestingly, this tyrosine is the principal substrate for Src kinase action (49), and the ability of E2 to activate Src at the membrane (8,17) may therefore contribute to assembling the mature signalsome upon ER ligation by E2. Caveolin-1 can bind to and promote the assemblage of G proteins, Src, Grb7, Raf, Ras, MEK, and the EGFR at the plasma membrane (48). Caveolin-1 also facilitates ER translocation to the plasma membrane and localization within the caveolae microdomain (5). In this way, localizing ER and the signaling molecules to a confined area could augment the ability of E2/ER to transactivate EGFR, resulting in the stimulation of FIG. 5. G␣ subunit protein activation is required for E2-induced ERK activation in MCF-7 cells. A, expression of dominant negative mini-genes for G␣ q and G␣i but not G␣ s , G 12 , or G 13 prevents E2-induced ERK. The cells were transfected to express truncated G␣ subunits, serving as dominant negatives, then recovered, and exposed to E2 10 nM for 8 min. ERK activity was determined. Lanes 1 and 2 are nontransfected cells; lane 3 is E2-stimulated ERK after control plasmid transfection. The bar graph represents three experiments combined. *, p Ͻ 0.05 for control versus E2, or control versus E2-incubated, G␣ irtransfected cells. ϩ, p Ͻ 0.05 for E2-incubated, G␣ ir -transfected cells versus E2 in G␣ i or G␣ q mini-gene expressing cells. B, G␤␥ contributes to E2-induced ERK activation. The cells were transiently transfected to express a dominant negative, C-terminally truncated ␤-adrenergic receptor kinase (BARK1-CT), the cells were recovered for 24 h, and then ERK activation by E2 was determined. A representative experiment, repeated twice, is shown. ERK activity (this work and Ref. 25). However, upon GPCR ligation, caveolin probably dissociates from binding to the EGFR, leading to the activation of this receptor tyrosine kinase (50).
Although some details of the mechanisms of EGFR transactivation by ER (or any GPCR) are known, there are several aspects that are not clear. We found that the ability of membrane ER to activate G␣ q and G␣ i , but not the ␣ subunits of G s , G 12 , and G 13 , was important to the subsequent (but still rapid) signaling events upstream of EGFR activation (Fig. 9). G␤␥ inhibition also prevented the full transactivation of EGFR and ERK up-regulation in response to E2. This underscores the ideas that E2 activates several G proteins (6) and that there are specific functions for each but with some degree of redundancy. The partial redundancy we demonstrate may be related to the requirement that full signaling by E2 requires multiple G protein activations. Supporting this idea, we found that coexpressing dominant negative mini-genes for G␣ q and G␣ i (but not co-expression with G␣S) added to the inhibition of E2/ER signaling to MMP activation and EGFR activation, compared with the inhibition of single G␣ subunits. 2 This may be related to the necessity for complete activation of Src and Src-induced signaling to MMP activation (see below).
FIG. 6. ER is necessary for E2-induced proximal signaling. A, specific G protein subunits are required for ER-induced ERK. HCC-1569 cells were co-transfected to express ER␣ or pcDNA3 and truncated G␣ subunits or the C-terminal truncated ␤-adrenergic receptor kinase (BARK1-CT). The cells were recovered and then incubated with 10nME2 for 10 min, and ERK activity was determined. The bar graph represents two experiments combined. B, HB-EGF secretion in response to E2. ER␣ or pcDNA3 was expressed, and the cells were incubated with E2 with or without MMPI or ICI182780 for 3 min. Electrophoresed proteins were then subjected to Western blot. The bar graph is three experiments combined. C, MMPI activation by E2 requires ER. ER␣-transfected HCC-1569 cells were incubated with E2 for 2 min, and the cell lysate was used to determine MMP activity by spectroflurometry. EDTA is a calcium chelator, U-73122 is a PLC inhibitor, and calphostin C is a PKC inhibitor.
We also found that MMP-2 and MMP-9 were necessary for E2 to stimulate the secretion of HB-EGF and the transactivation of EGFR. First, E2 activated these two enzymes, as determined by gelatin zymography and substrate cleavage studies. However, E2 did not up-regulate MMP-3 and MMP-13 activity, thus showing the specificity of our results. Second, E2 induced the release of HB-EGF into the cell culture medium after only 3 min of incubation, and this was substantially prevented by the specific antisense (but not missense) constructs for MMP-2 and MMP-9, with the effects being additive. Shedding of HB-EGF is a complicated process, and involvement of Ras-Raf-Mek (51), Rac (52), or PKC␦ and the metalloprotease-disintegrin, MDC9 (37), has been proposed. In some cellular contexts, unknown metalloproteinase(s) mediates this shedding (53). Recently, TACE/ADAM17 has been shown to cleave expressed HB-EGF at 24 h in fibroblasts (54). However, our results suggest that MMP-2 and MMP-9 are sufficient. cipitated from the cell lysate(s) was used for in vitro kinase activity assays, with ATF-1 as substrate. The bar graphs are from three experiments combined.
FIG. 7. Additional signaling pathways that depend upon ER to EGFR cross-talk. A, E2-induces AKT activation in MCF-7 cells, dependent upon MMP activation and EGFR tyrosine kinase function. MCF-7 cells were incubated with E2 with or without GM6001 or tyrphostin AG1478 for 10 min, and AKT phosphorylation at serine 473 was determined. B, p38␤ activation in endothelial cells by E2 is significantly prevented by inhibition of MMP activity or the EGFR tyrosine kinase. EC were incubated with 10 nM E2 for 15 min, and the p38 immunopre-  2) or to the plasma membrane (lanes 3 and 4), followed by 2 min of treatment with E2 and determination of MMP activity. A representative study is shown, repeated once. B, expressing the E domain in the membrane leads to the transactivation of EGFR by E2. The transfected cells were assayed for EGFR phosphorylation by Western Blot, using an antibody against tyrosine 1138 (lane 3 versus lane 4). EGF-induced transactivation of its receptor serves as a positive control. The study was repeated twice.
The specific signal from GPCRs that leads to MMP activation is not well understood. In this regard, we report the novel finding that Src is necessary for E2 to activate MMP-2 and MMP-9 and subsequent HB-EGF shedding. A previous study implicated Src as upstream from HB-EGF, but its role was undetermined (29). The precise mechanisms by which Src accomplishes MMP secretion and activation is unknown but is under investigation. It should be appreciated that this kinase is also downstream of EGFR, either through Src binding this receptor or through potential cross-talk of EGFR to G protein-coupled receptors, leading to Src activation (55,56).
Our novel identification of MMP-2 and MMP-9 secretion and activation as being involved in estrogen signaling underlies the overall contribution of these MMPs to breast cancer biology. MMP-2 and MMP-9 have been implicated in the aggressive behavior of breast cancer (57,58). The ability of breast cancer cells to migrate or invade/metastasize is importantly dependent on the degradation of cell matrix by MMPs. However, these proteases also play additional important roles to mediate cell survival, differentiation, and angiogenesis (reviewed in Ref. 59). Recently, MMP-2 production in response to E2 was found to be dependent on ERK signaling to the up-regulated activity of the AP-2 transcription factor in mesangial cells (60). In our model, MMP activation is necessary for E2 to stimulate HB-EGF secretion into the culture media, and HB-EGF but not TGF␣ transactivates the EGFR to signal to ERK.
The ability of EGFR to underlie E2/ER-induced ERK may represent only a single example of the wider signaling interactions of these two growth modulatory systems. We therefore asked whether other important signaling pathways that originate from membrane ER are also dependent on EGFR. We report here that E2 activates protein kinase B in breast cancer cells and p38␤ MAP kinase in EC and that both pathways are also dependent upon transactivation of the EGFR. Utilization of EGFR to activate ERK is relatively common for a variety of GPCRs (38,61); however, GPCRs can also activate ERK by pathways apart from EGFR (62). In this respect, we previously showed that E2 stimulates G␣ s and cAMP, as well as ERK in CHO cells that are transiently transfected to express ER but that lack endogenous EGFR (6). Other EGF receptor family members might facilitate GPCR signaling to distinct pathways and thereby contribute to the specificity of signaling. For instance, the ability of muscarinic receptors to activate AKT is dependent upon the transactivation of the ErbB3 member of the EGFR family (63). In other situations, platelet-derived growth factor or insulin-like growth factor-1 receptors may be necessary for GPCR effects (64 -66). Thus, the tyrosine kinase receptor milieu in a particular cell may specifically control the panoply of signaling typically enacted by a GPCR ligand.
These interactions extend to cross-talk in both directions, including from the growth factor receptor tyrosine kinase to ER (67). Insulin-like growth factor-1 and EGF can signal to transcription via ER, independently of E2 (68,69). This occurs through growth factor receptor-induced phosphorylation of the nuclear sex steroid receptor (70) or co-accessory proteins (71), and the induction of several kinase cascades is important in this regard (70,72). These complex interactions are important in that they may contribute to the ability of breast cancer cells to proliferate or survive via ER, even when circulating levels of E2 are low, as in the post-menopausal woman.
An additional important issue is whether ER is required for E2 to activate signaling pathways from the membrane. It has previously been shown that E2 can transactivate the EGFR and signal in breast cancer cells that do not express ER (24). This purportedly occurs through a nondefined interaction with the orphan GPCR, GPR 30, and can nonspecifically be activated by estrogen receptor antagonists (ICI182780) and relatively inactive steroisomers (17-␣-E2). We report here that in the absence of ER, E2 can not activate ERK in HCC-1569 cells that lack this receptor. Expressing ER (or the E domain targeted to the cell membrane) allows E2 to signal through specific G proteins, MMP activation, and HB-EGF secretion that activates EGFR. Although some cells have been reported to respond rapidly to E2 in a nontraditional ER-related or ERindependent fashion (10,73), the mechanisms underlying these reports remain unknown. Furthermore, the majority of studies indicate the requirement of ER for E2 action (5,6,8,20,27,28), and these studies identify typical receptor pharmacology for the nongenomic actions of this sex steroid (reviewed in Ref. 43).
What part of ER is necessary for signal transduction at the membrane? Tyrosine 537 in the AF-2 portion of the E domain is essential for the interaction of ER with Src and functional up-regulation of ERK in MCF-7 cells (8). Recently, an interaction between the AF-1 domain of ER␣ and the phosphotyrosine binding and SH2 domains of the adapter protein Shc was postulated to mediate ERK activation in MCF-7 cells (20). However, Razandi et al. (5) recently showed that targeting the E domain of ER␣ to the plasma membrane of CHO cells is sufficient for robust ERK activation by E2, and Kousteni et al. (17) showed that this was sufficient to rescue HeLa cells from apoptosis. Here, we show that targeting the E domain to the membrane (and not to the nucleus) of HCC-1569 cells results in MMP-2 activation, EGFR transactivation, and ERK up-regulation. Thus, it is not clear what the significance of the AF-1 region of ER might be for signaling from the membrane. We propose that elements in the E domain, such as AF-2, allows for the complex interactions with G proteins, caveolin, Src, and other signaling molecules.
In summary, E2 activation of ERK is dependent on several G␣ and G␤␥ subunits of small GTP-binding proteins. Src-dependent stimulation of MMP-2 and MMP-9 activity in response to E2/ER releases HB-EGF, leading to EGFR transactivation, and signaling to MAP kinase. The E domain of ER␣ appears to be sufficient to activate these mechanisms. The assemblage of signal transduction complexes probably platformed on caveolin or growth factor receptor tyrosine kinase proteins (EGFR and insulin-like growth factor receptor) accounts for much of the ability of E2 to signal through membrane-localized ER to different pathways. This mechanism is increasingly appreciated to play important roles in the cellular biology of E2 actions, and manipulation of these