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 Gαq and Gαi blocked E2-induced, EGFR-dependent ERK activation, and Gβγ 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 antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38β MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERα to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer. 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 Gαq and Gαi blocked E2-induced, EGFR-dependent ERK activation, and Gβγ 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 antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38β MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERα to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer. estradiol estrogen receptor(s) epidermal growth factor EGF receptor(s) G protein-coupled receptor(s) matrix metalloproteinase(s) phospholipase C protein kinase C heparin-binding epidermal growth factor transforming growth factor endothelial cell(s) Chinese hamster ovary antisense oligonucleotide(s) scrambled antisense oligonucleotide(s) 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 (1Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2699) Google Scholar). 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Endocrinology. 1999; 140: 3805-3814Crossref PubMed Scopus (159) Google Scholar). In CHO cells, expression of a single cDNA for either ERα or ERβ produces both membrane and nuclear receptor populations and results in E2 activation of signal transduction from the membrane (6Razandi M. Pedram A. Greene G.L. Levin E.R. Mol. Endocrinol. 1999; 13: 307-319Crossref PubMed Scopus (1008) Google Scholar). In many cell types, endogenous membrane ER have been identified (15Morey A.K. Pedram A. Razandi M. Prins B.A., Hu, R.-M. Biesiada E. Levin E.R. Endocrinology. 1997; 138: 3330-3339Crossref PubMed Scopus (144) Google Scholar, 18Pappas T.C. Gametchu B. Yannariello-Brown J. Collins T.J. Watson C.S. FASEB J. 1995; 9: 404-410Crossref PubMed Scopus (477) Google Scholar, 20Russell K.S. Haynes M.P. Sinha D. Clerisme E. Bender J.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5930-5935Crossref PubMed Scopus (338) Google Scholar) 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 (5Razandi M., Oh, P. Pedram A. Schnitzer J. Levin E.R. Mol. Endocrinol. 2002; 16: 100-115Crossref PubMed Scopus (298) Google Scholar), leading to cell survival (17Kousteni S. Bellido T. Plotkin L.I. O'Brien C.A. Bodenner D.L. Han L. Han K. DiGregorio G.B. Katzenellenbogen J.A. Katzenellenbogen B.S. Roberson P.K. Weinstein R.S. Jilka R.L. Manolagas S.C. Cell. 2001; 104: 719-730Abstract Full Text Full Text PDF PubMed Google Scholar). Additionally, the AF-1 domain of ERα has been identified to interact with the adapter protein, Shc, in whole cell homogenates (21Song R.X. McPherson R.A. Adam L. Bao Y. Shupnik M. Kumar R. Santen R.J. Mol. Endocrinol. 2002; 16: 116-127Crossref PubMed Scopus (386) Google Scholar). 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 ERα or ERβ co-precipitates with and activates Gαs and Gαq proteins (6Razandi M. Pedram A. Greene G.L. Levin E.R. Mol. Endocrinol. 1999; 13: 307-319Crossref PubMed Scopus (1008) Google Scholar). 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 (22Aronica S.M. Kraus W.L. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8517-8522Crossref PubMed Scopus (609) Google Scholar,23Lieberherr M. Grosse B. Kachkache M. Balsan S. J. Bone Min. Res. 1993; 8: 1365-1376Crossref PubMed Scopus (152) Google Scholar). In EC, endogenous membrane ER physically associates with Gαi and activate endothelial nitric-oxide synthase; this probably takes place within caveolae (7Wyckoff M.H. Chambliss K.L. Mineo C. Yuhanna I.S. Mendelsohn M.E. Mumby S.M. Shaul P.W. J. Biol. Chem. 2001; 276: 27071-27076Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 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 (24Filardo E.J. Quinn J.A. Frackelton A.R., Jr. Bland K.I. Mol. Endocrinol. 2002; 16: 70-84Crossref PubMed Scopus (563) Google Scholar, 25Filardo E.J. Quinn J.A. Bland K.I. Frackelton A.R. Mol. Endocrinol. 2000; 14: 1649-1660Crossref PubMed Google Scholar). This occurs through the activation of Gβγ, 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 (26Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). 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 (25Filardo E.J. Quinn J.A. Bland K.I. Frackelton A.R. Mol. Endocrinol. 2000; 14: 1649-1660Crossref PubMed Google Scholar). 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 (5Razandi M., Oh, P. Pedram A. Schnitzer J. Levin E.R. Mol. Endocrinol. 2002; 16: 100-115Crossref PubMed Scopus (298) Google Scholar, 6Razandi M. Pedram A. Greene G.L. Levin E.R. Mol. Endocrinol. 1999; 13: 307-319Crossref PubMed Scopus (1008) Google Scholar, 8Migliaccio A. Castoria G., Di Domenico M. de Falco A. Bilancio A. Lombardi M. Vitorria Barone M. Ametrano D. Zannini M.S. Abbondanza C. Bontempo P. Auricchio F. EMBO J. 2000; 19: 5406-5417Crossref PubMed Google Scholar, 20Russell K.S. Haynes M.P. Sinha D. Clerisme E. Bender J.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5930-5935Crossref PubMed Scopus (338) Google Scholar, 27Singer C.A. Figueroa-Masot X.A. Batchelor R.H. Dorsa D.M. J. Neurosci. 1999; 19: 2455-2463Crossref PubMed Google Scholar, 28Zhu Y. Bian Z., Lu, P. Karas R.H. Bao L. Cox D. Hodgin J. Shaul P.W. Thoren P. Smithies O. Gustafsson J.A. Mendelsohn M.E. Science. 2002; 295: 505-508Crossref PubMed Scopus (420) Google Scholar). 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 (29Pierce K.L. Tohgo A. Ahn S. Field M.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2001; 22: 23155-23160Abstract Full Text Full Text PDF Scopus (204) Google Scholar), 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. 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 (30Pedram A. Razandi M. Levin E.R. J. Biol. Chem. 1998; 273: 26722-26728Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). 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). For ERK or p38β 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 (30Pedram A. Razandi M. Levin E.R. J. Biol. Chem. 1998; 273: 26722-26728Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 33Razandi M. Pedram A. Levin E.R. J. Biol. Chem. 2000; 275: 38540-38546Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). 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. 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 μg (each well of 6-well plates) or 10 μg of fusion plasmid DNA (100-mm dishes). Plasmids included wild type mouse ERα (31Couse J.F. Curtis S.W. Washburn T.F. Lindzey J. Golding T.S. Lubahn D.B. Smithies O. Korach K.S. Mol. Endocrinol. 1995; 9: 1441-1454Crossref PubMed Google Scholar) (kindly provided by Dr. Ken Korach) PRK5-HER, a dominant negative EGF receptor construct (kindly provided by Dr. A. Ullrich (32Redemann A. Holzmann B. von Ruden T. Wagner E.F. Schlessinger J. Ullrich A. Mol. Cell. Biol. 1992; 12: 491-498Crossref PubMed Scopus (120) Google Scholar), a dominant negative Src construct, pRC-csrc-K298M (kindly provided by Drs. Louis Luttrell and Robert Lefkowitz (26Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar), a dominant negative, truncated β-adrenergic receptor kinase plasmid (BARK1-CT pRK5) from Dr. Walter Koch (34Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar), and truncated Gα subunit plasmids, serving as specific dominant negative constructs for Gs, Gq, Gi, G12, and G13 (35Gilchrist A., Li, A. Hamm H.E. Methods Enzymol. 2002; 344: 58-69Crossref PubMed Scopus (21) Google Scholar). 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. 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 CO2incubator. 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 0.05m Tris, pH 7.5, 5 mm CaCl2, 0.02% NaN3). 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 mmTris, pH 7.5, 100 nm NaCl2) containing 5 μm of the Mca-Pro-Leu-Dpa-Ala-Arg-NH2substrate 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, CAGGGGCTGCCAGAGGCTCAT, 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. 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-β-E2 concentrations, ICI 182780 (1 μm), 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). 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. 1 A, lanes 1 and 2). When ERα was expressed in these cells, 17-β-E2 (lane 4), but not 17-α-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 (6Razandi M. Pedram A. Greene G.L. Levin E.R. Mol. Endocrinol. 1999; 13: 307-319Crossref PubMed Scopus (1008) Google Scholar). 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. 1 B, left andcenter panels). Importantly, tyrphostin AG1478 also substantially prevented the ability of E2 to activate ERK in the three cell types (Fig. 1 B, all panels, lanes 2 versus lanes 6). To corroborate this finding, we expressed a dominant negative EGFR (31Couse J.F. Curtis S.W. Washburn T.F. Lindzey J. Golding T.S. Lubahn D.B. Smithies O. Korach K.S. Mol. Endocrinol. 1995; 9: 1441-1454Crossref PubMed Google Scholar) in MCF-7 cells, and E2 was much less effective in stimulating this MAP kinase, compared with cells expressing the empty vector (control) (Fig. 1 C). 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 (36Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1499) Google Scholar). To examine this, we first determined whether E2 could stimulate the secretion of HB-EGF, determined by Western blot. As seen in Fig. 2 A, 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. 2 B). In contrast, antibody to TGFα-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 TGFα-1) prevented E2-induced phosphorylation of the EGFR (Fig. 2 C). 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. 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 (36Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1499) Google Scholar, 37Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar). 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. 3 A). 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. 3 B, first andsecond 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. 3 C,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. 4 A,left panel). Similarly, we validated the function of the MMP-2 or MMP-9 to specifically inhibit only the intended protein target (Fig. 4 A, 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 down-regulated 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, an