Modulation of Estrogen Receptor α Function and Stability by Tamoxifen and a Critical Amino Acid (Asp-538) in Helix 12
2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês
10.1074/jbc.m211129200
ISSN1083-351X
AutoresSandra Timm Pearce, Hong Liu, V. Craig Jordan,
Tópico(s)Ubiquitin and proteasome pathways
ResumoEstrogen receptor α (ER) is a ligand-activated transcription factor implicated in breast cancer growth. Selective estrogen receptor modulators (SERMs), such as tamoxifen (4-OHT), bind to the ER and affect the position of helix 12, thereby influencing coregulator binding and ER transcriptional activation. Previous studies have shown that a triple mutation in helix 12 (3m; D538A/E542A/D545A) caused a change in ER stability and obliterated 4-OHT action (Liu, H., Lee, E. S., de los Reyes, A., Zapf, J. W., and Jordan, V. C. (2001) Cancer Res. 61, 3632–3639). Two approaches were taken to determine the role of individual mutants (D538A, L540Q, E542A, and D545A) on the activity and stability of the 4-OHT·ER complex. First, mutants were evaluated using transient transfection into ER-negative T47D:C4:2 cells with an ERE3-luciferase reporter, and second, transforming growth factor α (TGFα) mRNA was used as a gene target in situ for stable transfectants of MDA-MB-231 cells. Transcriptional activity occurred in the presence of estrogen in all of the mutants, although a decreased response was observed in the L540Q, 3m, and D538A cells. The 3m and D538A mutants lacked any estrogenic responsiveness to 4-OHT, whereas the other mutations retained estrogen-like activity with 4-OHT. Unlike the other mutants, the ER was degraded in the D538A mutant with 4-OHT treatment. However, increasing the protein levels of the mutant with the proteasome inhibitor MG132 did not restore the ability of 4-OHT to induce TGFα mRNA. We suggest that Asp-538 is a critical amino acid in helix 12 that not only reduces the estrogen-like actions of 4-OHT but also facilitates the degradation of the 4-OHT·D538A complex. These data further illustrate the complex role of specific surface amino acids in the modulation of the concentration and the estrogenicity of the 4-OHT·ER complex. Estrogen receptor α (ER) is a ligand-activated transcription factor implicated in breast cancer growth. Selective estrogen receptor modulators (SERMs), such as tamoxifen (4-OHT), bind to the ER and affect the position of helix 12, thereby influencing coregulator binding and ER transcriptional activation. Previous studies have shown that a triple mutation in helix 12 (3m; D538A/E542A/D545A) caused a change in ER stability and obliterated 4-OHT action (Liu, H., Lee, E. S., de los Reyes, A., Zapf, J. W., and Jordan, V. C. (2001) Cancer Res. 61, 3632–3639). Two approaches were taken to determine the role of individual mutants (D538A, L540Q, E542A, and D545A) on the activity and stability of the 4-OHT·ER complex. First, mutants were evaluated using transient transfection into ER-negative T47D:C4:2 cells with an ERE3-luciferase reporter, and second, transforming growth factor α (TGFα) mRNA was used as a gene target in situ for stable transfectants of MDA-MB-231 cells. Transcriptional activity occurred in the presence of estrogen in all of the mutants, although a decreased response was observed in the L540Q, 3m, and D538A cells. The 3m and D538A mutants lacked any estrogenic responsiveness to 4-OHT, whereas the other mutations retained estrogen-like activity with 4-OHT. Unlike the other mutants, the ER was degraded in the D538A mutant with 4-OHT treatment. However, increasing the protein levels of the mutant with the proteasome inhibitor MG132 did not restore the ability of 4-OHT to induce TGFα mRNA. We suggest that Asp-538 is a critical amino acid in helix 12 that not only reduces the estrogen-like actions of 4-OHT but also facilitates the degradation of the 4-OHT·D538A complex. These data further illustrate the complex role of specific surface amino acids in the modulation of the concentration and the estrogenicity of the 4-OHT·ER complex. estrogen receptor α activation functions 1 and 2 ligand binding domain selective estrogen receptor modulator estrogen diethylstilbestrol 4-hydroxytamoxifen transforming growth factor α raloxifene phosphate-buffered saline bovine serum albumin estrogen response element Estrogen receptor α (ER)1 is a member of the steroid hormone superfamily of nuclear receptors, which are gene regulatory transcription factors. Similar structural domains, designated A–F, are shared between the nuclear receptors (for review, see Refs. 1MacGregor J.I. Jordan V.C. Pharmacol. Rev. 1998; 50: 151-196PubMed Google Scholar and 2Nilsson S. Makela S. Treuter E. Tujague M. Thomsen J. Andersson G. Enmark E. Pettersson K. Warner M. Gustafsson J.A. Physiol. Rev. 2001; 81: 1535-1565Crossref PubMed Scopus (1592) Google Scholar). Two transcriptional activation functions, activation function 1 (AF1) and activation function 2 (AF2), are present in the ER (see Fig. 1). AF1 is a constitutive activation function located in the A/B region, and AF2 is a ligand-dependent activation function in the E region or ligand binding domain (LBD). The activity of AF1 and AF2 is largely mediated by the cell and promoter context (3Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (889) Google Scholar, 4Tzukerman M.T. Esty A. Santiso-Mere D. Danielian P. Parker M.G. Stein R.B. Pike J.W. McDonnell D.P. Mol. Endocrinol. 1994; 8: 21-30Crossref PubMed Scopus (612) Google Scholar) and can be independent or synergistic (5Kraus W.L. McInerney E.M. Katzenellenbogen B.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12314-12318Crossref PubMed Scopus (183) Google Scholar). The ER is an important therapeutic target for the treatment and prevention of breast cancer. Selective estrogen receptor modulators (SERMs) are compounds that bind to the ER and exert tissue-specific effects. Tamoxifen was the first SERM approved clinically for the treatment and prevention of breast cancer. Tamoxifen acts as an antiestrogen in the breast but has estrogenic properties in that it maintains bone density (6Love R.R. Mazess R.B. Barden H.S. Epstein S. Newcomb P.A. Jordan V.C. Carbone P.P. DeMets D.L. N. Engl. J. Med. 1992; 326: 852-856Crossref PubMed Scopus (1007) Google Scholar), lowers circulating cholesterol (7Love R.R. Wiebe D.A. Newcomb P.A. Cameron L. Leventhal H. Jordan V.C. Feyzi J. DeMets D.L. Ann. Intern. Med. 1991; 115: 860-864Crossref PubMed Scopus (342) Google Scholar, 8Fisher B. Costantino J.P. Wickerham D.L. Redmond C.K. Kavanah M. Cronin W.M. Vogel V. Robidoux A. Dimitrov N. Atkins J. Daly M. Wieand S. Tan-Chiu E. Ford L. Wolmark N. J. Natl. Cancer Inst. 1998; 90: 1371-1388Crossref PubMed Scopus (4834) Google Scholar) and causes an increased risk of endometrial cancer in women over 50 (8). Raloxifene is a chemically related SERM that is used for the prevention of osteoporosis but also lowers cholesterol and reduces the risk of both breast cancer and endometrial cancer (9Cummings S.R. Eckert S. Krueger K.A. Grady D. Powles T.J. Cauley J.A. Norton L. Nickelsen T. Bjarnason N.H. Morrow M. Lippman M.E. Black D. Glusman J.E. Costa A. Jordan V.C. JAMA. 1999; 281: 2189-2197Crossref PubMed Scopus (1848) Google Scholar). ICI 182,780 is considered to be a pure antiestrogen in that it displays no agonist activity at the ER (10Wakeling A.E. Dukes M. Bowler J. Cancer Res. 1991; 51: 3867-3873PubMed Google Scholar). This occurs because ICI 182,780 interferes with receptor dimerization (11Fawell S.E. White R. Hoare S. Sydenham M. Page M. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6883-6887Crossref PubMed Scopus (345) Google Scholar) and increases ER protein turnover (12Dauvois S. Danielian P.S. White R. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4037-4041Crossref PubMed Scopus (431) Google Scholar). Analysis of the crystal structure of the ligand·ER complex has been instrumental in understanding ER conformation at the molecular level and highlights the importance of helix 12 in modulating estrogenic and antiestrogenic actions. Helix 12 is located in the LBD of the ER, but the composition and orientation of helix 12 differs depending on the ligand bound to the ER (13Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar). When the ER LBD is complexed with the ER agonists estrogen (E2) or diethylstilbestrol (DES), helix 12 is positioned over the ligand binding pocket (see Fig.2 A) (13Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar, 14Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2949) Google Scholar). This proper positioning generates AF2 and forms a surface for the recruitment of coactivators. However, when 4-hydroxytamoxifen (4-OHT, the active metabolite of tamoxifen) or raloxifene is bound to the ER LBD, the antiestrogenic side chain displaces helix 12 from its normal position, thereby preventing the formation of a functional AF2 (Fig. 2 B) (13Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2254) Google Scholar, 14Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2949) Google Scholar). Having excluded AF2, the reported partial agonist activity of 4-OHT can only be mediated by AF1 (15Berry M. Metzger D. Chambon P. EMBO J. 1990; 9: 2811-2818Crossref PubMed Scopus (664) Google Scholar). In a previous study, a binding site responsible for the estrogen-like action of 4-OHT was defined that is referred to as AF2b (16MacGregor Schafer J. Liu H. Bentrem D.J. Zapf J.W. Jordan V.C. Cancer Res. 2000; 60: 5097-5105PubMed Google Scholar). This site contains two critical components: Asp-351 and a portion of helix 12 (Asp-538, Glu-542, and Asp-545). AF2b is proposed to be a docking site for coactivators or corepressors that modulate the estrogenicity of the 4-OHT or raloxifene ER complex (17Yamamoto Y. Wada O. Suzawa M. Yogiashi Y. Yano T. Kato S. Yanagisawa J. J. Biol. Chem. 2001; 276: 42684-42691Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Liu H. Lee E.S. de los Reyes A. Zapf J.W. Jordan V.C. Cancer Res. 2001; 61: 3632-3639PubMed Google Scholar, 19Liu H. Park W.C. Bentrem D.J. McKian K.P. de los Reyes A. Loweth J.A. Schafer J.M. Zapf J.W. Jordan V.C. J. Biol. Chem. 2002; 277: 9189-9198Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Therefore, different ligands induce different receptor conformations, and the positioning of helix 12 is the key event that permits discrimination between ER agonists and antagonists by influencing the interaction of the ER with coregulators. The estrogenic or antiestrogenic action of ligands at the ER depends on the subtle changes in ER shape that programs the ER to form an active or inactive transcription complex or to be degraded by the proteasome. The amount of available ER in the cell is controlled by a balance between synthesis and degradation. ER stability is influenced by the nature of the bound ligand such that ligand-induced conformational changes modulate the ability of the ER to interact with proteins involved in the degradation process (20Preisler-Mashek M.T. Solodin N. Stark B.L. Tyriver M.K. Alarid E.T. Am. J. Physiol. 2002; 282: E891-E898Crossref PubMed Scopus (80) Google Scholar). The transcriptional activity of the resulting ER pool is also influenced by the ligands present. The ER is activated if the ligand is estrogenic, and the established estrogens can be classified as class I or class II (21Jordan V.C. Schafer J.M. Levenson A.S. Liu H. Pease K.M. Simons L.A. Zapf J.W. Cancer Res. 2001; 61: 6619-6623PubMed Google Scholar). Class I estrogens, such as DES or E2, are planar compounds that use the AF2 site for optimal action. Class II estrogens, represented by angular triphenylethylene compounds such as 4-OHT and fixed ring 4-hydroxy triphenyl pentene, utilize AF2b for activity. However, ligands such as SERMs or pure antiestrogens can block the activity of the ER by creating a ligand·ER complex that is inactive. Overall, the complex decision-making network depends upon the protein recognition sequences exposed on the external surface of the relevant SERM·ER complex in response to ligand binding. Analysis of the helix 12 region of AF2b using the 3m mutation (D538A/E542A/D545A) yielded important insight into mechanism of 4-OHT agonism. The transforming growth factor α (TGFα) gene is recognized as a target of estrogen action and is involved in cell growth stimulation by estrogen (22Bates S.E. Davidson N.E. Valverius E.M. Freter C.E. Dickson R.B. Tam J.P. Kudlow J.E. Lippman M.E. Salomon D.S. Mol. Endocrinol. 1988; 2: 543-555Crossref PubMed Scopus (395) Google Scholar, 23Lee D.C. Fenton S.E. Berkowitz E.A. Hissong M.A. Pharmacol. Rev. 1995; 47: 51-85PubMed Google Scholar), so the biological activity of the 4-OHT·ER complex can be assessed using Northern blotting for TGFα mRNA. Expression of TGFα mRNA is normally induced by E2 and 4-OHT treatment in MDA-MB-231 human breast cancer cells stably transfected with the wild type ER (S30 cells) (24Jiang S.Y. Jordan V.C. J. Natl. Cancer Inst. 1992; 84: 580-591Crossref PubMed Scopus (243) Google Scholar). The 3m mutation resulted in a decreased induction of TGFα in response to E2 and no response to 4-OHT (18Liu H. Lee E.S. de los Reyes A. Zapf J.W. Jordan V.C. Cancer Res. 2001; 61: 3632-3639PubMed Google Scholar). Therefore, the 3m mutation abolished the agonist activity of 4-OHT and decreased the agonist activity of E2. In addition, a slight degradation of the ER was observed when the 3m mutant stable cell line was treated with E2, 4-OHT, and ICI (18Liu H. Lee E.S. de los Reyes A. Zapf J.W. Jordan V.C. Cancer Res. 2001; 61: 3632-3639PubMed Google Scholar). This is in contrast to stable cell lines containing the wild type ER, which displayed a large down-regulation of the ER in the presence of E2 and ICI, but an increase in ER protein with 4-OHT treatment. Although the effect of E2 on the 3m mutation and the three individual amino acids comprising the 3m mutation has been studied using ERE-luciferase assays (4Tzukerman M.T. Esty A. Santiso-Mere D. Danielian P. Parker M.G. Stein R.B. Pike J.W. McDonnell D.P. Mol. Endocrinol. 1994; 8: 21-30Crossref PubMed Scopus (612) Google Scholar, 25Danielian P.S. White R. Lees J.A. Parker M.G. EMBO J. 1992; 11: 1025-1033Crossref PubMed Scopus (721) Google Scholar, 26Mahfoudi A. Roulet E. Dauvois S. Parker M.G. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4206-4210Crossref PubMed Scopus (113) Google Scholar, 27Montano M.M. Ekena K. Krueger K.D. Keller A.L. Katzenellenbogen B.S. Mol. Endocrinol. 1996; 10: 230-242Crossref PubMed Scopus (85) Google Scholar), the majority of the studies were not performed in breast cancer cell lines and in a comprehensive manner. In addition, the precise interaction between 4-OHT and the individual mutations is not known. Amino acid L540 is a nearby amino acid of interest on the underside of helix 12 when it is sealing estrogen in the hydrophilic pocket of the LBD. The L540Q mutation was initially generated by random chemical mutagenesis and is a dominant negative ER mutant (28Ince B.A. Zhuang Y. Wrenn C.K. Shapiro D.J. Katzenellenbogen B.S. J. Biol. Chem. 1993; 268: 14026-14032Abstract Full Text PDF PubMed Google Scholar, 29Ince B.A. Montano M.M. Katzenellenbogen B.S. Mol. Endocrinol. 1994; 8: 1397-1406PubMed Google Scholar, 30Ince B.A. Schodin D.J. Shapiro D.J. Katzenellenbogen B.S. Endocrinology. 1995; 136: 3194-3199Crossref PubMed Google Scholar, 31Schodin D.J. Zhuang Y. Shapiro D.J. Katzenellenbogen B.S. J. Biol. Chem. 1995; 270: 31163-31171Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Previous studies in MDA-MB-231 breast cancer cells have shown that an ERE-CAT reporter is activated by 4-OHT and ICI 164,384, but not by E2, in the presence of the L540Q mutant (27Montano M.M. Ekena K. Krueger K.D. Keller A.L. Katzenellenbogen B.S. Mol. Endocrinol. 1996; 10: 230-242Crossref PubMed Scopus (85) Google Scholar). Therefore, the L540Q mutation reverses the pharmacology of E2 and ICI 182,780 that is normally observed at the wild type ER in MDA-MB-231 cells. We have stably transfected individual mutant ER cDNAs into MDA-MB-231 human breast cancer cells to create an in vitromodel to address the contribution of specific amino acids in helix 12 (D538A, E542A, D545A, and L540Q) to the agonist activity of 4-OHT at the AF2b site. We have found that Asp-538 is the critical amino acid in helix 12 that not only reduces the estrogen-like actions of 4-OHT but also enhances the degradation of the ER upon 4-OHT treatment. Stable cell lines were maintained in phenol red-free minimum essential media supplemented with 5% calf serum treated 3× with dextran-coated charcoal, 0.5 mg/ml G418 (Geneticin, Invitrogen, Carlsbad, CA), 2 mml-glutamine, 0.1 mm non-essential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin, and 6 μg/ml insulin. This media is referred to as stripped media, indicating that it is free of E2. S30 cells are MDA-MB-231 human breast cancer cells stably transfected with wild type ERα (24Jiang S.Y. Jordan V.C. J. Natl. Cancer Inst. 1992; 84: 580-591Crossref PubMed Scopus (243) Google Scholar) and are referred to as wild type cells. These cells are grown in stripped media. T47D:C4:2 cells are ERα-negative human breast cancer cells (32Pink J.J. Bilimoria M.M. Assikis J. Jordan V.C. Br. J. Cancer. 1996; 74: 1227-1236Crossref PubMed Scopus (70) Google Scholar) that were propagated in phenol red-free RPMI media containing 10% fetal serum calf serum treated 3× with dextran-coated charcoal, as well as the concentrations of amino acids, penicillin, streptomycin, and insulin described above. ER− represents a G418-resistant clone that is ER-negative. The 3m stable cell line (18Liu H. Lee E.S. de los Reyes A. Zapf J.W. Jordan V.C. Cancer Res. 2001; 61: 3632-3639PubMed Google Scholar) containing the triple mutation (D538A/E542A/D545A) was also grown in stripped media. 4-OHT and E2 were purchased from Sigma (St. Louis, MO). ICI 182,780 was obtained from AstraZeneca (Macclesfield, England). Raloxifene was a generous gift from Eli Lilly and Co. (Indianapolis, IN). All drugs were dissolved in ethanol and stored at −20 °C. MG132 was dissolved in Me2SO and obtained from Calbiochem(San Diego, CA). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit according to the manufacturer's instructions (Stratagene, La Jolla, CA). The ERαPSG5 plasmid (HEGO, kindly provided by P. Chambon) was used as a template for PCR. Primers used were as follows: D538A (5′-GCA TCT CCA GCA GCA GGG CAT AGA GGG GCA CCA CG-3′ and 5′-CGT GGT GCC CCT CTA TGC CCT GCT GCT GGA GAT GC-3′), L540Q (5′-GGC GTC CAG CAT CTC CAG CTG CAG GTC ATA GAG GGG-3′ and 5′-CCC CTC TAT GAC CTG CAG CTG GAG ATG CTG GAC GCC-3′), E542A (5′-GGG CGT CCA GCA TCG CCA GCA GCA GGT C-3′ and 5′-GAC CTG CTG CTG GCG ATG CTG GAC GCC C-3′), and D545A (5′-GTA GGC GGT GGG CGG CCA GCA TCT CCA GC-3′ and 5′-GCT GGA GAT GCT GGC CGC CCA CCG CCT AC-3′). Miniprep DNA was isolated from the resulting bacterial colonies using the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The Miniprep DNA was sequenced for the presence of the mutation. A larger scale DNA preparation was made from a chosen mutant using the Qiagen Maxiprep kit, and the entire ER DNA was sequenced. Each mutant was then cloned into the pIRESneo2 plasmid (Clontech, Palo Alto, CA) using theEcoRI site flanking the ER cDNA. MDA-MB-231 (clone 10A) cells (24Jiang S.Y. Jordan V.C. J. Natl. Cancer Inst. 1992; 84: 580-591Crossref PubMed Scopus (243) Google Scholar) were grown in stripped media for 3–4 days prior to transfection. The cells were transfected with 10 μg of the ER mutant in the pIRESneo2 plasmid. 5 × 106 cells were electroporated in a 0.4-cm cuvette (Bio-Rad, Hercules, CA) at a voltage of 0.250 kV and a high capacitance of 0.95 microfarad in phenol red-free minimal essential media with no additives. The cells were transferred to a 10-cm plate and incubated overnight in 10 ml of stripped media without G418, and the media was changed the next day. The following day, media containing 0.5 mg/ml G418 was added, and the cells were subsequently maintained in this media. Individual colonies appeared ∼1 month after transfection, and these were isolated and screened for stable expression of the ER by Western blotting. The cells were transfected with 1 μg of the ERE3-luciferase plasmid (33Catherino W.H. Jordan V.C. Cancer Lett. 1995; 92: 39-47Crossref PubMed Scopus (67) Google Scholar) and 1 μg of the mutant or wild type ERαPSG5 plasmid. To normalize for transfection efficiency, 0.2 μg of the PCMVβ plasmid (Clontech, Palo Alto, CA) were also transfected. 5 × 106 cells were electroporated in a 0.4-cm cuvette (Bio-Rad, Hercules, CA) at a voltage of 0.320 kV and a high capacitance of 0.95 microfarad in serum-free media. The cells were transferred to 12-well plates and incubated overnight. The next day, the cells were treated with the appropriate compound for 24 h. The cells were washed once with cold PBS, and 100 μl of extraction buffer (0.1 m potassium phosphate (pH 7.5), 1% Triton X-100, 100 μg/ml BSA, 2.5 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol) was added to each well. The cells were incubated on ice for 2 min, dislodged from the plates, and transferred to an Eppendorf tube. The lysate was centrifuged for 2 min at top speed in a microcentrifuge, and the supernatant was used for the assay. 50 μl of the lysate was mixed with 350 μl of reaction buffer (160 mm MgCl2, 75 mm glycylglycine (pH 7.8), 0.5 mg/ml BSA, 19 mg/ml ATP, and 15 mm Tris-HCl (pH 7.5)) and 100 μl of luciferin (0.4 mg/ml). Luminescence was measured in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) for 10 s. β-Galactosidase activity was measured using 10 μl of each sample and the Galacto-Light Plus detection system (Applied Biosystems, Bedford, MA). Data are reported as relative light units, which is the luciferase reading divided by the β-galactosidase reading. Stable transfectants were treated with compounds for 24 h. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. 20 μg of RNA was loaded per lane in a 1% agarose/0.66m formaldehyde gel. The RNA was transferred to a MagnaGraph nylon transfer membrane (Osmonics, Minnetonka, MN) overnight in 10× SSPE buffer (20× SSPE is 3.6 m NaCl/0.2 mNaH2PO4/0.02 m EDTA (pH 7.4)). The RNA was fixed to the membrane by UV-cross-linking. The membrane was prehybridized in hybridization solution (0.5 m sodium phosphate, 10 mm EDTA, 1% BSA, 7% SDS (pH 7.2)) for a minimum of 2 h at 60 °C. The TGFα probe (a gift from Dr. R. Derynck, Genentech, South San Francisco, CA) or the ERα probe (theEcoRI fragment from the ERαPSG5 plasmid) was labeled with [32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences, Piscataway, NJ), and the labeled probe was separated from free 32P using Microspin columns (AmershamBiosciences) according to the manufacturer's instructions. The probe was heated at 95 °C for 5 min, added to the hybridization buffer, and incubated at 60 °C overnight. The next day, the membrane was washed for 30 min at 60 °C with 1× SSPE/0.1% SDS, 30 min with 0.5× SSPE/0.05% SDS, and 2 × 15 min with 0.1× SSPE/0.1% SDS. To visualize TGFα, the membrane was exposed to film overnight. Equal loading of samples was verified by stripping the membrane and reprobing with β-actin. Cells were treated for 24 h with compound. To harvest protein, cells were washed once with PBS, scraped using a cell scraper into 10 ml of PBS, and transferred to a 15-ml conical tube. The cells were pelleted, and the supernatant was aspirated. The cell pellet was resuspended in 100 μl of extraction buffer (50 mm HEPES, 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 10% glycerol, 0.5% Nonidet P-40, 10 mm β-glycerophosphate (pH 8), containing a 1:100 dilution of a freshly added protease inhibitor mixture (Sigma P8340, St. Louis, MO)), passed through a 22-gauge needle, and incubated on ice for 30 min. The cell lysate was centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant was transferred to a new tube. Samples were quantitated using the Bio-Rad protein assay kit. 20 μg of cell lysate were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked in blotto (2.5% dry milk/0.05% Tween/0.5× PBS) for 1 h to overnight. Blots were probed with polyclonal ERα antibodies at 1:200 (G20, Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal β-actin antibodies at 1:20,000 (Sigma A5441, St. Louis, MO) for 1 h at room temperature. The membrane was then washed 3 × 5 min with wash buffer (0.5× PBS/0.05% Tween). The blot was incubated in a 1:3000 dilution of horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h. The membrane was washed 3 × 5 min with wash buffer, and bands were visualized using chemiluminescence (ECL, Amersham Biosciences, Piscataway, NJ). Western and Northern blots were quantitated using the gel plot feature in Scion Image version 4.0.2. The results were statistically analyzed using SPSS 9.0. Previous results in our laboratory showed that a triple mutation in helix 12 of the ER (3m, D538A/E542A/D545A) caused a change in ER stability and eliminated 4-OHT agonist activity (18Liu H. Lee E.S. de los Reyes A. Zapf J.W. Jordan V.C. Cancer Res. 2001; 61: 3632-3639PubMed Google Scholar). To analyze the role of individual amino acids in helix 12, multiple mutations were generated. These mutations include single point mutations of the 3m mutation (D538A/E542A/D545A) and the L540Q mutation (Figs. 1 and 2). The transcriptional activity of the ER mutants was first tested by transient transfection of ER-negative T47D:C4:2 cells with the mutant ER cDNA and an ERE3-luciferase reporter (33Catherino W.H. Jordan V.C. Cancer Lett. 1995; 92: 39-47Crossref PubMed Scopus (67) Google Scholar). Transfection with the empty vector PSG5 showed no induction of luciferase activity with any of the treatments used (Fig. 3). In addition, none of the mutants exhibited any response to treatment with the vehicle control ethanol (EtOH). E2 treatment resulted in a statistically significant induction of luciferase activity with the wild type ER and all of the mutants when compared with the EtOH control. The greatest induction was observed with the E542A mutant. The wild type, D538A, D545A, and 3m mutants displayed an intermediate level, and the L540Q mutant displayed the smallest induction. The L540Q mutant was the only mutant that showed a slight induction of luciferase activity during ICI 182,780 treatment, but this was not statistically significant. In addition, the wild type, E542A, and D545A ERs displayed an induction of luciferase activity upon 4-OHT treatment, whereas the D538A, L540Q, and 3m mutants did not. When wild type, 3m, and D538A cells were treated with E2 plus 4-OHT, the response of the cells was the same as that observed with 4-OHT alone, indicating that 4-OHT acts as a complete antiestrogen in these cells (data not shown). Therefore, of the three mutations present in the 3m mutant, the D538A mutation is responsible for the elimination of the agonist activity of 4-OHT at an ERE in T47D:C4:2 cells. To further evaluate these mutants in a reproducible manner, stable transfectants were generated in ER-negative MDA-MB-231 cells. At least five clones were obtained representing each mutation, and the clones were screened for the presence of the ER using Western blotting. Two clones harboring each mutation were initially screened using Northern blotting for TGFα mRNA levels. Both of the clones studied showed similar TGFα levels in response to various treatments, so a single representative clone was chosen for further analysis. Each of the stable clones was screened to ensure that the proper mutation was present using reverse transcription-PCR and sequencing. ER protein levels were compared between each of the stable cell lines (Fig. 4). All of the cell lines contained similar levels of ER protein, so the characteristics observed in each cell line were not a result of varying ER levels. A clone that was stably transfected but ER-negative by Western blot analysis was used as a control and designated ER−. The transcriptional activity of the ER mutants was also analyzed using Northern blot analysis of TGFα mRNA. The advantage of this assay is that the TGFα gene is an endogenous gene in MDA-MB-231 cells, and induction of TGFα mRNA levels reflects a process that is inherent to these cells. Cells from the ER− clone were treated with EtOH, E2, 4-OHT, and E2 plus 4-OHT, and no induction of TGFα mRNA was observed (data not shown). Wild type cells showed an induction of TGFα mRNA in response to E2 and 4-OHT, but 4-OHT did not act as an antiestrogen in these cells, because it was not able to significantly block the E2 response (Fig. 5). A similar pattern of mRNA expression was observed in the E542A and D545A mutants. Although the 3m and D538A mutants showed an increase in TGFα mRNA in response to E2 treatment, the level of induction was less than that observed for the other mutants. In addition, no induction occurred with the 4-OHT treatment. This is in agreement with ERE-luciferase assay results and suggests that Asp-538 is the single amino acid within the 3m mutation required for the agonist activity of 4-OHT at the ER. The L540Q mutant produced an induction of TGFα mRNA with ICI 182,780 and 4-OHT treatment but not with E2 treatment. In addition, raloxifene (Ral) treatment had no effect on TGFα mRNA level
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