Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor
1998; Springer Nature; Volume: 17; Issue: 7 Linguagem: Inglês
10.1093/emboj/17.7.2008
ISSN1460-2075
AutoresAntimo Migliaccio, Domenico Piccolo, Gabriella Castoria, Marina Di Domenico, Antonio Bilancio, Maria Lombardi, Wenrong Gong, Miguel Beato, Ferdinando Auricchio,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoArticle1 April 1998free access Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor Antimo Migliaccio Antimo Migliaccio Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Domenico Piccolo Domenico Piccolo Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Gabriella Castoria Gabriella Castoria Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Marina Di Domenico Marina Di Domenico Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Antonio Bilancio Antonio Bilancio Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Maria Lombardi Maria Lombardi Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Wenrong Gong Wenrong Gong Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Miguel Beato Miguel Beato Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Ferdinando Auricchio Corresponding Author Ferdinando Auricchio Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Antimo Migliaccio Antimo Migliaccio Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Domenico Piccolo Domenico Piccolo Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Gabriella Castoria Gabriella Castoria Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Marina Di Domenico Marina Di Domenico Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Antonio Bilancio Antonio Bilancio Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Maria Lombardi Maria Lombardi Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Wenrong Gong Wenrong Gong Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Miguel Beato Miguel Beato Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany Search for more papers by this author Ferdinando Auricchio Corresponding Author Ferdinando Auricchio Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy Search for more papers by this author Author Information Antimo Migliaccio1, Domenico Piccolo1, Gabriella Castoria1, Marina Di Domenico1, Antonio Bilancio1, Maria Lombardi1, Wenrong Gong2, Miguel Beato2 and Ferdinando Auricchio 1 1Istituto di Patologia Generale e Oncologia, Facoltà di Medicina e Chirurgia, II Università di Napoli, Largo S.Aniello a Caponapoli, 2, 80138 Napoli, Italy 2Institut für Molekularbiologie und Tumorforschung, Philipps-Universität Marburg, Emil-Mannkopff-Strasse 2, D-35037 Marburg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2008-2018https://doi.org/10.1093/emboj/17.7.2008 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21ras/Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PRB), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21ras/Erk pathway signals received from the agonist-activated PRB. These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells. Introduction Ovarian hormones stimulate cell proliferation in several female tissues (Clarke and Sutherland, 1990), in particular in the mammary gland, where estrogens and progesterone increase alveolar formation and ductal branching, and are required for full lobulo-alveolar development (Warner et al., 1978). A positive role for progestins in mammary gland cell proliferation has been observed in a variety of systems, including the mouse mammary gland (Bresciani, 1968), mammary tumors of rodents (Kiss et al., 1986; Manni et al., 1987), primary culture of human mammary epithelial cells (Longman and Buehring, 1987) and human breast cancer lines (Braunsberg et al., 1987; Hisson and Moore, 1987). In spite of this widely acknowledged importance of steroids as mitogens, the mechanisms by which they stimulate cell proliferation have not been deciphered yet. Regulation of growth factor production or stimulation by steroids of expression of genes required for cell division have been proposed (Weisz and Bresciani, 1993; Dickson and Lippman, 1995). Among the possible targets are the so-called early genes, such as c-fos and jun-B, but estrogens also induce c-myc expression, and this could be related to sustained growth (Weisz and Bresciani, 1993). In addition to early genes, cell cycle-controlling genes, such as cyclin D1, are also regulated by steroid hormones and could be targets of estrogens and progesterone (Musgrove et al., 1994). The activity of the G1 cyclins could also be influenced through an effect of ovarian hormones on cyclin-dependent kinase (cdk) inhibitors (Musgrove et al., 1995; Planas-Silva and Weinberg, 1997; Prall et al., 1997). Moreover, estrogens could also participate in proliferation in an indirect way by inducing the production of growth factors (Dickson and Lippman, 1995). Recently, participation of the Src/Ras/MAP kinase pathway in estrogen-induced growth has been postulated (Di Domenico et al., 1996; Migliaccio et al., 1996). Targeted disruption of the estrogen receptor α (ERα) gene clearly demonstrates a role for this receptor in the post-natal development of the uterus and the mammary gland, as homozygous ERα−/− female mice exhibit hypoplastic uteri and undeveloped mammary glands, with only vestigial ducts present at the nipples (Lubahn et al., 1993; Korach, 1994). On the contrary, PR−/− mice have normally developed uteri at puberty but respond to repeated administration of estrogens with a dramatic hyperplasia and inflammation of the uterus, suggesting that progesterone receptor (PR) is involved in controlling the proliferative response to estrogens and in preventing uterine inflammation (Lydon et al., 1995). Progesterone is the actual proliferative hormone in the post-puberty growth of the mammary gland (Lydon et al., 1995). While both stromal and epithelial cells participate in mammary gland development, it is the PR of epithelial cells which is essential for lobulo-alveolar development (Humphreys et al., 1997). It is very likely that the effect of PR is mediated through induced expression of cyclin D1, since a disruption of this gene yields a very similar mammary phenotype, namely lack of post-puberty development (Fantl et al., 1995; Sicinski et al., 1995). Steroids, in addition to regulating gene transcription (Beato et al., 1995), produce rapid, non-transcriptional responses which in some cases are reminiscent of those evoked by peptide growth factors, suggesting an interaction of steroids with cell surface receptors (Pappas et al., 1995). Vitamin D rapidly activates phospholipase C of intact enterocytes (Liebereherr et al., 1989), and estrogen stimulates adenylate cyclase in different cells (Aronica et al., 1994). Progesterone stimulates oocyte maturation in Xenopus by a mechanism involving activation of protein kinases (Sagata et al., 1989; Muslin et al., 1993). Progestins also activate calcium influx in sperm (Blackmore et al., 1990) and induce tyrosine phosphorylation of a protein (Mendoza et al., 1995). We have found that in MCF-7 mammary tumor cells, estradiol stimulates Src kinase and the MAP kinase pathway very rapidly (Di Domenico et al., 1996; Migliaccio et al., 1996). To investigate whether the proliferative activity of progestins is mediated by a similar mechanism, we have used T47D mammary tumor cells which are rich in PR (Chalbos et al., 1982). Here we report that progestins rapidly and reversibly stimulate the transducing pathway c-Src/p21ras/Erk-2 (Schlessinger, 1993) in these cells. Surprisingly, the activation of this pathway by progestins not only requires the PRB but also ligand-free ER, as shown in T47D cells treated with anti-estrogens and in Cos-7 cells transfected with the appropriate receptors. In contrast to ER, PRB does not interact with c-Src, but it interacts with ER via its N-terminal region which is absent in PRA. Interestingly, a PR point mutant ineffective in transcriptional activation still activates the signaling transduction pathway. The relevance of this activation to cell proliferation is suggested by experiments with c-Src inhibitors of different specificity. Our data show that in addition to cross-talk with peptide growth factors (Ignar-Trowbridge et al., 1992; Zhang et al., 1994) and neurotransmitter receptors (Power et al., 1991), steroid receptors can also cross-talk with each other. Results Progestin stimulation of Erk-2 activity in T47D cells In order to study the effect of progestins on the MAP kinase pathway, we used T47D mammary carcinoma cells, which contain ER and constitutive high levels of PR (Figure 2C). To reduce the levels of activated ER, the cells were maintained for 1 week in the presence of charcoal-treated serum and in the absence of phenol red, a substance with a weak estrogenic activity (Berthois et al., 1986). The Erk-2 activity in T47D cells treated for different times with 10 nM of the synthetic progestin R5020 was evaluated by immunoprecipitation of cell lysates with anti-Erk-2 antibody, followed by measurement of the myelin basic protein (MBP) phosphorylating activity in the immunoprecipitates. R5020 treatment resulted in a rapid stimulation of Erk-2 activity which was already detectable after 2 min, reached maximal values after 5 min and returned to normal levels after 60 min. The anti-progestin RU486 (1 mM) inhibited the stimulatory effect of the agonist treatment, indicating that binding to the PR is required for the progestin effect. No MBP phosphorylation was detectable in the control immunoprecipitates with the same anti-Erk-2 antibody but in the presence of an excess of the Erk-2 peptide (C14 peptide), against which this antibody has been raised (Figure 1A). Five minutes of R5020 treatment, in addition to stimulating Erk-2, also enhanced Erk-1 activity weakly, measured on proteins immunoprecipitated from lysates by anti-Erk-1 antibody using MBP as a substrate. This effect is also inhibited by the anti-progestin (not shown). Surprisingly, the stimulatory effect of the progestin on Erk-2 was inhibited by different anti-estrogens. The pure steroidal anti-estrogen ICI 182,780 strongly inhibited the progestin induction at a concentration of 1 μM, while 10 μM ICI 182,780 reduced the Erk-2 activity even below the values observed prior to hormone induction (Figure 1B). The non-steroidal anti-estrogen OH-tamoxifen abolished Erk-2 stimulation by R5020 at concentrations of 0.1–10 mM (Figure 1C). Figure 1.Activation of Erk-2 in progestin-treated T47D cells. (A) Cells were left untreated or were treated for the indicated times with 10 nM R5020 in the absence or presence of the anti-progestin RU486 (1 μM). Cells were also not treated or treated for 5 min with R5020 in the absence or presence of 1 or 10 μM ICI 182,780 (B) or increasing concentrations (0.1, 1 and 10 μM) of OH-tamoxifen (C). Cell lysates were immunoprecipitated using anti-Erk-2 antibody in the absence or presence of the C14 peptide against which the antibody had been raised. The immunoprecipitates were assayed for Erk activity using MBP as a substrate. Download figure Download PowerPoint Figure 2.Activation of Erk-2 in progestin- and estrogen-treated T47D and T47D-Y cells. (A) T47D cells were treated for 2 and 5 min with 10 nM of either estradiol (E2) or R5020. (B) T47D cells were treated for 2 min with either E2 or R5020 in the absence or presence of 0.1 μM OH-tamoxifen; T47D-Y cells were treated with either E2 or R5020. Cell lysates were immunoprecipitated using anti-Erk–2 antibody in the absence or in presence of the C14 peptide, and the immunoprecipitates were assayed for Erk activity using MBP as a substrate. (C) Western blot with anti-hPR or anti-ER (H222) antibodies of lysates from T47D and T47D-Y cells left untreated or treated with either E2 or R5020. M indicates the marker lane. Molecular weights on the right are expressed in kDa. The arrows on the left indicate the positions of PRA, PRB and ER. Download figure Download PowerPoint A comparison of the effects of estradiol and progestin on Erk-2 activity in T47D cells showed a similar stimulation by the two steroids, detectable after 2 and 5 min of hormone treatment (Figure 2A). It is perhaps more relevant that the progestin R5020 stimulated Erk-2 activity in T47D but not in T47D-Y cells (Figure 2B), which lack PR but are endowed with high ER levels (Sartorius et al., 1994) (Figure 2C). This finding confirms that Erk-2 activation by the progestin requires the PR. Estradiol stimulated Erk-2 activity in T47D as well as in T47D-Y cells (Figure 2B), showing that the estrogen effect is independent of PR. Progestins increase GTP-bound p21ras and stimulate c-Src activity in T47D cells We next analyzed the effect of progestins on steps of the signal transduction pathway upstream of MAP kinases, such as p21ras and c-Src. We measured the guanine nucleotides bound to p21ras in [32P]orthophosphate-labeled T47D cells treated for 4 min with 10 nM R5020, in the absence or presence of anti-progestins or anti-estrogens, and calculated the GTP:GTP + GDP ratio using a PhosphorImager. R5020 treatment increased the GTP: GTP + GDP ratio 2.6-fold, and this increase was strongly reduced or abolished by the addition of either the anti-progestin RU486 or the anti-estrogen ICI 182,780 (Figure 3A). These findings demonstrate that progestin activates p21ras by a mechanism that probably requires both PR and ER. Figure 3.Effect of progestins on p21ras and c-Src activity in T47D cells. (A) Cells were radiolabeled with [32P]orthophosphate and incubated for 4 min in the absence or presence of 10 nM R5020 either alone or together with 1 μM RU486 or 10 μM ICI 182,780. Nucleotides bound to p21ras were analyzed. The ratio GTP/GTP + GDP averaged from two experiments is shown. (B) Cells were left untreated or were treated with 10 nM R5020 for the indicated times. (C) Cells were not treated or treated for 2 min with R5020 in the absence or presence of either RU486 or ICI 182,780. In (B) and (C), lysates were incubated with either anti-c-Src 327 antibody or control antibody. The immunoprecipitates were assayed for Src kinase activity using acid-treated enolase as a substrate. Download figure Download PowerPoint Treatment of T47D cells with 10 nM R5020 for different times enhanced c-Src kinase activity on enolase as measured in cell lysates immunoprecipitated with monoclonal 327 anti-c-Src antibody (Figure 3B). The activity was stimulated 2 and 5 min after hormone addition and decreased to uninduced levels thereafter. Immunoprecipitates obtained with a control antibody were devoid of c–Src kinase activity (Figure 3B). T47D cells were also treated with 10 nM R5020 for 2 min in the presence of either 1 μM of the anti-progestin RU486 or 10 μM of the anti-estrogen ICI 182,780 (Figure 3C). The stimulatory effect of R5020 on the c-Src kinase activity was almost completely abolished by the anti-progestin, indicating a requirement for PR occupancy by the agonist. As in the case of Erk-2 and p21ras, stimulation of c-Src activity by R5020 was strongly reduced by the pure anti-estrogen ICI 182,780, suggesting that progestin activation of the entire pathway requires ER. Comparison of c-Src and Erk-2 responses to EGF and progestin To gain an impression of the relative magnitudes of activation of the signaling cascade by progestin and by a classical growth factor, T47D cells were stimulated with either 10 nM R5020 or 100 ng/ml epidermal growth factor (EGF), and the effects on c-Src and Erk-2 followed (Figure 4A and B). The growth factor and the hormone stimulated c-Src and Erk-2 with similar intensities and kinetics. We conclude that progestin activation of the signaling pathway is comparable with that following growth factor treatment. Figure 4.Effect of EGF and the progestin R5020 on c-Src and Erk-2 of T47D cells. Cells were left untreated or were treated for the indicated times with 10 nM R5020 or 100 ng/ml EGF. Cell lysates were either incubated with control or anti-c-Src antibodies and the precipitates assayed for c-Src activity using enolase as a substrate (A) or immunoprecipitated with anti-Erk-2 antibodies in the presence or absence of the C14 peptide and the precipitates assayed for Erk-2 activity using MBP as a substrate (B). Download figure Download PowerPoint Effect of tyrosine kinase inhibitors on progestin-stimulated cell growth and Erk activation It is known that activation of the c-Src/Ras/Erk pathway can lead to cell growth (Schlessinger and Ullrich, 1992). We explored the role of c-Src in the progestin stimulation of Erk-2 kinase activity and the link between the signal transduction pathway activation and cell growth stimulation using two c-Src kinase inhibitors, genistein (Akiyama and Ogavara, 1991) and PP1, a new, specific Src family kinase inhibitor (Hanke et al., 1996). The inhibitors were added to the medium together with R5020, and cell growth was followed for 4 days. Both inhibitors prevented cell growth stimulation by progestin to the same extent as the anti-estrogen ICI 182,780 (Figure 5A). To assay the effect of the two inhibitors on Erk-2 activity, cells were pre-incubated with either genistein or PP1 for 18 h and Erk-2 activity measured after 5 min of progestin treatment. Both inhibitors reduced progestin stimulation of Erk-2 activity, but the effect of PP1 was stronger, leading to a reduction of Erk-2 activity below the level in uninduced cells (Figure 5B). The specificity of the action of PP1 on c-Src is supported by the fact that 10 min and 18 h of PP1 treatment are equally effective in abolishing Erk-2 stimulation by R5020 (Figure 5B and C). These results suggest a causal role of c-Src stimulation by progestin on both Erk-2 activation and cell proliferation. Figure 5.Effect of tyrosine kinase inhibitors on progestin-induced cell growth and Erk-2 activity in T47D cells. (A) T47D cells were left untreated or were treated for 4 days in the absence or presence of 10 nM R5020 alone (control) or in the presence of either 40 μM genistein, 10 μM PP1 or 10 μM ICI 182,780, then counted. (B) Cells left untreated or treated for 18 h with 40 μM genistein or 10 μM PP1 were incubated in the absence or presence of R5020 for 5 min. Cell lysates were immunoprecipitated by anti-Erk 2 antibodies and assayed for Erk-2 activity. Reduction of PP1 treatment from 18 h to 120, 60, 30 or even 10 min did not affect the inhibition by PP1 of Erk-2 activity stimulated by the progestin (C). Download figure Download PowerPoint Progestin stimulation of Erk-2 and c-Src activities in COS-7 cells transiently transfected with hormone receptors The inhibitory effect of anti-progestins and anti-estrogens on R5020 activation of the Src/p21ras/Erk pathway suggests that progestin action is mediated by PR but also depends on antagonist-free ER. To test this notion, Cos-7 cells, which lack ER and PR, were transfected with either pSG5-HEGO plasmid encoding the human ER (Tora et al., 1989a), pSG5-hPRB encoding the human PRB (Kastner et al., 1990) or with both plasmids, and the effects of progestins on MAP kinase activity were assayed. Both receptors appear to be co-expressed at a similar level (Figure 6C). Figure 6A shows that in cells transfected with either ER or PRB expression vectors individually, progestin treatment for 5 min did not significantly affect the basal level of Erk-2 activity. In contrast, in cells co-transfected with both receptors, R5020 enhanced Erk-2 activity. The stimulation of c-Src activity by R5020 also required the simultaneous expression of both receptors (Figure 6B). These findings demonstrate that the progestin effects on the signaling pathway in Cos cells require not only binding to the PR but also expression of ER. They also support the interpretation that the inhibitory effect of anti-estrogens on the progestin stimulation of c-Src, p21ras and Erk-2 in T47D cells (Figures 1,2,3) is a consequence of their binding to ER and the consequent blockade of ER function. Figure 6.Effect of progestin on Erk-2 and c-Src in Cos-7 cells transfected with hER and hPRB cDNA. (A and B) Cos-7 cells were transfected with either empty pSG5 vector, pSG5-hPRB, pSG5-HEG0 or both the pSG5-hPRB and pSG5-HEG0 vectors. Thereafter, cells were left unstimulated or were stimulated with 10 nM R5020. Lysates from cells stimulated for 5 min were incubated with anti-Erk-2 antibody (A) whereas lysates from cells stimulated for 2 min were incubated with anti-c-Src antibodies (B). The immunoprecipitates were assayed for Erk-2 (A) or c-Src (B) activity, respectively. (C) Panels a and b show blots of lysates from the experiments in (A) and (B) with anti-PR or H222 anti-ER antibodies, respectively. Download figure Download PowerPoint Progestin- and estradiol-induced association of ER with c-Src in Cos-7 cells transiently transfected with hER and hPRB cDNA To investigate the molecular basis of the ER requirement for progestin action on the signaling pathway, we transfected Cos cells with ER and PRB expression vectors, treated the cells with R5020 or estradiol and immunoprecipitated the cell lysates with anti-ER, anti-PR or anti-c-Src antibodies. Proteins from each immunoprecipitate were blotted separately with anti-ER, anti-PR and anti-c-Src antibodies (Figure 7A–C). In the immunoprecipitates obtained with anti-ER antibody (Figure 7A), we detected PRB independently of the hormonal treatment, whereas c-Src was detected only when cells were treated for 2 min with either progestin or estradiol. This finding is consistent with the notion that, in the presence of hormones, c-Src interacts with ER pre-associated with PR, forming a stable ternary complex, but could also be explained by the existence of two independent complexes containing ER associated with either PRB or c-Src. Immunoprecipitation by anti-PR antibody (Figure 7B) showed association of ER with PR in the absence and presence of hormones, and failed to detect c-Src associated with PRB. Furthermore, when proteins immunoprecipitated by anti-c-Src antibodies were analyzed (Figure 7C), c-Src was found associated with ER only in the presence of hormones, while no association with PRB was found, independently of the hormonal treatment. Therefore, the results of immunoprecipitation with anti-c-Src and anti-PR antibodies do not support the existence of a stable ternary complex including PRB, ER and c-Src. Figure 7.Interaction between PR, ER and c-Src in Cos-7 cells transfected either with both hER and hPRB cDNA or separately with hER cDNA or hPRBcDNA. (A–C) Cos-7 cells were transfected with either empty pSG5 vector or both the pSG5-HEG0 and pSG5 hPRB vectors. Cells were left untreated or were treated with either 10 nM R5020 for 2 and 5 min or 10 nM estradiol (E2) for 2 min. Cell lysates in (A–C) were immunoprecipitated with H222 anti-ER, anti-PR and anti-c-Src antibodies, respectively. Each immunoprecipitate was blotted with anti-ER, anti-PR or anti-c-Src antibodies, and the expected position of the proteins in the blot is indicated by an arrow. (D and E) Cos-7 cells were transfected with either empty pSG5 vector, pSG5-HEG0 or pSG5-hPRB. Cells were left untreated or were treated with E2 or R5020 for 2 min, and lysates were incubated with either anti-ER (D) or anti-PR (E) antibodies. The immunoprecipitates were blotted with H222 anti-ER and anti-c-Src in (D) and with anti-PR and anti-c-Src in (E). (F) Cos cells transfected with either empty pSG5 vector or both the pSG5-HEG0 and pSG5-hPRB vectors were left untreated or were treated for 2 min with 10 nM R5020 in the absence or presence of either 1 μM RU486 or 10 μM ICI 182,780, or for 2 min with 10 nM E2 alone or together with 10 μM ICI 182,780. Cell lysates were immunoprecipitated with anti-c-Src antibodies and blotted with anti-c-Src and anti-ER antibodies. Download figure Download PowerPoint The experiments with Cos cells co-transfected with PRB and ER expression vectors leave open the possibility that PRB is not found associated with c-Src because it is competed by ER. To test this possibility, Cos cells were transfected separately with either hER or hPRB expression vectors and stimulated by estradiol or progestin, respectively. Cell lysates were precipitated with anti-ER (Figure 7D) or with anti-PR (Figure 7E) antibodies, and proteins in the precipitates were analyzed by blotting with anti-c-Src antibodies and either anti-ER (Figure 7D) or anti-PR (Figure 7E) antibodies. PRB was not found associated with c-Src in the absence of ER (Figure 7E), thus excluding a competition between the two hormone receptors for association with c-Src. In contrast, ER still associated with c-Src after estradiol treatment, in the absence of PRB (Figure 7D). Moreover, progestin in the absence of its receptor did not induce ER–c-Src association (Figure 7D), confirming that progestin-induced ER–c-Src association requires PR. To explore further the functional significance of the interaction between ER and c-Src, we investigated the effect of anti-hormones. The anti-estrogen ICI 182,780 prevented the association of ER and c-Src induced by estradiol (Figure 7F). Furthermore, the stimulation of the interaction between ER and c-Src observed 2 min after R5020 treatment was prevented by either the anti-estrogen or the anti-progestin RU486 (Figure 7F). The observation that in this experiment there is less ER–c-Src complex in the presence of anti-hormones than in their absence is probably due to some residual estradiol still present in the medium in spite of the charcoal treatment of the serum. Interaction between PR, ER and c-Src in T47D cells We next attempted to define more precisely the interaction between PR and ER using immunoprecipitation and gene transfection experiments. When T47D cell lysates were immunoprecipitated by anti-PR antibodies, which recognize both the A and B forms of PR (Figure 8A, panel a), as in Cos cells (Figure 7B) the co-immunoprecipitation of ER was observed before as well as after treatment with progestin or estrogen (Figure 8A, panel b). The association between PR and ER appeared to be independent of ligand as it was not inhibited by anti-progestins or anti-estrogens (Figure 8A, panel b). As in transfected Cos cells (Figure 7B, C and E), PR did not associate with c-Src in T47D cells (Figure 8A, panel c). As expected from the experiment with Cos cells (Figure 7A), when anti-ER antibodies were used to immunoprecipitate T47D cell lysates, PR was co-precipitated in the absence or
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