The EMBO Journal vol.10 no.8 pp.2237-2245, 1991

Unregulated expression of c-Jun or c-Fos proteins but not Jun D inhibits oestrogen receptor activity in human breast cancer derived cells Vassilis Doucas, Giannis Spyrou and Moshe Yaniv Unite des Virus Oncogenes, UA 1149 du CNRS, Departement des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Communicated by M.Yaniv

We present evidence that oestrogen receptor activity in human MCF-7 breast cancer cells is reduced by overexpression of c-Jun or c-Fos proteins and to a lesser extent by Jun B overexpression. In contrast, overexpression of Jun D protein does not affect the activity of the oestrogen receptor. A region of c-Jun found to be required for repression of oestrogen receptor activity is located outside the DNA binding domain and is not conserved among the three Jun proteins. Finally, we suggest that c-Jun and c-Fos act independently to inactivate the oestrogen receptor. Key words: c-Jun/c-Fos/Jun D/oestrogen receptor

Introduction Several recent reports have demonstrated that functional interactions occur between members of two families of transcription factors, the steroid and steroid-related hormone receptors and c-Jun and c-Fos constituents of activator protein 1 (API). The Jun-Fos heterodimer is implicated in repression of transcription from the human osteocalcin gene, both the basal and the vitamins A and D3 induced levels, by recognizing, together with the corresponding receptor, a common regulatory element within the promoter region (Schule et al., 1990a). Another mechanism was shown to be involved in the inhibition of glucocorticoid hormone dependent transcription by the same nuclear protooncogenes. Inhibition in this case does not seem to involve binding to a common target. Rather, c-Fos or c-Jun may form a complex with the hormone receptor, decreasing its activity. The effect is reciprocal since hormone bound receptor also inhibits gene activation by c-Jun and c-Fos (Jonat et al., 1990; Lucibello et al., 1990; Schule et al., 1990b; Yang-Yen et al., 1990). A different pattern was observed with a DNA element found upstream of the mouse proliferin gene. In this case, c-Jun was essential for activation of this element by the glucocorticoid receptor while the receptor conferred a negative effect in the presence of cJun and relatively high levels of c-Fos (Diamond et al., 1990). Finally, direct cooperation between c-Jun, c-Fos and the oestrogen receptor in transcriptional activation was observed with the ovalbumin gene promoter; binding of the oestrogen receptor to DNA was not required (Gaub et al., 1990). MCF-7 human breast cancer cells containing oestrogen receptor, are frequently used for the study of oestrogen Oxford University Press

responsive genes (Nunez et al., 1987). This cell line is dependent on the presence of exogenous oestrogen for both in vitro proliferation and tumour formation in the nude mouse (Dickson et al., 1986). The human oestrogen receptor (hER), whose action is triggered by oestradiol, controls initiation of transcription by binding to its cognate oestrogen responsive element (ERE) which contains the consensus palindromic sequence GGTCANNNTGACC (Kumar et al., 1986, 1987; Klein-Hitpass et al., 1986; Evans, 1988; Kumar and Chambon, 1988). In the liver of Xenopus laevis the expression of vitellogenin genes, which code for the yolk precursor proteins, is under strict oestrogen control. Hormonal activation involves both the induction of transcription and the stabilization of the vitellogenin mRNA (Shapiro et al., 1983; Tata et al., 1983; Wahli and Ryffel, 1985). When the Xenopus vitellogenin A2 gene, which contains a perfect ERE in the 5' flanking region, is transfected into MCF-7 cells transcription is modulated by oestrogen levels. (Klein-Hitpass et al., 1986; Kumar et al., 1987). In the present study, we examined the hormonal response of this element in the presence of elevated levels of Jun and Fos proteins. Is hormonal stimulation activated by Jun or Fos as in the case of the ovalbumin promoter or do Jun and Fos inhibit this receptor similarly to their action on some glucocorticoid response elements? Using transient transfections in MCF-7 cells, we show that overexpression of c-Jun and c-Fos proteins suppresses oestrogen dependent transcription of an ERE-containing reporter gene (pA2), without affecting its basal level of expression. Among the other members of the jun family, Jun B gave only partial suppression while overexpressed Jun D protein had no effect on oestrogen dependent transcription. TPA treatment that increases the endogenous API activity also suppressed oestrogen induced transcription of the ERE-containing reporter gene. Deletion of either one or two regions of c-Jun, one adjacent to the basic DNA binding domain and the other containing the leucine repeat blocked repression. Finally, overexpression of the human oestrogen receptor in MCF-7 cells abolished c-Fos induced repression of the pA2 transcription, but had no effect on cJun induced repression.

Results Suppression of oestrogen induced transcription by c-Jun or c-Fos proteins The chimeric pA2 (vit -tk-CAT) construction contains the oestrogen responsive element of the X. laevis vitellogenin A2 gene upstream of the HSV 1 thymidine kinase promoter, linked to the bacterial CAT reporter gene (Klein-Hitpass et al., 1986). When transfected into human MCF-7 cells, CAT activity is strongly stimulated by addition of hormone to the growth medium (Figure 1, lanes 1 and 2). To investigate the effect of Jun and Fos proteins on the oestrogen receptor activity, we tested the transcriptional efficiency of 2237

V.Doucas, G.Spyrou and M.Yaniv

this reporter plasmid in the presence of increasing concentrations of c-jun or c-fos coding plasmids. The expression of both c-jun (Figure 1, lane 5) and c-fos (Figure 1, lane 6) lead to a marked decrease in oestrogen induced transcription of the pA2 construction. This inhibition by c-Jun and c-Fos was dependent on the concentration of the expression plasmids used and therefore on the concentration of these proteins (Figure 2, see also Figure 7 below). The highest level of inhibition of the pA2 oestrogen induced activity was

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demonstrate that the effect observed is not due to the RSV LTR and Fos expression plasmids. The cells RSV-c-jun and 0.8 Ag RSV-c-fos plasmids (lanes 5,6). Estrogen (E2) stimulated the vit-tk-CAT dependent activity 13-fold in this experiment. This activity was reduced 8-fold in the presence of c-Jun or c-Fos.

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obtained with final concentrations of 1.6 jig and 0.8 ug per plate for c-jun and c-fos transfected expression plasmids, respectively. These levels of c-Fos and c-Jun did not change the basal, hormone independent expression of the vitellogenin construction (Figure 2 and data not shown). Since the intracellular levels of Jun and Fos proteins were not accurately determined, it is not clear if Fos is more active at repressing oestrogen receptor activity than Jun on a molar ratio. To exclude the possibility that overexpression of Jun and Fos produced by the transfected plasmids may nonspecifically inhibit transcription by sequestering general transcription factors, we examined the activity of several promoters which are not regulated by Jun and Fos. We have checked the SV40 early promoter with or without its enhancer region and the thymidine kinase minimal promoter, each of them linked to the bacterial CAT reporter gene, in cotransfection experiments. Increasing concentrations of the c-jun and c-fos expression plasmids had no effect on the activity of these constructs (data not shown). To test if endogenous Jun and Fos were also capable of suppressing oestrogen receptor activity, we treated cells with TPA (12-O-tetradecanolphorbol-13-acetate) which is known to stimulate endogenous expression of both jun and fos (Greenberg and Ziff, 1984; Angel et al., 1987; Lee et al., 1987; Lamph et al., 1988; Piette et al., 1988). Indeed, oestrogen induced transcription of the vit-tk-CAT construction was suppressed -8-fold by TPA treatment (Figure 3). We also tested the effect of TPA on a reporter plasmid construction which contains a smaller portion of the vitellogenin A2 promoter (from -331 to -295) linked to tk-CAT. However, this shorter element, which also contains the ERE, is known to confer oestrogen respoi[ns-

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Fig. 2. Effect of increasing concentrations of the c-jun and c-fos expression vectors on pA2 transcription. Transient transfection experiments were done as in Figure 1, using the RSV-Luciferase, RSV-c-jun and RSV-c-fos expression vectors at different concentrations (numbers given in the figure are ptg used) and the reporter vit-tk-CAT plasmid construction at 1.4 Ag. The values are the average of at least three experiments. CAT activity is given in arbitrary units.

2238

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Inhibition of oestrogen receptor by Jun and Fos

with c-fos and c-jun coding plasmids (see below). These results show that the Jun-Fos complex can activate transcription in MCF-7 cells and that the vit-tk-CAT reporter plasmid probably does not contain cryptic API binding sites. In order to understand better the molecular basis of the suppression of oestrogen receptor activity by c-Jun or c-Fos and to investigate whether repression involves titration of the endogenous oestrogen receptor, we overexpressed the human oestrogen receptor by cotransfections with an expression vector containing the human oestrogen receptor coding sequences. Increasing the amount of human oestrogen receptor in the MCF-7 cells only slightly reversed the inhibitory effect of c-Jun (Figure 4). In contrast, overexpression of oestrogen receptor largely abolished c-Fos

repression (Figure 4). These results suggest that the two components of the API transcription factor, which share homology in their dimerization and DNA binding domains, probably act through different mechanisms to suppress the oestrogen induced transcriptional activity of the vit -tk -CAT construction. No synergy between c-Jun and c-Fos is observed in inhibition of oestrogen activation It is well established that cooperativity between c-Jun and c-Fos in transcriptional activation depends to a large part on the increase in affinity for the DNA target of the Jun-Fos heterodimers relative to Jun homodimers (Curran and Franza, 1988; Vogt and Bos, 1990). To investigate whether the repression of pA2 hormonal induction may be mediated by Jun - Fos heterodimers we tested the efflciency of suppression by cotransfecting the two corresponding expression plasmids. MCF-7 cells were transfected with pA2 and increasing concentrations ofjun andfos expression vectors. As shown in Figure 5A, cotransfection of c-jun and c-fos

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Fig. 4. Overexpression of the human oestrogen receptor differentiates between c-Jun and c-Fos inhibitory effects. A cDNA encoding the oestrogen receptor, driven by the early SV40 promoter (HEO) or a deleted derivative (AHEO), was cotransfected into MCF-7 cells along with c-jun or c-fos expression vectors. The vt-tk-CAT construction was used as reporter plasmid at 1.4 jig. The amounts (ug) of the expression plasmids used are indicated. CAT activity is given in arbitrary units. pA2 transcription was stimulated 8.5-fold by oestrogen in this experiment. The repression efficiency by c-Fos and c-Jun was 7.7- and 5.7-fold respectively.

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Fig. 5. No cooperativity occurs between c-Jun and c-Fos proteins in suppressing with vit-tk-CAT oestrogen induced transcription. In parallel experiments, cells were transfected in the presence of oestrogen, with increasing concentrations of c-jun, c-fos or of both expression vectors. In the cotransfection experiment the concentration of the c-fos expression plasmid was constant at 0.2 Ag while we used increasing amounts of c-jun. In the abscissae are represented the final amounts (ug) of the c-jun and/or c-fos transfected expression plasmids. In panel A, we used as reporter plasmid the vit-tk-CAT construction at 1.4 ytg. Activity is expressed as a percentage relative to the initial hormone dependent vit-tk-CAT activity. In panel B, the reporter plasmid was the PK3 -tk-CAT, used at 2.0 ytg. The basal activity of this construction was 0.2, given in arbitrary units.

2239

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Fig. 6. Inhibitory effect of Jun B and Jun D proteins. MCF-7 cells were transfected with vit-tk-CAT construction as reporter plasmid at 1.4 tg. The different expression vectors were used at increasing concentrations (pg) as indicated. Taking the c-Jun induced repression as 100%, Jun B repressed -70%, while Jun D did not repress at all.

did not drastically increase the inhibitory effect compared with the effect due to either c-jun or c-fos alone. In parallel experiments cotransfection of jun -fos expression plasmids acted synergistically to induce transcription of a construction containing three API sites upstream of the CAT gene (Figure 5B). These results argue that suppression of oestrogen receptor activity by c-Jun and c-Fos is not dependent on heterodimer formation. Effect of Jun B and Jun D on oestrogen induced transcription Three members of the jun proto-oncogene family have been identified in murine and human cells, c-Jun, Jun B and Jun D. All three share high homology in the C-terminal region which is involved in dimerization and DNA binding, but are less conserved in their N-terminal sequences (Ryder et al., 1988, 1989; Ryseck et al., 1988; Lamph et al., 1988; Angel et al., 1988; Hirai et al., 1989). All three form heterodimers among themselves or with the different members of the fos family (Chiu et al., 1988; Halazonetis et al., 1988; Landschulz et al., 1988; Nakabeppu et al., 1988; Cohen et al., 1989; Hirai et al., 1989; Zerial et al. 1989). The three jun proteins differ, however, in some of their properties. These include tissue distribution, transcriptional stimulatory activity, and sensitivity to different signal transduction pathways (Vogt and Bos, 1990). We have investigated whether Jun B and Jun D resemble c-Jun in its effect on oestrogen activated transcription. Increasing amounts of expression vectors for jun B or jun D were cotransfected with pA2 into MCF-7 cells. As shown in Figure 6, increasing amounts of jun B partially inhibited oestrogen dependent activity of pA2, reaching a plateau value of 70% of the suppression obtained with c-jun. No further increase in repression was observed with higher amounts (5 ug) of jun B expression plasmid. Surprisingly, expression ofjun D in MCF-7 cells did not suppress oestrogen induced transcription at all (Figure 6). Even large amounts of jun D expression vector (5 and 10 Itg, data not shown) did not change the CAT reporter activity. Thus, the three Juns vary in their -

2240

ability to interfere with oestrogen induced transcription in MCF-7 cells. To verify that the absence of suppression by jun D is not caused by the failure of the transfected cells to synthesize murine Jun D, we analysed transfected cell extracts in immunoblots probed with anti-Jun D antibodies (see Materials and methods). As shown in Figure 7, increasing concentrations of transfected jun D expression plasmid gave rise to a parallel increase in the intensity of the Jun D immunoblot signal. Expression of Jun and Fos proteins in transfected cells was also verified by immunofluorescence of fixed cells. Approximately 5 % of the cells were transfected and showed a clear immunofluorescent signal in the nucleus. No staining was observed with antibodies not specific for the transfected gene product (data not shown and Figure 7). A region of c-Jun located outside its DNA binding domain is required for repression of oestrogen activity In order to localize the domain(s) of the c-Jun protein that is responsible for inhibition of pA2 transcription, we tested a series of c-Jun deletion mutants in transient cotransfection experiments (Figure 8). For each mutant we verified that the modified protein was synthesized and transported to the nucleus by immunofluorescence and/or Western blotting with

affinity purified polyclonal antibodies raised against the Cterminal 143 amino acids of c-Jun (Figure 9). Repression of oestrogen receptor activity was dependent on the presence of the N-terminal half of the c-Jun protein. Deletion of the N-terminal 168 amino acids abolished repression, even though the protein was still synthesized and transported to the nucleus (Figure 9B). Furthermore, this mutant retains its capacity to homodimerize and heterodimerize with c-Fos and to bind DNA specifically (Hirai and Yaniv, 1989; Hirai et al., 1990). The N-terminal region of c-Jun has been shown previously to be involved in transcription activation in vivo (Chiu et al., 1988; Hirai et al., 1990). Additional mutants were tested to localize more precisely the protein segment involved in repression. The mutant

Inhibition of oestrogen receptor by Jun and Fos

continued to repress (Figure 8). The repression efficiency was slightly higher than that of the wild type c-Jun, possibly due to a higher efficiency of expression (compared with the c-Jun wild type) in the transfected cells (Figure 9A). We also tested the deletion A284-3 11 which removes the leucine zipper domain. Repression by this Jun mutant was only 10-30% of the wild type c-Jun activity (Figure 8). With this mutant also we found an abnormal distribution by immunofluorescence; the protein accumulated as dots in the nucleus (Figure 9). Furthermore, the anti-Jun signals in Western blots with this mutant were lower. However, we cannot exclude the possibility that a large fraction of our antibody preparation was directed against this region. For these reasons it is not yet entirely clear whether the leucine zipper domain of the Jun protein is necessary for repression of oestrogen activity.

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Fig. 7. Immunoblot and immunofluorescence analysis of MCF-7 transfected cells. Proteins isolated from cells transfected with increasing concentrations of RSV-c-jun and RSV-jun D were separated by 10% SDS-PAGE blotted to nitrocellulose filters and probed with anti-Jun (A) or aPepD antibodies (B). Cells were transfected with 1.6 Ag of RSV-c-jun (C), RSV-jun B (D), RSV-jun D (E), and RSV-c-fos (F). For immunolocalization, cells were stained with anti-Jun (C-D), aPepD (E) and anti-Fos (F) antibodies. Magnification: 300x.

A 132 -146 efficiently repressed the oestrogen induced pA2 activity, while the deletion A132 -220 was unable to repress. Both were synthesized and correctly transported to the nucleus (Figure 9C and D). These data suggest that repression is dependent on the region from amino acids 147 -220. In order to analyze further the region around amino acid 168 we used two chimeric constructions of c-Jun and Jun D proteins. As shown in Figure 8, the first construction jun cD (see Materials and methods) possessed 30% of the c-Jun repressive activity, while the second one (jun Dc) did not repress at all. These results suggest that sequences on both sides of amino acid 168 are implicated in the repression. Another deletion mutant of c-Jun, A2 -220, which maintains the DNA binding domain, gave a minimal (10%) repression effect. However, since this mutant protein accumulated in the nucleolus of the transfected cells (Figure 9F), we cannot exclude the possibility that this aberrant localization may have resulted in the lowered repression. A deletion mutant in the C-terminal region, from amino acid 319 to 334 which maintained an intact leucine zipper, -

We report here that increased intracellular concentrations of c-Jun or c-Fos strongly inhibited the hormone dependent stimulation of the A2 vitellogenin -tk-CAT chimeric gene by the oestrogen receptor present in human MCF-7 cells. However, no effect of Jun or Fos was observed on the basal activity of the promoter. The effect of c-Jun and c-Fos did not seem to be cooperative. Since heterodimer formation strongly increases the affinity of c-Jun to its target sequence (TRE) (Chiu et al., 1988; Halazonetis et al., 1988; SassoneCorsi et al., 1988; Hirai and Yaniv, 1989), whereas c-Fos is unable to bind this site in the absence of Jun (Kouzarides and Ziff, 1988; Gentz et al., 1989; Schuermann et al., 1989; Zerial et al., 1989), these results suggest that direct DNA binding is not a prerequisite for repression of the oestrogen activity. The absence of cooperativity could mean that both proto-oncogene products interfere in the same step of transcriptional activation by the receptor. However, this does not seem to be the case. Increase in the intracellular level of the oestrogen receptor by cotransfecting with an appropriate expression vector largely relieved the c-Fos but not the c-Jun mediated inhibition. These results are compatible with a model in which c-Fos interacts directly with the oestrogen receptor or with a complex of the receptor with other proteins, whereas c-Jun could block DNA bound receptor molecules or a potential cellular co-activator essential for receptor function (Lewin, 1990). Further indications of the nature of the c-Jun sequences involved in trans-repression came from analysis of several c-Jun deletion mutants. Removal of the N-terminal 168 amino acids or an internal deletion removing amino acids 132 -220, deletions that do not affect DNA binding, abolished trans-repression. The critical role of sequences in the N-terminal part of c-Jun is further supported by the results obtained with Jun B or Jun D. Jun B and Jun D are almost identical to c-Jun in their C-terminal part including the dimerization and DNA binding domains, but diverge in their N-terminal parts (see Results). Transfection of a Jun B coding plasmid gave partial inhibition, the plateau value reached did not change upon further increase in Jun B coding plasmid. Finally, synthesis of excess Jun D did not affect oestrogen dependent transcription. Looking for sequences that are common to c-Jun and Jun B but are absent from Jun D, we have identified a glycine rich region (between amino acids 147 and 220 of the c-Jun), as a potential critical

2241

V.Doucas, G.Spyrou and M.Yaniv

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Fig. 8. Deletion analysis of c-Jun. MCF-7 cells were transfected as described. The vit-tk-CAT construction was used at 1.4 ,tg as reporter plasmid. The cells were treated with oestrogen at a final concentration of 10 nM. The wild type expression vector, RSV-c-jun, and mutants A2-168, A2-220, A284-311, A132-146, A132-220, A319-334, jun Dc jun cD were transfected at 1.6 fg. Taking the transrepression caused by wild type c-Jun as 100%, the relative repression obtained with the different c-Jun mutants is given as a percentage of this activity. The values given are the mean of four experiments, with the bar corresponding to half the standard deviation. The boxes from the right to the left, represent respectively the basic domain and the leucine zipper of the jun construct.

element. Whether this part of the protein is indeed responsible for the suppression activity remains to be studied. Another mutant that destroyed most c-Jun repression carried a deletion of the leucine zipper domain, A284 -311. However, immunolocalization of this mutant protein, distinct from the uniform nuclear distribution of the normal protein, exhibited a punctate pattern. Thus, we cannot rule out the possibility that the loss of repression is due to this aberrant localization. However, since this deleted region is nearly identical between the Jun proteins, it seems unlikely to be the basis for the specificity in repression of the oestrogen activity. It is also plausible that c-Jun dimerization is required to form the exact three dimensional structure that is involved in inhibition. c-Jun forms c-Jun-c-Jun homodimers or c-Jun-c-Fos heterodimers with roughly equal efficiencies (S.I.Hirai and M.Yaniv, unpublished observations). The c-Jun protein overexpressed in transfected cells will thus likely accumulate as homodimers. In this respect, c-Jun action may be different from that of c-Fos which is incapable of forming homodimers. Our results with c-Jun pointing to the importance of a region adjacent to the DNA binding domain is reminiscent of the results obtained for the inhibition of the glucocorticoid receptor by c-Fos. Here also a segment preceding the basic domain was found to be crucial for trans-repression (Lucibello et al., 1990). What is the biological significance of the inhibition of the oestrogen receptor by Jun and Fos? High intracellular concentrations of c-Jun and c-Fos accumulate in the nuclei when quiescent cells are replenished with growth factors. It is plausible that under these conditions the cell will enter into a pathway that will decrease the rate of synthesis of proteins involved in highly differentiated functions that are dependent on steroid hormones. It has been demonstrated that tumour promoter (TPA) treatment induced growth arrest and changes in cell morphology of MCF-7 cells (Roos et al., 1986; Valette et al., 1987). Since immediate early genes, like c-fos or c-jun are stimulated by TPA (Greenberg and Ziff, 1984; Angel et al., 1987; Lee et al., 1987; Lamph

2242

et al., 1988), we can suppose that inhibition of hormone dependent growth of these cells by TPA could involve these proteins. Indeed, we showed that a gene containing an ERE binding site is suppressed by jun/fos overexpression and also by TPA treatment. Several recent reports (Jonat et al., 1990; Lucibello et al., 1990; Schule et al., 1990b; Yang-Yen et al., 1990), as well as our present study establish that c-jun and c-fos gene products, two of the components of the API transcription factor, alter the activity of several steroid hormone receptors. These studies show that some cross talk can occur between the regulatory processes involving steroid hormones and those involving the Jun and Fos nuclear oncogenes. It has also been demonstrated that steroid hormone receptors compete for factors that mediate their enhancer function (Meyer et al., 1989). Accepting the hypothesis of Meyer et al. that this cross-talk between steroid hormone receptors could reflect transcriptional interference, we would like to propose that the function of c-Jun or c-Fos protein in repressing the oestrogen receptor activity and thereby affecting the growth of MCF-7 cells, could reflect common mechanisms in transcriptional activation between receptors and the two components of the API transcription factor. In conclusion, our present study demonstrates that excess intracellular c-Jun or c-Fos protein inhibits the hormone dependent activation of transcription from a perfect oestrogen responsive element. We show that DNA binding alone is not sufficient for this inhibitory function and that a segment in the N-terminal half of c-Jun is involved in mediating this repression. Finally, Jun B partially represses while Jun D does not interfere with the oestrogen receptor activity.

Materials and methods Cell culture MCF-7 cell lines (Soule et al., 1973) were maintained at 37°C, 7% CO2 atmosphere in Dulbecco's modified Eagle's medium supplemented with 6% fetal calf serum. At least 4 days prior to each experiment the medium was

Inhibition of oestrogen receptor by Jun and Fos

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Fig. 9. Immunoblot and immunolocalization of c-Jun deletions. MCF-7 cells were transfected with 1.6 1tg of different c-Jun deletion mutants. AntiJun antibodies were used for protein analysis (A) as well as for immunofluorescence (B-G) as described in Materials and methods. Shown are fluorescence micrographs of A2-168 (B), A.132-146 (C), A132-220 (D), Jun cD (E), A2-220 (F) A284-311 (G). Magnification: 300x and 600x for B-D and F, G respectively. removed, the cells were washed with phosphate buffered saline (PBS) and medium was replaced with phenol-red-less medium, supplemented with 0.6 g/l glutamine, 1 1tg/m1 insulin and 6% fetal calf serum treated with activated carbon (Reddel et al., 1984), in order to deprive the medium of the oestrogen.

Antisera An antisera (anti-Jun) reacting with all three mouse jun gene products (c-Jun, Jun B and Jun D) was prepared as follows. A cDNA encoding the 143 C-terminal amino acids of the mouse c-Jun protein beginning at the AAGLAFP, was cloned into a vector containing the gene coding for Staphylococcus aureus protein A (Nilsson et al. 1985). The resulting fusion protein, produced after induction of the bacterial culture with IPTG, was purified by preparative SDS-PAGE. Female rabbits were immunized by injecting subcutaneously at six sites - 700 jig of fusion protein in Freund's complete adjuvant. Four weeks later a 'booster' dose (150 itg) prepared with Freund's incomplete adjuvant was administrated. Another two 'booster' doses at 2 week invervals were performed. After an additional 14 days, blood was obtained and the antibodies were affinity purified by using bacterially produced mouse c-Jun (unpublished results) immobilized on nitrocellulose filters (Ausubel et al., 1989). Rabbit polyclonal antibodies against Jun D (aPepD) were generated by immunization with a synthetic peptide coupled to keyhole limpet hemocyanin (KLH), following the immunization procedure described above. The peptide, GCQLLPQHQVPAY (PepD), corresponded to the 13 C-terminal amino acids of the mouse Jun D protein. BSA-PepD was coupled to activated ultrogel (IBF) and the column was used for affinity purification of the antisera (Ausubel et al., 1989).

The specific 455 anti-Fos antibody (Verrier et al., 1986) was the kind

gift of Dr B.Verrier.

Immunoblotting A volume of MCF-7 cells, transfected with the appropriate plasmid, corresponding to 10 Ag of total protein were resuspended in sample buffer (60 mM Tris-HCI pH 6.8, 1% SDS, 20% glycerol, 140 mM j3-mercaptoethanol, 0.01% bromophenol blue), boiled for 5 min followed by brief sonication. The proteins were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with TBST (150 mM NaCl, 10 mM Tris-HCI pH 8, 0.05% Tween 20) + 2% BSA for 45 min at room temperature. The filters were incubated with affinity purified antibodies for 1 h at room temperature, washed three times in TBST, incubated with an anti-rabbit IgG alkaline phosphatase conjugate and developed by the protocol supplied by the blotting detection kit (Promega).

Immunofluorescence Cells transiently transfected with the RSV -jun construct were used for immunofluorescence analysis. Cells grown in the absence of oestrogen for several days were seeded onto glass coverslips. Culture and transfection conditions were the same as the ones described below for the CAT expression analysis. The cells were transfected with 1.6 Mg of expression plasmids and at the end of the 24 h incubation in fresh medium were fixed in 3.7% formaldehyde in PBS for 25 min at room temperature. The coverslips were washed with PBS, incubated with 50 mM NH4CI for 15 min at room temperature and permeabilized with 0.5% Triton X-100 for 1 min. After washing with PBS each coverslip was incubated with affinity purified primary

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V.Doucas, G.Spyrou and M.Yaniv antiserum in a humidified chamber for 60 min at 37°C. After three washes in PBS the coverslips were incubated with a fluorescein conjugated secondary antibody for 30 min. At the end of the incubation the coverslips were washed three times with PBS and mounted in glycerol buffered with PBS. Transfection of cells and CAT expression analysis Transfection of plasmid DNA into MCF-7 cells was performed using the standard calcium phosphate coprecipitation technique (Wigler et al., 1977). Cells were grown in the absence of oestrogen for at least 4 days and seeded onto 6 cm Petri dishes. Transfections were performed when the cells reached -70% confluency. Typically, 1.5-3.0 ,g of reporter plasmid and 1 itg of RSV-fl-galactosidase expression plasmid as an internal control for transfection efficiency were used. Sonicated and denaturated salmon sperm DNA was used to adjust the total amount of transfected DNA to 10 jg. As an internal control for transfected promoters we used the expression plasmid RSV - luciferase (de Wet et al., 1987) at concentrations equal to those used for the other expression plasmids, under RSV promoter regulation (c-jun, c-fos, c-jun mutants, etc.). Rous sarcoma virus LTR sequence was derived from RSV-CAT plasmids (Gorman et al., 1982). After transfection, oestrogen (E2) was added to a final concentration of 10 nM. The cells were exposed to the precipitate for 16-20 h, washed before incubation for an additional 24 h in fresh medium as above with the appropriate additions. Cells were collected and CAT assay and calculation of percentage conversion for CAT activity was performed after correcting the efficiency of transfections for the ,B-galactosidase activity (Herbomel et al., 1984). All experiments were repeated at least three times and the values given are the means. Plasmid construction The vit-tk-CAT construction (pA2), containing a perfect oestrogen receptor binding site (ERE) at -331/-320, has been described and tested by Klein-Hitpass et al. (1986). For an expression vector for the human oestrogen receptor we used the clone described by Green et al. (1986). Deletion of the coding sequence of the human receptor produced the LHEO expression vector (plasmid pSG5), which was used as a control, in the cases where the human receptor was transfected. The construction RSV -c-jun, RSV -jun D, RSV -jun B, RSV -c-fos and the c-jun mutants are under RSV control. A2- 168 (CJ169), A284-31 1 (CDL) and the PK3 -tk-CAT construction containing three API sites upstream of the tk promoter have been described elsewhere (Hirai et al., 1990). The other mutants of the c-jun expression vector were constructed as follows: A2 -220: the c-jun mutant A2 -168, was cut with AccI and SacII (corresponding to nucleotides 7 and 1021 in the coding region), repaired with Klenow and T4 DNA polymerase and re-ligated. zv319-334: the wild type c-jun expression vector was cut with HpaI (positions:1305 nt in the coding region, 335 nt further in the non-coding region) and ligated with an oligonucleotide, producing a stop codon just after amino acid 318. A132 - 146 the wild type c-jun expression vector was cut with Sacd (positions: 749 nt, 788 nt in the coding region) and religated. A 132 -220 the wild type c-jun expression vector was cut with SacII (positions: 749 nt, 1020 nt in the coding region) and religated with the double strand oligo, GCA ACT GTC TAC CCG/CGT TGA CAG ATG GGC, adding in frame three additional amino acids: T, V, Y. jun cD is a chimeric construction containing the first 168 amino acids of the c-Jun protein (position AccI) and the C-terminal (amino acids 182-341) of the Jun D protein. jun Dc is a chimeric construction containing the first 182 amino acids (position AccI) of the Jun D protein and the C-terminal (amino acids 168 -334) of c-Jun.

Acknowledgements We thank P.Chambon for the kind gifts of the pA2 construction and expression vectors of human oestrogen receptor, H.Rochefort for valuable discussion, B.Verdier for anti-Fos antibodies, M.Crepin for MCF-7 cells, F.Bourgade for help in antibody preparation and the Louvard laboratory for advice in immunological techniques. We are grateful to C.Pfarr and J.Ham for valuable comments on the manuscript. This work was supported by grants from INSERM, ARC, LNFCC, FRMF and EEC. V.D. was supported by a fellowship from the Greek government (IKY) and G.S. by a fellowship from EMBO.

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