The EMBO Journal vol.10 no.12 pp.3839-3849, 1991
Cell-specific inhibitory and stimulatory effects of Fos and Jun on transcription activation by nuclear receptors
Lirim Shemshedini, Rudolf Knauthe, Paolo Sassone-Corsi, Astrid Pornon and Hinrich Gronemeyer Laboratoire de Genetique Mol6culaire des Eucaryotes, CNRS, Unite 184 de Biologie Moleculaire et de Gednie Genetique de 1'INSERM, Institut de Chimie Biologique, Faculte de Medecine, 11, rue Humann, 67085 Strasbourg Cedex, France Communicated by P.Chambon
We investigated the effect of c-Fos and/or c-Jun coexpression on transcription activation by the progesterone (PR), glucocorticoid (GR) or androgen (AR) receptors using three different reporter genes and four different cell lines. We found that c-Fos could only inhibit, while c-Jun could either inhibit or further stimulate receptorinduced transcription. All these effects were receptor, promoter, and cell type specific, and, importantly, the steroid receptors had non-reciprocal effects on the transactivation ability of c-Jun in the presence or absence of c-Fos. Collectively, these results argue against heterodimer formation as a mechanism to explain the phenomena. Transactivation by the endogenous PR in T47D cells could be inhibited by increasing the intracellular c-Fos level with forskolin as well as by coexpressing c-Fos; no such effect was seen in MCF-7 cells. The inhibition by c-Fos of PR-induced transcription involves a competitive mechanism, which requires the presence of the intact c-Fos leucine zipper and is directed mainly at the transcription activation function (TAF) located in the PR and GR hormone binding domains (TAF-2). However, the co-expression of c-Fos did not alter the 'squelching/transcriptional interference' by the PR of estrogen receptor (ER)-induced transcription. Multiple mechanisns are discussed which may be involved in the crosstalk between the two signal transduction pathways. Key words: Fos/Jun/nuclear receptors/transcription activation
Introduction Steroid receptors are ligand-inducible trans-acting transcription factors. Their major domains, a DNA (DBD) and a hormone binding domain (HBD), have been identified as segments of the receptor primary structure (Beato, 1989; Evans, 1988; Green and Chambon, 1988). While the DBD has been investigated in great detail (Danielsen et al., 1989; Mader et al., 1989; Umesono and Evans, 1989), the function of the HBD is, due to its complexity, less well understood. Apart from binding the hormone, the estrogen receptor HBD harbours a dimerization domain (Fawell et al., 1990; Kumar and Chambon, 1988), and in the case of the PR it has been demonstrated that hormone binding induces receptor dimerization in vivo (Guiochon-Mantel et al., 1989). We and (©) Oxford University Press
others have shown that one (TAF-2) of the two transcription activation functions (TAFs) of the estrogen (ER), progesterone (PR) and glucocorticoid receptor (GR) is located in the HBD (Bocquel et al., 1989; Hollenberg and Evans, 1988; Meyer et al., 1990; Webster et al., 1988a). A second function, TAF-1, is present in the N-terminal regions A/B (Berry et al., 1990; Bocquel et al., 1989; Godowski et al., 1988; Hollenberg and Evans, 1988; Tora et al., 1989b). Both TAFs operate in a cell- and promoterspecific fashion (Berry et al., 1990; Bocquel et al., 1989; Tora et al., 1989b). AP- 1, a transcription factor whose components are nuclear proteins encoded by c-fos and c-jun proto-oncogenes, is implicated in diverse aspects of cell growth, differentiation and development and was originally identified as interacting with phorbol ester (TPA)-response promoter elements (TPAREs or TREs) (Angel et al., 1987; Lee et al., 1987). AP-1 consists of dimers formed between protein members of the fos and jun gene families. A TRE is recognized by c-Jun homodimers or c-Jun/c-Fos heterodimers; the binding of c-Fos to the TRE is dependent on heterodimerization with c-Jun. Homo- and heterodimerization are mediated through non-covalent interactions facilitated by so-called 'leucine zippers' (LZs; Landschulz et al., 1988). A basic region (BR) adjacent to the LZs of c-Fos and c-Jun is believed to mediate DNA binding of the AP- 1 complex (Kouzarides and Ziff, 1989; Verma and Ransone, 1989; Vogt and Morgan, 1990). It has been reported previously that the expression of certain oncogene products may alter the ability of the GR to activate transcription of target genes (Hamilton and DeFranco, 1989; Jaggi et al., 1986), but the mechanism by which this was achieved was unclear. Recently, 'cross-talks' between the signal transduction pathways employing steroid receptors and the c-fos/c-jun oncogene products have been reported and attempts have been made to understand the underlying molecular events. Apparently, three phenomenologically distinct classes of effects have been observed: (i) in the case of the ovalbumin promoter, the DNA binding sites for the estrogen receptor and c-Jun/c-Fos overlap, but estrogen-mediated induction of transcription did not require the ER DBD (Gaub et al., 1990); (ii) in the case of the proliferin gene promoter, a 'composite' GRE could bind both the GR and the c-Fos/c-Jun complex and the relative levels of the two oncogene products determined whether the transcriptional response in the presence of hormone was positive or negative (Diamond et al., 1990); and (iii) mutual transcriptional inhibition was observed between the GR and c-Jun and/or c-Fos, even when the inhibitor was unable to bind to the response element of the activator (Jonat et al., 1990; Lucibello et al., 1990; Schuele et al., 1990; Yang-Yen et al., 1990). In the latter case, some of the groups have reported that overexpressed or in vitro synthesized c-Jun and GR can reciprocally inhibit each other's DNA binding ability in vitro (Schuele et al., 1990; Yang-Yen et al., 1990). In addition, some reported the formation of c-Jun/GR com3839
L.Shemshedini et al.
plexes in vitro (Diamond et al., 1990; Jonat et al., 1990; Yang-Yen et al., 1990), while others could not observe complexes between the GR and c-Jun (Schuele et al., 1990) or c-Fos (Lucibello et al., 1990). In some of these reports, no clear distinctions have been made between the effects exerted by c-Jun and c-Fos, fostering the impression that the two oncogene products act similarly. In the initial phase of this study, we observed that the transcription activation by the PR was inhibited when c-Fos was co-expressed, while the co-expression of c-Jun had no effect. Since this result was unexpected in view of the observations described above for the GR, we systematically analysed the effects of c-Jun and/or c-Fos on the ability of the PR, GR and androgen receptor (AR), all of which can bind the same response elements (Beato, 1989; Ham et al., 1988; Simental et al., 1991), to activate different target genes in various cell types. We further designed experiments to try to determine the type of mechanism(s) underlying the c-Fos effect on PR-induced transcription, and show that none of the previously proposed mechanisms can satisfactorily explain our observations.
Results Hormone-dependent transcription activation by the progesterone, glucocorticoid and androgen receptors is differently affected by co-expression of c-Fos or c-Jun Co-expression in HeLa cells of c-Fos strongly inhibited transcription activation of the MMTV promoter by the hPR form B/R5020 complex (Figure IA, compare lanes 8 and 9; for a description of the hPR isoforms A and B, see Kastner et al., 1990 and refs therein). In contrast, co-expression of c-Jun did not affect hPR-induced MMTV transcription (compare lanes 8 and 10). The simultaneous presence of c-Fos and c-Jun had the same effect on hPR activity as did c-Fos alone (data not shown). Transcriptional activation by hPR form A was similarly inhibited by c-Fos (data not shown). Since MMTV-CAT is a reporter gene also for the glucocorticoid and androgen receptors (Ham et al., 1988), we tested the effect of c-Fos and c-Jun co-expression on the ability of these receptors to activate transcription. Under identical conditions to those for the hPR, both c-Fos and c-Jun inhibited hGR-induced MMTV transcription [lanes 12-14; inhibition of the endogenous hGR in HeLa cells was seen as well (lanes 4-6), although it was more evident when higher amounts of extracts were used for the CAT assay]. However, in striking contrast to the data obtained with hPR or hGR, MMTV transcription induced by the androgen receptor (hAR) was strongly stimulated when c-Jun was coexpressed (compare lanes 16 and 18). Moreover, hAR activity was not (or very weakly) affected when c-Fos was co-expressed at levels which strongly inhibited hPR or hGR activity (compare lanes 16 and 17). No effect on hPRinduced MMTV transcription was observed with parental expression vectors, or when another nuclear proto-oncogene, c-myc, was co-expressed (data not shown). Thus, we conclude that increased levels of c-Fos or c-Jun have very different effects on the abilities of hPR, hGR and hAR to activate transcription from the MMTV-CAT reporter recombinant. Furthermore, it seems unlikely that this effect is mediated by c-Fos/c-Jun heterodimers, since one of the
3840
two proto-oncogenes suffices to produce the respective
receptor-specific effect. Cell- and promoter-specific differences Similar effects to those described above were seen when transfections were done in CVI (Figure 1B), 3T3 (Figure IC) or P19 (Figure ID) cells, with the exceptions that c-Fos clearly inhibited hAR-induced MMTV-CAT transcription in these cells (compare Figures lB and D, lanes 14 and 15, and Figure IC, lanes 16 and 17) and that hGR activity in 3T3 cells was, relative to the inhibition by c-Fos, much less inhibited by c-Jun (compare Figures lA and C, lanes 12-14). However, the pattern of effects exerted by the two proto-oncogenes diverged significantly from that seen with the MMTV-CAT when different promoters, all of which are responsive to hPR, hGR and hAR (see the respective lanes in Figure 2), were used in the four cell lines. With GRE-tk, hPR-induced transcription was inhibited by c-Fos in HeLa and CVl cells (Figure lA and B, compare lanes 8 and 9, and lanes 6 and 7, respectively), as well as in 3T3 and P19 cells (Table I), while co-expression of c-Jun enhanced the activity of hPR in CVI, 3T3 and P19 cells (Figure 2B, compare lanes 6 and 8; Table I) but not in HeLa cells (Figure 2A compare lanes 8 and 10). No effect of c-Fos was seen on hGR-dependent stimulation of GRE-tk-CAT transcription when the hGR expression vector was co-transfected (Figure 2A, compare lanes 12 and 13). This is, however, very likely due to high levels of hGR overcoming the inhibitory effect of c-Fos, since the activation by the endogenous hGR is inhibited by c-Fos (Figure 2A, compare lanes 4 and 5; note that with the same batch of HeLa cells GRE-tk is more strongly activated by the endogenous hGR than is the MMTV promoter). These results suggest already that the effect may be dependent on the relative abundance of activating receptor and c-Fos (see below) and demonstrate that the cell type is important for the type of response (inhibition, no effect, stimulation) that will occur in the presence of these protooncogenes. It is important to note that the basal transcription of the MMTV, GRE-TATA or tk promoter was not affected by c-Fos or c-Jun (data not shown). The androgen receptor is a very weak activator of GREtk-CAT and thus no induction of transcription could be detected when HeLa cells were exposed to dihydrotestosterone (DHT) (Figure 2A, compare lanes 15 and 16); only weak activation was seen in CVl cells (Figure 2B, compare lanes 13 and 14). However, c-Jun strongly stimulated DHT-induced transcription of GRE-tk-CAT in all four cell lines tested (Figure 2A and B, compare lanes 16 and 18, and lanes 14 and 16, respectively; Table I), although c-Jun itself was unable to activate the GRE-tk promoter (data not shown). In order to investigate whether the observed effects could be related to the complexity of the MMTV or tk promoters, we used as reporter gene GRE-TATA-CAT (see Materials and methods) containing a minimal promoter. Activation of GRE-TATA-CAT by hPR, hGR and hAR was, in general, less affected by c-Fos or c-Jun than that of the more complex promoters (Table I). While c-Fos inhibited activation by all three receptors in CV1 cells (Figure 2D, compare lanes 6 and 7, 10 and 11, and 14 and 15), it was without effect in P19 cells (Table I; hAR-induced transcription has not been analysed). In HeLa cells, only hPR-dependent activation was
Nuclear receptors, Fos and Jun: transcriptional interaction
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Fig. 1. Co-expression of c-Fos or c-Jun results in cell- and receptor-specific effects on the MMTV-CAT transcription induced by the progesterone, glucocorticoid or androgen receptor. (A) HeLa cells were transfected with 1 isg MMTV-CAT, expression vectors encoding hPR form B (0.5 jsg hPR1; lanes 7-10), hGR (1 1tg HGO; lanes 11-14) or hAR (1 yg hAR1; lanes 15-18) and with vectors (1.5 /ig) expressing c-Fos (lanes 9, 13 and 17) or c-Jun (lanes 10, 14 and 18). The effects of c-Fos (lane 5) and c-Jun (lane 6) on transactivation by the endogenous GR of HeLa cells (lane 4) were investigated as well. Cognate steroid hormones (20 nM dihydrotestosterone, DHT; 20 nM dexamethasone, DEX; 20 nM R5020) were administered as illustrated at the top. Controls involve the effect of progestin (R5020, lane 2) and androgen (DHT, lane 3) on MMTV-CAT transcription (lane 1) in the absence of co-transfected receptors. In the absence of cognate hormones, reporter gene transcription is not stimulated by the recombinant hPR (lane 7), hGR (lane 11) or hAR (lane 15). The following inhibition of receptor-induced transcription was measured in the presence of c-Fos or c-Jun: lane 9, 59%; lane 13, 67%; lane 14, 58%. c-Jun increased hAR-induced transcription by 650% (lane 18). (B-D) Similar analysis to A, except that CV1 (B), 3T3 (C) and P19 cells (D) were co-transfected and exposed to hormone as illustrated at the top. CV1 and P19 cells apparently do not express sufficient levels of endogenous GR to activate MMTV-CAT transcription (lanes 4 in B and D). Therefore, in these cells the effect of c-Fos and c-Jun co-expression on GR-induced transcription was assayed only with exogenously added receptor. Note that the weak stimulation by c-Jun of hPR-induced transcription in CV1 cells (Figure lB, lane 8) was not seen in two other experiments. The following inhibition of receptor-induced transcription was measured in the presence of c-Fos or c-Jun: (B) lane 7, 33%; lane 11, 64%; lane 12, 45%; lane 15, 43%; (C) lane 9, 33%; lane 13, 87%; lane 14, 27%; lane 17, 24%; (D) lane 7, 35%; lane 11, 74%; lane 12, 63%; lane 15, 40%. c-Jun increased hAR-induced transcription by 200% (B, lane 16); 206% (C, lane 18); 120% (D, lane 16).
c-Fos sensitive (Figure 2c, compare lanes 8 and 9) and only hGR-induced activation was inhibited by c-Jun (compare lanes 12 and 14), but in both cases the inhibition was much less pronounced than with the complex promoters. While in HeLa cells hAR/DHT-induced activation of the minimal promoter was not affected by either c-Fos or c-Jun (Figure 2C, lanes 15- 17; Table I), in CV 1 cells it was inhibited by c-Fos and stimulated by c-Jun. In summary, we have presented evidence that (i) c-Fos and c-Jun have different effects on the same receptor in the same cell type, (ii) c-Fos can inhibit and (iii) c-Jun can either inhibit or further stimulate receptor-induced transcription, (iv) and that these effects are cell specific and (v) related to the nature of the respective target gene promoter. That only the induced, but not the basal, activity of the target gene promoters is affected suggests that c-Fos and c-Jun may exert their negative effect directly on the receptor (by somehow
disabling it from activating transcription) or indirectly on transcriptional intermediary factors (TIFs) and/or certain promoter-associated factors. Exogenous c-Fos or exposure to forskolin inhibits activation of GRE-tk-CAT by the endogenous progesterone receptor in T47D, but not in MCF-7 cells We tested whether the endogenous hPR would also be affected by transfecting c-fos expression vectors or, alternatively, by increasing the endogenous c-Fos levels by exposing cells to forskolin (Sassone-Corsi et al., 1988b) [note that forskolin does not induce c-Jun transcription (Sassone-Corsi et al., 1990)]. Interestingly, only one of the two breast cancer cell lines tested responded to changes in the intracellular c-Fos levels. Transcriptional activation of GRE-tk-CAT, induced by exposing T47D cells to R5020, 3841
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Fig. 2. Cell- and promoter-specific effects of co-expressed c-Fos or c-Jun on the transcription activation by steroid hormone receptors. As described Figure for Figure 1, HeLa (A and C) or CVI cells (B and D) were transfected with steroid receptor (amounts of receptor expression vectors as inamounts the at illustration top; see nM; hormones (20 various the of or presence in the absence either vectors JLg), (3 IA) and oncogene expression and concentrations as in Figure 1), except that the reporter genes were GRE-tk-CAT (1 yg in A and B) or GRE-TATA-CAT (1 ,ug in C and D). The following inhibition of receptor-induced transcription was measured in the presence of c-Fos or c-Jun: (A) lane 9, 73%; lane 14, 49%; (B) 15, lane 7, 67%; lane 11, 76%; lane 12, 35%; lane 15, 43%; (C) lane 9, 67%; lane 14, 30%; (D) lane 7, 78%; lane 11, 96%; lane 12, 83%, lane 74%. c-Jun increased hPR- or hAR-induced transcription by 147% (A, lane 18); 220% (B, lane 8); 60% (B, lane 16); 41% (D, lane 8); 152% (D, lane 16).
could be entirely inhibited when the c-fos expression vector was co-transfected (Figure 3, compare lanes 8 and 10). Importantly, a significant reduction of R5020-induced transcription was also observed when T47D cells were exposed to forskolin (compare lanes 8 and 12). In contrast, in MCF-7 cells neither exogenously added c-Fos nor exposure to forskolin inhibited R5020-induced transcription activation of GRE-tk-CAT (compare lanes 2, 4 and 6). Identical results were obtained with transfected c-Fos when the hPR concentration in MCF-7 or T47D cells was increased by co-transfecting hPR1 (data not shown). Together, these results show (i) that the cell-specific inhibition of PR-induced transcription by c-Fos can be reproduced with two breast cancer cell lines and (ii) that the effect observed with co-transfected hPR and c-Fos can be reproduced with endogenous receptor and proto-oncogene product. Therefore, we conclude that variations in the endogenous levels of c-Fos may have a profound effect on gene activation by progestins. Inhibition of hPR-induced activation by c-Fos involves a competitive mechanism
The inhibition of hPR-mediated GRE-tk-CAT transcription by c-Fos is a dose-dependent process. The activation induced
3842
with 20 ng of hPR1 steadily decreased with increasing amounts of c-fos expression vector, with almost no activation seen in the presence of 2 Atg of c-fos (Figure 4A, black bars). When the hPRI amount was increased to 100 ng, an increased amount of c-fos was required for the same level of inhibition, suggesting that the relative concentrations of hPR and c-Fos are important for the inhibitory effect. Indeed, increasing the amount of co-expressed hPR relieved this inhibition in a dose-dependent fashion. The hPR expressed from 4 or 10 Itg hPRI completely outcompeted the c-Fos effect [Figure 4B, compare the black and striped bars; note that due to 'squelching' or 'auto-inhibition' (Bocquel et al., 1989) higher amounts of hPR1 activate less]. We conclude that the inhibitory action of c-Fos on hPR-induced transcription is based on a truly competitive mechanism and it is tempting to speculate that it involves either the receptor itself or a critical component of the transactivation process that can be used/sequestered by both c-Fos and hPR. Fos mutants with a disabled leucine zipper are inefficient inhibitors of hPR-mediated transcriptional stimulation To determine which region(s) of c-Fos could be involved in the inhibition of hPR-induced transcription, we tested a
Nuclear receptors, Fos and Jun: transcriptional interaction Table I. Schematic summary of the effects of co-expressed c-Jun or c-Fos on the ability of the progesterone (PR), glucocorticoid (GR) and androgen receptor (AR) to activate three promoters (MMTV, GRE-tk and GRE-TATA) in four different cell lines (given at the top) HeLa fos jun
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MMTV
Cell-specific inhibitory and stimulatory effects of Fos and Jun on transcription activation by nuclear receptors
Lirim Shemshedini, Rudolf Knauthe, Paolo Sassone-Corsi, Astrid Pornon and Hinrich Gronemeyer Laboratoire de Genetique Mol6culaire des Eucaryotes, CNRS, Unite 184 de Biologie Moleculaire et de Gednie Genetique de 1'INSERM, Institut de Chimie Biologique, Faculte de Medecine, 11, rue Humann, 67085 Strasbourg Cedex, France Communicated by P.Chambon
We investigated the effect of c-Fos and/or c-Jun coexpression on transcription activation by the progesterone (PR), glucocorticoid (GR) or androgen (AR) receptors using three different reporter genes and four different cell lines. We found that c-Fos could only inhibit, while c-Jun could either inhibit or further stimulate receptorinduced transcription. All these effects were receptor, promoter, and cell type specific, and, importantly, the steroid receptors had non-reciprocal effects on the transactivation ability of c-Jun in the presence or absence of c-Fos. Collectively, these results argue against heterodimer formation as a mechanism to explain the phenomena. Transactivation by the endogenous PR in T47D cells could be inhibited by increasing the intracellular c-Fos level with forskolin as well as by coexpressing c-Fos; no such effect was seen in MCF-7 cells. The inhibition by c-Fos of PR-induced transcription involves a competitive mechanism, which requires the presence of the intact c-Fos leucine zipper and is directed mainly at the transcription activation function (TAF) located in the PR and GR hormone binding domains (TAF-2). However, the co-expression of c-Fos did not alter the 'squelching/transcriptional interference' by the PR of estrogen receptor (ER)-induced transcription. Multiple mechanisns are discussed which may be involved in the crosstalk between the two signal transduction pathways. Key words: Fos/Jun/nuclear receptors/transcription activation
Introduction Steroid receptors are ligand-inducible trans-acting transcription factors. Their major domains, a DNA (DBD) and a hormone binding domain (HBD), have been identified as segments of the receptor primary structure (Beato, 1989; Evans, 1988; Green and Chambon, 1988). While the DBD has been investigated in great detail (Danielsen et al., 1989; Mader et al., 1989; Umesono and Evans, 1989), the function of the HBD is, due to its complexity, less well understood. Apart from binding the hormone, the estrogen receptor HBD harbours a dimerization domain (Fawell et al., 1990; Kumar and Chambon, 1988), and in the case of the PR it has been demonstrated that hormone binding induces receptor dimerization in vivo (Guiochon-Mantel et al., 1989). We and (©) Oxford University Press
others have shown that one (TAF-2) of the two transcription activation functions (TAFs) of the estrogen (ER), progesterone (PR) and glucocorticoid receptor (GR) is located in the HBD (Bocquel et al., 1989; Hollenberg and Evans, 1988; Meyer et al., 1990; Webster et al., 1988a). A second function, TAF-1, is present in the N-terminal regions A/B (Berry et al., 1990; Bocquel et al., 1989; Godowski et al., 1988; Hollenberg and Evans, 1988; Tora et al., 1989b). Both TAFs operate in a cell- and promoterspecific fashion (Berry et al., 1990; Bocquel et al., 1989; Tora et al., 1989b). AP- 1, a transcription factor whose components are nuclear proteins encoded by c-fos and c-jun proto-oncogenes, is implicated in diverse aspects of cell growth, differentiation and development and was originally identified as interacting with phorbol ester (TPA)-response promoter elements (TPAREs or TREs) (Angel et al., 1987; Lee et al., 1987). AP-1 consists of dimers formed between protein members of the fos and jun gene families. A TRE is recognized by c-Jun homodimers or c-Jun/c-Fos heterodimers; the binding of c-Fos to the TRE is dependent on heterodimerization with c-Jun. Homo- and heterodimerization are mediated through non-covalent interactions facilitated by so-called 'leucine zippers' (LZs; Landschulz et al., 1988). A basic region (BR) adjacent to the LZs of c-Fos and c-Jun is believed to mediate DNA binding of the AP- 1 complex (Kouzarides and Ziff, 1989; Verma and Ransone, 1989; Vogt and Morgan, 1990). It has been reported previously that the expression of certain oncogene products may alter the ability of the GR to activate transcription of target genes (Hamilton and DeFranco, 1989; Jaggi et al., 1986), but the mechanism by which this was achieved was unclear. Recently, 'cross-talks' between the signal transduction pathways employing steroid receptors and the c-fos/c-jun oncogene products have been reported and attempts have been made to understand the underlying molecular events. Apparently, three phenomenologically distinct classes of effects have been observed: (i) in the case of the ovalbumin promoter, the DNA binding sites for the estrogen receptor and c-Jun/c-Fos overlap, but estrogen-mediated induction of transcription did not require the ER DBD (Gaub et al., 1990); (ii) in the case of the proliferin gene promoter, a 'composite' GRE could bind both the GR and the c-Fos/c-Jun complex and the relative levels of the two oncogene products determined whether the transcriptional response in the presence of hormone was positive or negative (Diamond et al., 1990); and (iii) mutual transcriptional inhibition was observed between the GR and c-Jun and/or c-Fos, even when the inhibitor was unable to bind to the response element of the activator (Jonat et al., 1990; Lucibello et al., 1990; Schuele et al., 1990; Yang-Yen et al., 1990). In the latter case, some of the groups have reported that overexpressed or in vitro synthesized c-Jun and GR can reciprocally inhibit each other's DNA binding ability in vitro (Schuele et al., 1990; Yang-Yen et al., 1990). In addition, some reported the formation of c-Jun/GR com3839
L.Shemshedini et al.
plexes in vitro (Diamond et al., 1990; Jonat et al., 1990; Yang-Yen et al., 1990), while others could not observe complexes between the GR and c-Jun (Schuele et al., 1990) or c-Fos (Lucibello et al., 1990). In some of these reports, no clear distinctions have been made between the effects exerted by c-Jun and c-Fos, fostering the impression that the two oncogene products act similarly. In the initial phase of this study, we observed that the transcription activation by the PR was inhibited when c-Fos was co-expressed, while the co-expression of c-Jun had no effect. Since this result was unexpected in view of the observations described above for the GR, we systematically analysed the effects of c-Jun and/or c-Fos on the ability of the PR, GR and androgen receptor (AR), all of which can bind the same response elements (Beato, 1989; Ham et al., 1988; Simental et al., 1991), to activate different target genes in various cell types. We further designed experiments to try to determine the type of mechanism(s) underlying the c-Fos effect on PR-induced transcription, and show that none of the previously proposed mechanisms can satisfactorily explain our observations.
Results Hormone-dependent transcription activation by the progesterone, glucocorticoid and androgen receptors is differently affected by co-expression of c-Fos or c-Jun Co-expression in HeLa cells of c-Fos strongly inhibited transcription activation of the MMTV promoter by the hPR form B/R5020 complex (Figure IA, compare lanes 8 and 9; for a description of the hPR isoforms A and B, see Kastner et al., 1990 and refs therein). In contrast, co-expression of c-Jun did not affect hPR-induced MMTV transcription (compare lanes 8 and 10). The simultaneous presence of c-Fos and c-Jun had the same effect on hPR activity as did c-Fos alone (data not shown). Transcriptional activation by hPR form A was similarly inhibited by c-Fos (data not shown). Since MMTV-CAT is a reporter gene also for the glucocorticoid and androgen receptors (Ham et al., 1988), we tested the effect of c-Fos and c-Jun co-expression on the ability of these receptors to activate transcription. Under identical conditions to those for the hPR, both c-Fos and c-Jun inhibited hGR-induced MMTV transcription [lanes 12-14; inhibition of the endogenous hGR in HeLa cells was seen as well (lanes 4-6), although it was more evident when higher amounts of extracts were used for the CAT assay]. However, in striking contrast to the data obtained with hPR or hGR, MMTV transcription induced by the androgen receptor (hAR) was strongly stimulated when c-Jun was coexpressed (compare lanes 16 and 18). Moreover, hAR activity was not (or very weakly) affected when c-Fos was co-expressed at levels which strongly inhibited hPR or hGR activity (compare lanes 16 and 17). No effect on hPRinduced MMTV transcription was observed with parental expression vectors, or when another nuclear proto-oncogene, c-myc, was co-expressed (data not shown). Thus, we conclude that increased levels of c-Fos or c-Jun have very different effects on the abilities of hPR, hGR and hAR to activate transcription from the MMTV-CAT reporter recombinant. Furthermore, it seems unlikely that this effect is mediated by c-Fos/c-Jun heterodimers, since one of the
3840
two proto-oncogenes suffices to produce the respective
receptor-specific effect. Cell- and promoter-specific differences Similar effects to those described above were seen when transfections were done in CVI (Figure 1B), 3T3 (Figure IC) or P19 (Figure ID) cells, with the exceptions that c-Fos clearly inhibited hAR-induced MMTV-CAT transcription in these cells (compare Figures lB and D, lanes 14 and 15, and Figure IC, lanes 16 and 17) and that hGR activity in 3T3 cells was, relative to the inhibition by c-Fos, much less inhibited by c-Jun (compare Figures lA and C, lanes 12-14). However, the pattern of effects exerted by the two proto-oncogenes diverged significantly from that seen with the MMTV-CAT when different promoters, all of which are responsive to hPR, hGR and hAR (see the respective lanes in Figure 2), were used in the four cell lines. With GRE-tk, hPR-induced transcription was inhibited by c-Fos in HeLa and CVl cells (Figure lA and B, compare lanes 8 and 9, and lanes 6 and 7, respectively), as well as in 3T3 and P19 cells (Table I), while co-expression of c-Jun enhanced the activity of hPR in CVI, 3T3 and P19 cells (Figure 2B, compare lanes 6 and 8; Table I) but not in HeLa cells (Figure 2A compare lanes 8 and 10). No effect of c-Fos was seen on hGR-dependent stimulation of GRE-tk-CAT transcription when the hGR expression vector was co-transfected (Figure 2A, compare lanes 12 and 13). This is, however, very likely due to high levels of hGR overcoming the inhibitory effect of c-Fos, since the activation by the endogenous hGR is inhibited by c-Fos (Figure 2A, compare lanes 4 and 5; note that with the same batch of HeLa cells GRE-tk is more strongly activated by the endogenous hGR than is the MMTV promoter). These results suggest already that the effect may be dependent on the relative abundance of activating receptor and c-Fos (see below) and demonstrate that the cell type is important for the type of response (inhibition, no effect, stimulation) that will occur in the presence of these protooncogenes. It is important to note that the basal transcription of the MMTV, GRE-TATA or tk promoter was not affected by c-Fos or c-Jun (data not shown). The androgen receptor is a very weak activator of GREtk-CAT and thus no induction of transcription could be detected when HeLa cells were exposed to dihydrotestosterone (DHT) (Figure 2A, compare lanes 15 and 16); only weak activation was seen in CVl cells (Figure 2B, compare lanes 13 and 14). However, c-Jun strongly stimulated DHT-induced transcription of GRE-tk-CAT in all four cell lines tested (Figure 2A and B, compare lanes 16 and 18, and lanes 14 and 16, respectively; Table I), although c-Jun itself was unable to activate the GRE-tk promoter (data not shown). In order to investigate whether the observed effects could be related to the complexity of the MMTV or tk promoters, we used as reporter gene GRE-TATA-CAT (see Materials and methods) containing a minimal promoter. Activation of GRE-TATA-CAT by hPR, hGR and hAR was, in general, less affected by c-Fos or c-Jun than that of the more complex promoters (Table I). While c-Fos inhibited activation by all three receptors in CV1 cells (Figure 2D, compare lanes 6 and 7, 10 and 11, and 14 and 15), it was without effect in P19 cells (Table I; hAR-induced transcription has not been analysed). In HeLa cells, only hPR-dependent activation was
Nuclear receptors, Fos and Jun: transcriptional interaction
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Fig. 1. Co-expression of c-Fos or c-Jun results in cell- and receptor-specific effects on the MMTV-CAT transcription induced by the progesterone, glucocorticoid or androgen receptor. (A) HeLa cells were transfected with 1 isg MMTV-CAT, expression vectors encoding hPR form B (0.5 jsg hPR1; lanes 7-10), hGR (1 1tg HGO; lanes 11-14) or hAR (1 yg hAR1; lanes 15-18) and with vectors (1.5 /ig) expressing c-Fos (lanes 9, 13 and 17) or c-Jun (lanes 10, 14 and 18). The effects of c-Fos (lane 5) and c-Jun (lane 6) on transactivation by the endogenous GR of HeLa cells (lane 4) were investigated as well. Cognate steroid hormones (20 nM dihydrotestosterone, DHT; 20 nM dexamethasone, DEX; 20 nM R5020) were administered as illustrated at the top. Controls involve the effect of progestin (R5020, lane 2) and androgen (DHT, lane 3) on MMTV-CAT transcription (lane 1) in the absence of co-transfected receptors. In the absence of cognate hormones, reporter gene transcription is not stimulated by the recombinant hPR (lane 7), hGR (lane 11) or hAR (lane 15). The following inhibition of receptor-induced transcription was measured in the presence of c-Fos or c-Jun: lane 9, 59%; lane 13, 67%; lane 14, 58%. c-Jun increased hAR-induced transcription by 650% (lane 18). (B-D) Similar analysis to A, except that CV1 (B), 3T3 (C) and P19 cells (D) were co-transfected and exposed to hormone as illustrated at the top. CV1 and P19 cells apparently do not express sufficient levels of endogenous GR to activate MMTV-CAT transcription (lanes 4 in B and D). Therefore, in these cells the effect of c-Fos and c-Jun co-expression on GR-induced transcription was assayed only with exogenously added receptor. Note that the weak stimulation by c-Jun of hPR-induced transcription in CV1 cells (Figure lB, lane 8) was not seen in two other experiments. The following inhibition of receptor-induced transcription was measured in the presence of c-Fos or c-Jun: (B) lane 7, 33%; lane 11, 64%; lane 12, 45%; lane 15, 43%; (C) lane 9, 33%; lane 13, 87%; lane 14, 27%; lane 17, 24%; (D) lane 7, 35%; lane 11, 74%; lane 12, 63%; lane 15, 40%. c-Jun increased hAR-induced transcription by 200% (B, lane 16); 206% (C, lane 18); 120% (D, lane 16).
c-Fos sensitive (Figure 2c, compare lanes 8 and 9) and only hGR-induced activation was inhibited by c-Jun (compare lanes 12 and 14), but in both cases the inhibition was much less pronounced than with the complex promoters. While in HeLa cells hAR/DHT-induced activation of the minimal promoter was not affected by either c-Fos or c-Jun (Figure 2C, lanes 15- 17; Table I), in CV 1 cells it was inhibited by c-Fos and stimulated by c-Jun. In summary, we have presented evidence that (i) c-Fos and c-Jun have different effects on the same receptor in the same cell type, (ii) c-Fos can inhibit and (iii) c-Jun can either inhibit or further stimulate receptor-induced transcription, (iv) and that these effects are cell specific and (v) related to the nature of the respective target gene promoter. That only the induced, but not the basal, activity of the target gene promoters is affected suggests that c-Fos and c-Jun may exert their negative effect directly on the receptor (by somehow
disabling it from activating transcription) or indirectly on transcriptional intermediary factors (TIFs) and/or certain promoter-associated factors. Exogenous c-Fos or exposure to forskolin inhibits activation of GRE-tk-CAT by the endogenous progesterone receptor in T47D, but not in MCF-7 cells We tested whether the endogenous hPR would also be affected by transfecting c-fos expression vectors or, alternatively, by increasing the endogenous c-Fos levels by exposing cells to forskolin (Sassone-Corsi et al., 1988b) [note that forskolin does not induce c-Jun transcription (Sassone-Corsi et al., 1990)]. Interestingly, only one of the two breast cancer cell lines tested responded to changes in the intracellular c-Fos levels. Transcriptional activation of GRE-tk-CAT, induced by exposing T47D cells to R5020, 3841
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Fig. 2. Cell- and promoter-specific effects of co-expressed c-Fos or c-Jun on the transcription activation by steroid hormone receptors. As described Figure for Figure 1, HeLa (A and C) or CVI cells (B and D) were transfected with steroid receptor (amounts of receptor expression vectors as inamounts the at illustration top; see nM; hormones (20 various the of or presence in the absence either vectors JLg), (3 IA) and oncogene expression and concentrations as in Figure 1), except that the reporter genes were GRE-tk-CAT (1 yg in A and B) or GRE-TATA-CAT (1 ,ug in C and D). The following inhibition of receptor-induced transcription was measured in the presence of c-Fos or c-Jun: (A) lane 9, 73%; lane 14, 49%; (B) 15, lane 7, 67%; lane 11, 76%; lane 12, 35%; lane 15, 43%; (C) lane 9, 67%; lane 14, 30%; (D) lane 7, 78%; lane 11, 96%; lane 12, 83%, lane 74%. c-Jun increased hPR- or hAR-induced transcription by 147% (A, lane 18); 220% (B, lane 8); 60% (B, lane 16); 41% (D, lane 8); 152% (D, lane 16).
could be entirely inhibited when the c-fos expression vector was co-transfected (Figure 3, compare lanes 8 and 10). Importantly, a significant reduction of R5020-induced transcription was also observed when T47D cells were exposed to forskolin (compare lanes 8 and 12). In contrast, in MCF-7 cells neither exogenously added c-Fos nor exposure to forskolin inhibited R5020-induced transcription activation of GRE-tk-CAT (compare lanes 2, 4 and 6). Identical results were obtained with transfected c-Fos when the hPR concentration in MCF-7 or T47D cells was increased by co-transfecting hPR1 (data not shown). Together, these results show (i) that the cell-specific inhibition of PR-induced transcription by c-Fos can be reproduced with two breast cancer cell lines and (ii) that the effect observed with co-transfected hPR and c-Fos can be reproduced with endogenous receptor and proto-oncogene product. Therefore, we conclude that variations in the endogenous levels of c-Fos may have a profound effect on gene activation by progestins. Inhibition of hPR-induced activation by c-Fos involves a competitive mechanism
The inhibition of hPR-mediated GRE-tk-CAT transcription by c-Fos is a dose-dependent process. The activation induced
3842
with 20 ng of hPR1 steadily decreased with increasing amounts of c-fos expression vector, with almost no activation seen in the presence of 2 Atg of c-fos (Figure 4A, black bars). When the hPRI amount was increased to 100 ng, an increased amount of c-fos was required for the same level of inhibition, suggesting that the relative concentrations of hPR and c-Fos are important for the inhibitory effect. Indeed, increasing the amount of co-expressed hPR relieved this inhibition in a dose-dependent fashion. The hPR expressed from 4 or 10 Itg hPRI completely outcompeted the c-Fos effect [Figure 4B, compare the black and striped bars; note that due to 'squelching' or 'auto-inhibition' (Bocquel et al., 1989) higher amounts of hPR1 activate less]. We conclude that the inhibitory action of c-Fos on hPR-induced transcription is based on a truly competitive mechanism and it is tempting to speculate that it involves either the receptor itself or a critical component of the transactivation process that can be used/sequestered by both c-Fos and hPR. Fos mutants with a disabled leucine zipper are inefficient inhibitors of hPR-mediated transcriptional stimulation To determine which region(s) of c-Fos could be involved in the inhibition of hPR-induced transcription, we tested a
Nuclear receptors, Fos and Jun: transcriptional interaction Table I. Schematic summary of the effects of co-expressed c-Jun or c-Fos on the ability of the progesterone (PR), glucocorticoid (GR) and androgen receptor (AR) to activate three promoters (MMTV, GRE-tk and GRE-TATA) in four different cell lines (given at the top) HeLa fos jun
CVi fos jun
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