We have shown in this study that the cytoplasmic localization of ERs correlates with efficient transactivation of the AP-1-regulated collagenase promoter in response to 17β-estradiol, while ERs present in the nucleus repress AP-1 activity under the same conditions. We suggest that this reversed 17β-estradiol-induced response is due to the ability of ERs to induce AP-1 activity via non-genomic signalling when they are present in the cytoplasm.
It is generally believed that ER-mediated regulation of AP-1-dependent transcription results from protein-protein interactions on the promoter. ERα interacts with c-Jun in vitro
] and the domain required for this interaction has been mapped to amino acids 259-302, which are located in the hinge domain of the receptor [22
]. The finding that NLSA, which lacks the entire hinge domain, efficiently activated the coll-73-luc reporter in response to 17β-estradiol (Fig. ) suggests that NLSA induces AP-1 activity through a distinct mechanism, independently of physical interaction with c-Jun on DNA. Furthermore, the coll-73-luc reporter was similarly induced in response to 17β-estradiol upon co-transfection with a cytoplasmic ERβ variant (ERβ 148-530 Flag) (Fig. ), suggesting that non-genomic signalling is involved. The finding that ERβ 148-530 Flag also activated the Stat-regulated β-casein reporter in response to 17β-estradiol (Fig. ), as has been previously shown for NLSA [17
], supports our hypothesis that the deletion mutant acts via non-genomic signalling in the cytoplasm. In contrast, however, a corresponding deletion mutant comprising a Flag tag upstream of the N-terminus (ERβ Flag 148-530) was present in the nucleus, and hence was unable to activate the coll-73-luc reporter via non-genomic signalling (Fig. ). Our results suggest that cytoplasmic re-localization of ERs and subsequent signaling through cytoplasmic signal transduction pathways is an additional mechanism by which ERs are able to modulate the transcriptional activity of AP-1. It should be noted that there are discrepancies in the literature regarding ER action at AP-1 sites. In accordance with our results, repression of the collagenase promoter in the presence of ERα and ERβ bound to 17β-estradiol has been described [9
]. In contrast, however, other studies have shown that ERα bound to 17β-estradiol induces activation of AP-1 [6
]. Differences in experimental conditions could explain some of the discrepancies obtained with ERs acting in the nucleus.
We recently reported that the introduction of various point mutations into the ERβ DBD results in an altered response by ER ligands on the collagenase promoter [11
]. The results presented here suggest a mechanism for this phenomenon. Disruption of the second zinc binding motif (as in the ERβ mutant C201A/C204A) resulted in altered localization of the receptor in the presence of 17β-estradiol. The coll-73-luc reporter was also efficiently activated under these conditions (Fig. and ). Furthermore, C201A/C204A activated the β-casein reporter in response to 17β-estradiol more efficiently than the wild-type-receptor, suggesting that C201A/C204A acts via non-genomic signalling in a manner similar to that in which both NLSA and ERβ 148-530 Flag act. Notably, all of the ERβ DBD mutants described in [11
] that reversed the response to 17β-estradiol on the collagenase promoter acted in the same manner (data not shown). Thus, we suggest that the introduction of various point mutations into the ERβ DBD enables the receptor to induce AP-1 activity via non-genomic signalling, due to re-localization of the receptor to the cytoplasm in the presence of 17β-estradiol. This phenomenon is not restricted to HC11 cells, since similar results were obtained in the human breast cancer cell line MCF-7, the human uterine cell line Ishikawa and the human hepatic cell line HepG2 (data not shown). In COS-7 cells, on the other hand, ERβ 148-530 Flag and the various ERβ DBD mutants, although present in the cytoplasm, only showed weak responses on the coll-73-luc reporter in the presence of 17β-estradiol, while NLSA efficiently activated the reporter (data not shown). These results are in accordance with our previous results [17
] and suggest that non-genomic signalling via ERβ is not as efficient in COS-7 cells as in HC11, MCF-7, Ishikawa, HepG2 and SYF cells, while ERα is active in all cells tested.
A natural splice variant of ERβ lacking the C-terminal part of the DBD (ERβδ3) localizes to discrete nuclear spots and activates transcription at AP-1 sites in the presence of 17β-estradiol [24
]. This agrees with the results presented here. Interestingly, however, we found that our ERβ DBD mutants, including one that lacked the entire DBD, not only localized to discrete nuclear spots, but also re-localized to the cytoplasm upon 17β-estradiol treatment. Furthermore, it has been suggested that a similar supernormal activity of an ERα DBD mutant (K206A) on the collagenase promoter arises from the inability of 17β-estradiol-bound K206A to recruite a putative co-repressor complex to the promoter. This inability would allow for effective target gene transcription [25
]. However, the finding that ERβ 148-530 Flag efficiently activated the coll-73-luc reporter in response to 17β-estradiol (Fig. ) demonstrates that the activity is not unique for receptors that have mutations within the DBD, and which would therefore be unable to interact with such a co-repressor complex through the DBD. Rather, our results suggest that ERβ 148-530 Flag and the various ERβ DBD mutants described in [11
] induce AP-1 activity via non-genomic signalling when present in the cytoplasm. We can not, however, rule out additional mechanisms by which ERs that have mutations within the DBD may act at AP-1 sites.
The ability of ER to modulate AP-1-dependent transcription in the presence of SERMs is specific for a particular subtype of ER, and this ability depends on the cellular localization of the receptor. While ERα wild-type was inactive in the presence of SERMs, ERβ wild-type activated the coll-73-luc reporter in response to tamoxifen, raloxifene, idoxifene and nafoxidene (Fig. ). In contrast, however, NLSA activated transcription in response to tamoxifen, while ERβ 148-530 Flag and C201A/C204A were inactive in the presence of all SERMs tested (Fig. and ). Notably, the inability of C201A/C204A to activate the coll-73-luc reporter in response to tamoxifen is not due to persistent nuclear localization of the protein, since the receptor re-localized to the cytoplasm also in the presence of estrogen antagonists (data not shown). Thus, tamoxifen-induced activation of AP-1 via non-genomic signalling appears to be specific for ERα. These results provide new insights into the mechanisms that underlie the tissue-specific actions of SERMs observed in vivo
. Non-genomic actions exerted by tamoxifen at AP-1 sites probably contribute to some of the responses, such as, for example, the responses in target tissues where pools of ERα are localized to the cytoplasm, such as the mammary gland, bone, brain and the cardiovascular system [26
]. Several genes involved in cell proliferation are regulated by AP-1, and thus the non-genomic actions of ERs at these sites may be highly important, and they may contribute to phenomena such as tamoxifen-resistance in ER-positive breast cancer cells. The full estrogen antagonist ICI 182,780 completely inhibited 17β-estradiol-induced activation of the coll-73-luc reporter in the presence of cytoplasmic ERs (Fig. and ), and this finding agrees with our previous results showing that non-genomic actions of ERs are inhibited in the presence of ICI 182,780 [17
]. It has been suggested that the synthetic compound estren distinguishes between genomic and non-genomic actions of ERα [32
]. Importantly, however, we found that the coll-73-luc reporter was repressed in the presence of ER wild-type receptors and estren (Fig. ), and we found that estren can induce AP-1-dependent transcription via non-genomic signalling (Fig. and ). Hence, estren affects the activity of ERs present both in the nucleus and in the cytoplasm.
MAP-kinase activity induced by 17β-estradiol results in an enhanced DNA-binding activity of AP-1 [33
] and 17β-estradiol-induced expression of cyclin D1
depends both on the AP-1 site within the promoter and on an intact MAP-kinase activity [34
]. We have shown that an intact MAP-kinase pathway is required for 17β-estradiol-induced activation of the coll-73-luc reporter, and we have shown that the activity does not depend on an intact PI3-kinase activity (Fig. ). These results agree with those of Dos Santos and those of Marino. Furthermore, Src-kinase is not required for 17β-estradiol-induced activation of AP-1, as shown by the intact responses on the coll-73-luc reporter upon co-transfection of cells devoid of Src-kinase with NLSA, ERβ 148-530 or C201A/C204A (Fig. ). We have previously shown that 17β-estradiol-induced transactivation of Stat-regulated promoters requires intact MAP-kinase, PI3-kinase and Src-kinase activities [17
]. Thus, our results suggest that distinct signal transduction pathways are involved in ER-dependent non-genomic actions targeting promoters regulated by AP-1 and by Stat proteins. The transcriptional activity of c-Jun depends only on serine phosphorylation [35
]. However, tyrosine phosphorylation is a pre-requisite for inducing nuclear translocation and DNA-binding activity of Stat proteins [36
], and this may explain why Src-kinase and PI3-kinase [16
] are required in the pathway targeting Stat-regulated promoters. Furthermore, the inability of 17β-estradiol-bound ERs to induce Src-kinase activity in the presence of estrogen antagonists [13
] might explain our previous finding that 17β-estradiol-induced activation of Stat proteins is inhibited in the presence of tamoxifen [17
]. In contrast, however, NLSA induced AP-1 activity in response to tamoxifen (Fig. ), independently of Src-kinase activity (Fig. ). Thus, the set of signal transduction proteins available in a given cell probably determines which target transcription factors are activated by ligand-bound ERs present in the cytoplasm, and hence the cellular outcome.