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Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Maria Sjöberg, Department of Cell and Molecular Biology, Karolinska Institute, S-171 Stockholm, Sweden. E-mail: maria.sjoberg{at}cmb.ki.se.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER) and ß (ERß) are able to induce transcriptional activation of signal transducer and activator of transcription (Stat)-regulated promoters via cytoplasmic signal transduction pathways. Stat5 and Stat3 are required for promoter induction, which correlates with cytoplasmic sublocalization of ERs and is independent of intact coactivator binding sites and DNA-binding domains. In endothelial cells, Stat5 and Stat3 are rapidly phosphorylated on both tyrosine and serine residues in response to 17ß-estradiol, and nuclear translocation is subsequently induced. 17ß-Estradiol-induced transactivation of a Stat-regulated promoter requires at least three different signal transduction pathways, including MAPK, Src-kinase, and phosphatidylinositol-3-kinase activities. In conclusion, this work identifies a novel pathway involving an agonist-bound ER-activated phosphorylation cascade, resulting in nuclear transcriptional activation of target transcription factors. These findings reveal novel targets for the development of drugs that modulate a nongenomic-to-genomic ER-dependent mechanism. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(ER) and ß (ERß). These receptors belong to a large superfamily of nuclear hormone receptors that share a well conserved DNA-binding domain (DBD), a structurally conserved ligand binding domain (LBD), and an N-terminal domain with no homology between the different receptors (1, 2, 3). The classical mechanism of activation depends on ligand binding, after which the receptor dimerizes and binds to estrogen response elements (EREs) located within the promoters of estrogen-responsive genes. Ligand binding also induces a conformational change in the LBD of the receptor, which allows the recruitment of coactivator proteins (4, 5). Estrogens also have very rapid effects in a variety of tissues including bone, brain, mammary gland, and vasculature. For example, estrogens have been shown to stimulate the activities of phospholipase C and adenylate cyclase, resulting in increased production of inositol lipid and cAMP, respectively (6, 7). In addition, they increase intracellular [Ca2+] and activate MAPK (8, 9, 10, 11, 12, 13) and phosphatidylinositol-3-kinase (PI3-kinase) signaling pathways (14, 15, 16, 17). Although it is not fully defined which receptors are responsible for mediating these rapid nongenomic effects of estrogens, some studies point to the existence of a separate membrane receptor unrelated to the classical nuclear receptors, while others implicate a subgroup of the classical ERs that is associated with cellular membranes (for review see Ref. 18).
Stat (signal transducers and activators of transcription) proteins are latent cytoplasmic transcription factors that are activated in response to a large number of cytokines, growth factors, and hormones. The binding of ligand to its cognate cytokine receptor induces activation of receptor-associated members of the Janus kinase (Jak) family, which in turn phosphorylate a conserved tyrosine residue in the C-terminal region of Stats. The tyrosine-phosphorylated Stats undergo dimerization and translocation to the nucleus where they regulate transcription by binding to specific DNA sequences (19). In addition to basic tyrosine phosphorylation, the transcriptional activity of Stats can be regulated by serine phosphorylation of the transactivation domain (20).
Due to the fact that Stats are activated through phosphorylation and because 17ß-estradiol is known to rapidly induce phosphorylation cascades in the cytoplasm, we were interested in investigating the possibility of Stats as downstream targets of nongenomic ER actions. In the present paper, we show that 17ß-estradiol-bound ERs activate cytoplasmic signal transduction pathways in a manner that is independent of receptor-DNA binding, resulting in activation of Stat-regulated promoters via Stat5 and Stat3 proteins. We show that tyrosine and serine phosphorylation of Stat5 and Stat3 are rapidly induced by 17ß-estradiol and that intact MAPK, PI3-kinase, and Src-kinase signaling pathways are required for 17ß-estradiol-induced activation of a Stat-regulated promoter. In summary, the mechanism described in this paper comprises a chain of reactions starting with activation of a cytoplasmic ER by 17ß-estradiol and resulting in nuclear transcriptional activation of target transcription factors, thus representing a novel pathway of nongenomic-to-genomic actions of ERs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and -ß proteins (data not shown), and we decided to analyze how 17ß-estradiol treatment would affect the phosphorylation status of Stat5 in these cells. Tyrosine and serine phosphorylation of Stat5 upon 50 nM 17ß-estradiol or 15% serum [fetal bovine serum (FBS)] treatment at various time points were analyzed by Western blot analyses of whole-cell extracts, using phospho-specific antibodies against tyrosine-694/699 and serine-725/731 of Stat5a/b, respectively. As shown in Fig. 1A
, both tyrosine and serine phosphorylation of Stat5 were rapidly induced upon 17ß-estradiol treatment and were inhibited in the presence of ICI 182,780, suggesting a requirement for an agonist-bound ER. Total levels of Stat5a and Stat5b proteins in the cell extracts are shown. An induced gel mobility shift upon 17ß-estradiol treatment is observed when immunoblotting with the Stat5b antibody (Fig. 1A, lower panel). This shift is known to be due to tyrosine phosphorylation of Stat5b, which was confirmed by the recognition of the upper band by the phospho-specific antibody.
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(upper section), there was an increased amount of Stat5 present in the nucleus when the cells had been exposed to 17ß-estradiol for 30 min, indicating that the steroid is able to promote translocation of Stat5 to the nucleus. However, the amount of Stat5 in the nucleus was almost back to basal level after 60 min, demonstrating that the steroid-induced translocation of Stat5 is a rapid and transient process. To investigate whether DNA-binding activity of Stat5 is induced upon 17ß-estradiol stimulation, an in vitro DNA-binding assay using a biotinylated Stat5 DNA-binding site was performed. As shown in Fig. 1B (lower section), in extracts prepared from cell nuclei of PAE cells treated with 17ß-estradiol or FBS for 30 min, binding of Stat5 to the consensus 
-interferon-activated sequence (GAS) element present in the ß-casein promoter was enhanced compared with untreated cells, whereas no interaction with a mutated DNA-binding site was observed.
To investigate whether the observed rapid phosphorylation of Stat5 in response to 17ß-estradiol correlated with a downstream event, we analyzed activation of a Stat5-regulated promoter after 17ß-estradiol treatment of PAE cells. As shown in Fig. 1C, activation of the ß-casein reporter gene was induced 2-fold in response to 17ß-estradiol compared with untreated cells, while FBS treatment resulted in a 2.5-fold induction.
In ER-negative COS-7 cells transiently transfected with the ß-casein reporter gene, no induction response was observed upon treatment with 17ß-estradiol alone. However, 17ß-estradiol induced promoter activity in these cells after cotransfection with ER expression plasmid (Fig. 1D). Stat5 is primarily activated by prolactin (PRL) and GH, whereupon tyrosine phosphorylation is induced by the Jak2 kinase. We therefore analyzed the requirement of Jak2 activity for 17ß-estradiol-induced activation of the ß-casein reporter gene. As shown in Fig. 1B, a dominant negative version of Jak2 (Jak2 KN) transfected into the cells did not interfere with 17ß-estradiol-induced reporter activity in the presence of ER, nor was the presence of the PRL receptor required since it is not expressed endogenously in these cells. However, as expected, Jak2 KN prevented activation of overexpressed Stat5 in response to PRL. Taken together, these results demonstrate that 17ß-estradiol rapidly induces phosphorylation, nuclear translocation, and DNA-binding activity of Stat5. Furthermore, these data show that an agonist-bound ER is able to induce ß-casein promoter activity through activation of the Stat5 transcription factor, independently of the PRL receptor and the Jak2 kinase.
Cytoplasmic Localization of ER and ß Is Favored for Activation of the ß-Casein Promoter in Response to 17ß-Estradiol
The involvement of nonnuclear ERs in mediating some of the described nongenomic actions of estrogens has been suggested (12, 21). To investigate whether the cellular localization of ER affected 17ß-estradiol-induced activation of the ß-casein promoter, an ER mutant with a deletion of important nuclear localization signals in the hinge domain (NLSA) was used (22). As shown in Fig. 2B, while the wild-type receptor showed nuclear staining, NLSA was predominantly present in the cytoplasm of cells. Interestingly, NLSA transiently transfected into COS-7 cells efficiently induced activation of the ß-casein reporter gene in response to 17ß-estradiol, with an 11-fold induction of the reporter observed while the wild-type receptor showed a 4-fold induction under the same conditions (Fig. 2C). To analyze the ability of ERß to exhibit the same effect on the ß-casein promoter if rendered cytoplasmic, a tag containing a fatty acylation site was incorporated in-frame upstream of the N terminus of ERß (ERß Mem). This site can be myristoylated and palmitoylated with subsequent membrane localization of the protein (23). As shown in Fig. 2B, ERß Mem localized to extranuclear sites and, interestingly, was more efficient in activating the ß-casein reporter gene compared with wild-type ERß (Fig. 2C). Taken together, these results demonstrate that cytoplasmic localization of ER and -ß is favored for activation of the ß-casein promoter in response to 17ß-estradiol.
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), confirming that endogenous Stat5 is targeted by the 17ß-estradiol-induced pathway. Furthermore, overexpression of a dominant-negative version of Stat5 (MGF
750), which lacks the transactivation domain (26), inhibited 17ß-estradiol-induced activation of the ß-casein reporter gene in a dose-dependent manner (Fig. 3B). To analyze whether Stat5 is the only target transcription factor induced by 17ß-estradiol on the promoter, the transfection experiments were repeated in mouse fibroblast cells devoid of functional Stat5a and Stat5b (Stat5ab-/- cells) (a gift from J. Ihle). In the presence of NLSA, 17ß-estradiol induced activation of the ß-casein reporter gene in these cells, indicating that Stat5 is not the only transcription factor to be activated. However, overexpression of Stat5 in cells lacking Stat5 proteins markedly increased the 17ß-estradiol-induced response (Fig. 3C), demonstrating that Stat5 can indeed be induced to activate the reporter in the presence of both NLSA and 17ß-estradiol.
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-interferon-activated sequence element present in the ß-casein promoter in vitro (data not shown) and, furthermore, Stat3 was shown to activate the ß-casein reporter gene in transient transfection experiments (data not shown and Ref. 27). The phosphorylation status of Stat3 upon 50 nM 17ß-estradiol or 15% serum (FBS) treatment was analyzed by Western blot analyses of PAE whole-cell extracts, using phospho-specific antibodies against tyrosine-705 and serine-727 of Stat3, respectively. As shown in Fig. 4A, both tyrosine and serine phosphorylation of Stat3 were induced within 15 min of 17ß-estradiol treatment and were inhibited in the presence of ICI 182,780. Total levels of Stat3 protein in the cell extracts are shown in Fig. 4A (lower panel).
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). Furthermore, in an in vitro DNA-binding assay, using the consensus Stat3-binding site present in the CRP promoter, binding of Stat3 to the element was observed in nuclear extracts from PAE cells treated with 17ß-estradiol or FBS for 30 min (Fig. 4C).
To determine whether the residual ß-casein promoter activity observed in Stat5ab-/- cells is due to Stat3 activity, a dominant negative version of Stat3 (Stat3ß) was transiently cotransfected with NLSA and the ß-casein reporter gene into these cells. Stat3ß is a naturally occurring splice variant with a deletion in the C-terminal transactivation domain (29). As shown in Fig. 4D, overexpression of Stat3ß efficiently suppressed 17ß-estradiol-induced activation of the reporter, demonstrating that the residual ß-casein promoter activity is due, at least in part, to activation of Stat3. However, the involvement of other Stat proteins cannot be excluded. Taken together, these results demonstrate that, in addition to Stat5, Stat3 is also activated via the pathway induced by 17ß-estradiol-bound ER.
Antiestrogens Block 17ß-Estradiol-Induced Activation of the ß-Casein Promoter
Next, the ability of antiestrogens to block activation of the ß-casein promoter by 17ß-estradiol-activated ER was analyzed. Both the partial ER antagonists, 4-hydroxy tamoxifen (OHT) and raloxifen, as well as the full antagonist ICI 182,780, completely blocked 17ß-estradiol-induced activation of the ß-casein reporter gene (Fig. 5). OHT and ICI 182,780 are thought to cause ER antagonism via interaction with the LBD, implying that an intact LBD in the agonist-bound conformation is important. 17-Estradiol, the transcriptionally inactive stereoisomer for 17ß-estradiol, was also tested, but did not induce the reporter. However, the ERß-specific agonist, genistein (30), was able to induce activation of the reporter at 100 nM in the presence of ERß wt (Fig. 5). In conclusion, these data demonstrate that binding of the natural hormone to ER is essential to induce activation of the ß-casein promoter.
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), showing that intact MAPK and Src-kinase signaling pathways are required. The involvement of Src-kinase was further demonstrated by transfection of embryonic fibroblast cells derived from Src-/- mice (SYF cells) (33). No, or minimal, activation of the reporter in response to 17ß-estradiol was observed in SYF cells transiently transfected with NLSA. However, coexpression of constitutively active Src-kinase (SrcY527F) restored induction of the reporter gene (Fig. 6B). Activation of the reporter was also prevented by pretreatment of the cells with the PI3-kinase inhibitor, LY 294002 (Fig. 6A), suggesting that not only MAPK and Src-kinase, but also PI3-kinase, is involved in the signaling pathway regulating 17ß-estradiol-induced ß-casein promoter activity. The involvement of the p38 MAPK and G protein-coupled receptors was also investigated, but, as shown in Fig. 4A, activation of the reporter was sustained in the presence of the p38 MAPK inhibitor, SB 203580, and the G-subunit inhibitor, pertussis toxin (Fig. 6A). Notably, a similar result was obtained when 17ß-estradiol-induced activation of the consensus LHRE reporter gene was analyzed in the presence of the various signaling inhibitors (data not shown), which confirms that the Src-kinase, MAPK, and PI3-kinase signaling pathways are required for 17ß-estradiol-induced transactivation of Stat5 and excludes the possibility that the kinases themselves activate the ß-casein promoter in a Stat5-independent manner.
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, ERK1/2 was indeed activated upon 17ß-estradiol treatment, but not in the presence of ICI 182,780, suggesting a requirement for an agonist-bound ER. Also shown in Fig. 6C, activation of ERK1/2 was prevented both by the MEK1/2 inhibitor, UO126, as expected, and by the Src-kinase inhibitor, PP1, suggesting a role for Src-kinase upstream of MAPK in the signal transduction pathway induced by 17ß-estradiol. Total levels of ERK1/2 proteins in the cell extracts are shown in Fig. 6C (lower panel). All together, these results demonstrate that the Src-kinase, MAPK, and PI3-kinase signaling pathways act downstream of 17ß-estradiol. In contrast, neither the p38 MAPK nor the G protein-mediated signal transduction pathways are involved in mediating 17ß-estradiol-induced ß-casein promoter activity.
Serine Phosphorylation of Stat5 Is Required for Optimal Transcriptional Activity in Response to 17ß-Estradiol
In addition to tyrosine phosphorylation, serine phosphorylation of Stat5 was also rapidly induced by 17ß-estradiol (Fig. 1A). Together with the establishment of serine phosphorylation as a regulator of Stat5 transcriptional activity in T lymphocytes (34), this observation prompted us to analyze the importance of specific serine residues within Stat5 for 17ß-estradiol-induced activation of the ß-casein promoter. Stat5ab-/- cells were transiently transfected with wild-type Stat5a or different point mutants, in which serine residues 725 and/or 779 in the C-terminal transactivation domain had been changed to alanines (S725A, S779A, and S725/779) (35), together with the ß-casein reporter gene and NLSA. Interestingly, the S725A, S779A, and S725/779A mutants all showed a lower induction of the reporter in response to 17ß-estradiol compared with wild-type Stat5a (Fig. 7). As expected, the Y694F mutant, in which the critical tyrosine residue 694 had been changed to phenylalanine, was unable to induce activation above basal level (Fig. 7). In conclusion, these results demonstrate that tyrosine phosphorylation is a prerequisite for 17ß- estradiol-induced transactivation of Stat5a, whereas intact serine residues in the transactivation domain are important for optimal transcriptional activity.
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. Figure 8A shows a schematic representation of the ER constructs used in the experiment. Figure 8B shows how the wild-type ER, in the presence of 17ß-estradiol, activated the reporter gene 4-fold compared with control cells without transfected ER. The importance of the DBD was analyzed by using a DNA-binding mutant in which two cysteine residues in the second zinc-finger had been changed to alanines. These mutations have previously been shown to inhibit DNA binding to an ERE (36). As expected, the DBD was found to be dispensable for activation of the reporter, demonstrating that the observed activity is not mediated through classical transcriptional activation by ER. The N-terminal domain was found to be dispensable, as shown using the N-terminal-deletion ER mutant (182–599), while deletion of the C-terminal domain (1–339) prevented reporter activation. An ER with mutations that abrogate interactions with nuclear hormone receptor coactivators, such as the ER AF2 mutant (L543A/L544A) (37) or the ER (K366A) (38), did not block activation of the reporter. However, a 50% reduction in activation was observed with the AF2 mutant, while the K366A mutant was as active as the wild-type receptor. An ER single-point mutant that is unable to bind 17ß-estradiol (G525R) (39) did not activate the reporter above background, indicating that the activity is mediated by hormone-bound ER. Furthermore, mutations in the helix 11 dimerization domain (L508/512/515E) was deleterious to the ability of ER to activate the reporter. This dimerization-defective mutant receptor is unable to bind DNA and is transcriptionally inactive (40). Deletion of the C-terminal-most F domain of ER did not affect reporter activation. Tyr 541, located in the N-terminal end of helix 12, has been shown to be important for maintaining the receptor in an inactive state in the absence of hormone (41). However, the constitutively active ER mutant, Y541-E, showed the same 17ß-estradiol-induced activation as that found for the wild-type receptor. Finally, NLSA, which contains a deletion of nuclear localization signals in the hinge domain and which is cytoplasmic in the absence of hormone (Fig. 2B and Ref. 22), activated the reporter gene 11-fold compared with control cells (see also Fig. 2C). Taken together, these results demonstrate that activation of the ß-casein promoter by 17ß-estradiol is mediated through the ligand binding domain(s) of an ER homodimer. Furthermore, cytoplasmic localization of the receptor aids efficient induction whereas interaction with coactivators in the nucleus is not essential.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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17ß-Estradiol was shown to rapidly induce tyrosine and serine phosphorylation, nuclear translocation, and DNA-binding activity of Stat5 in endothelial cells (Fig. 1, A and B) and to activate the ß-casein promoter in an ER-dependent manner (Fig. 1, C and D). Phosphorylation of Stat5 and induction of promoter activity were abrogated in the presence of antiestrogens (Figs. 1A and 5), suggesting a requirement for an agonist-bound ER. In addition to the true ß-casein promoter, the consensus Stat5 LHRE reporter gene and two Stat3-regulated reporter genes were efficiently induced (Figs. 3A and 4B), demonstrating that both Stat5 and Stat3 are targeted by the signal transduction pathway induced by 17ß-estradiol-bound ER. Tyrosine and serine phosphorylation and DNA-binding activity of Stat3 were also shown to be induced by 17ß-estradiol (Fig. 4, A and C). The ability of 17ß-estradiol to promote nuclear translocation of Stats has not been described previously, whereas it has been demonstrated with glucocorticoids (42) and progestins (43).
An intact MAPK signaling pathway was found to be required for 17ß-estradiol-induced activation of the ß-casein promoter, as shown by abrogated activity in the presence of the MEK1/2 inhibitor, UO126 (Fig. 6A). As further evidence for the involvement of the MAPK pathway, ERK1/2 was shown to be rapidly phosphorylated upon 17ß-estradiol treatment of endothelial cells (Fig. 6C), which is in agreement with previous studies that showed 17ß-estradiol-induced activation of ERK1/2 in endothelial cells (16) as well as in other cells and tissues (Refs. 8 , 9 , 12 , 13 , 44 , and 45 and our unpublished data). Consistent with the studies by Migliaccio et al. (8, 9) and Kousteni et al. (12), activation of ERK1/2 by 17ß-estradiol was found to be dependent on initial Src-kinase activity (Fig. 6C). Src-kinase was also found to be required for 17ß-estradiol-induced activation of the ß-casein promoter, since the activity was completely abrogated in the presence of the Src-kinase inhibitors, PP1 and genistein, and was not observed in cells lacking functional Src-kinase (Fig. 6, A and B). Phosphotyrosine 537 of human ER (corresponding to tyrosine 541 of mouse ER) was recently shown to interact with the SH2 domain of Src-kinase (46). However, tyrosine 541 was dispensable for 17ß-estradiol-induced activation of the ß-casein promoter (Fig. 8), suggesting that Src-kinase is activated downstream of ER by a distinct mechanism in this pathway. In addition to MAPK and Src-kinase activities, an intact PI3-kinase signaling pathway was also found to be required (Fig. 6A). Recent reports have shown that rapid, nongenomic activation of endothelial nitric oxide synthase by 17ß-estradiol and ER in endothelial cells involves the PI3-kinase signaling pathway (16, 17, 47) and that 17ß-estradiol increases PI3-kinase activity in MCF-7 cells (14). Furthermore, simultaneous PI3-kinase and MAPK activity has been shown to contribute to serine phosphorylation of Stat3, resulting in optimal transcriptional activity (48, 49). Although some of the described nongenomic actions of estrogens are mediated by modulation of G protein-coupled receptors (50), G protein-mediated signal transduction was shown not to be part of the 17ß-estradiol-induced pathway targeting Stats (Fig. 6A).
Cytoplasmic sublocalization of ERs was shown to correlate with a more efficient activation of the ß-casein promoter (Fig. 2C). In vivo, however, it is unclear whether the endogenous receptor exhibits this action through a specific pool of membrane-bound receptors or receptors that "pass by" the membrane during nucleocytoplasmic events (22). We believe, however, that the presence of ERs for a certain amount of time within plasma membrane domains facilitates signal transduction. Several members of the MAPK signaling pathway, as well as Src-kinase and PI3-kinase, are found clustered in caveolae, which are specialized membrane invaginations enriched in the scaffolding protein, caveolin-1 (51). ER has been shown to coimmunoprecipitate with caveolin-1 (52), and both ER (21) and Stat3 have been detected in caveolae (53). It is thus conceivable that the presence of ERs, Stats, and various signal transduction proteins in a defined cellular compartment, e.g. caveolae, facilitates signal transduction induced by an agonist-bound ER. This idea is supported by the finding that targeting of ERß to cellular membranes results in more efficient activation of the ß-casein promoter (Fig. 2C). In contrast to NLSA, which located to the nucleus after several hours of exposure to hormone (22), the cellular localization of ERß Mem was not affected by the addition of hormone (data not shown), further verifying that the initial action is mediated by an agonist-bound receptor present in the cytoplasm.
In transient transfection experiments, intact serine residues within the transactivation domain of Stat5a were shown to be required for optimal 17ß-estradiol-induced activation of the ß-casein promoter (Fig. 7). In addition to basic tyrosine phosphorylation, serine phosphorylation has been shown to affect the transcriptional activities of Stat1 and Stat3 in particular, and also that of Stat5 under certain conditions (34, 35, 54), although the mechanism has not been fully defined. However, members of the MAPK family have been implicated in the phosphorylation of serine residues conserved among the Stats (55, 56, 57, 58). Apart from the two characterized serine phosphorylation sites in Stat5a, residues 725 and 779 (57, 59), it is possible that additional serine residues within the molecule are preferentially phosphorylated and involved in 17ß-estradiol-induced transactivation.
Our analysis of functional domains in ER that are required for mediating activation of the ß-casein promoter, via the nongenomic mechanism described in this paper, revealed that the LBD is essential, whereas the DBD and the activation function 2 (AF-2) coactivator binding regions are dispensable. The findings, that an ER which is unable to bind ligand is inactive (Fig. 8) and that the activity is abrogated in the presence of ER antagonists (Fig. 5), confirm that the activity is mediated by an agonist-bound ER. The conformational change in the constitutively active Y541-E mutant, which is thought to generate an interacting surface allowing recruitment of coactivators in the absence of hormone (41), does not induce activation of the reporter (Fig. 8), implying that the surface targeting signal transduction pathways is different. The dimerization-defective mutant, which contains three mutated leucine residues in helix 11, is unable to activate the reporter (Fig. 8). This could be due to either a requirement for an ER dimer or, alternatively, the domain mutated being required for proper protein-protein interactions mediating the induction of signal transduction pathways.
Recently, we reported that ER and -ß can act as coactivators for PRL-activated Stat5 on the ß-casein promoter and that ERs are capable of interacting with Stat5 via the DBD/hinge domain (60). The deletion mutant NLSA is unable to interact with Stat5 and does not potentiate the transcriptional activity of PRL-activated Stat5 (our unpublished data). However, it markedly induces activation of the ß-casein promoter via the mechanism described in this paper, demonstrating that the transcriptional activity of Stat5 can be modulated via distinct ER-mediated actions.
Taken together, this study identifies a novel mechanism of gene activation by ERs in which 17ß-estradiol initiates cytoplasmic signaling events upon binding to the receptor, resulting in phosphorylation and subsequent transactivation of a target transcription factor. Interestingly, induction of the signaling cascade requires an agonist-bound receptor, but may dispense with other functional domains except for the LBD. We are now able to discriminate between three diverse actions of ERs: classical regulation of ERE-containing genes, cross-talk with PRL-activated Stat5b on the ß-casein promoter (60), and, as put forward in this paper, activation of Stats via induction of cytoplasmic signaling pathways. In contrast to the two latter actions, the classical mechanism of ER activation requires binding of coactivator proteins. This suggests that different conformations of the receptor are involved in various activities, potentially enabling ligand design to target specific actions of the receptor for therapeutic use.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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750 (26, 62); the long form of the Prl-R (63); the SrcY527F (64); the mouse ER expression vector pMT2-MOR (36); the ER DNA-binding mutant pMT2-MOR C241A/C244A (36); the ER N-terminal deletion mutant pMT2-MOR 182–595 (36); the ER C-terminal deletion mutant pMT2-MOR 1–339 (65); the ER AF-2 mutant pMT2-MOR L543A/L544A (66); the pMT2-MOR K366A (38); the pMT2-MOR Y541F (41); the pMT2-MOR 245–307 (NLSA) (22); and the human ERß expression vector, pSG5-ERß (67). The ER F mutant was prepared using the Gene Editor in vitro Site-Directed Mutagenesis System (Promega Corp., Madison, WI) with an oligonucleotide primer designed to delete the F domain (amino acids 550–595) of pMT2-MOR. The ERß LYN mutant was generated by subcloning a fatty acylation site into the coding sequence of pSG5-ERß wt. The DNA sequences of the new constructs were verified by automated sequencing.
Estrogens, Growth Factors, and Inhibitors
17ß-Estradiol, the inactive stereoisomer, 17-estradiol, genistein, 4-hydroxy tamoxifen, ovine PRL, recombinant human EGF, and pertussis toxin were purchased from Sigma (St. Louis, MO). The pure ER antagonist, ICI 182,780, was obtained from Tocris Cookson, Inc. (Ballwin, MO). The MEK1/2 inhibitor, UO126, and the PI3-kinase inhibitor, LY294002, were purchased from Cell Signaling Technology (Beverly, MA). The Src-family tyrosine kinase inhibitor, PP1, and the p38MAPK Inhibitor, SB203580, were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Raloxifen was a gift from Stefan Nilsson (Karo Bio AB, Huddinge, Sweden).
Cell Culture and Transient Transfection Techniques
COS-7 cells, Stat5ab-/- cells (a gift from J. Ihle), and SYF cells (purchased from ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% FBS (Life Technologies, Inc., Buckinghamshire, UK). PAE cells (a gift from Stefan Wennström) were cultured in DMEM/F-12 (Life Technologies, Inc.) supplemented with 10% FBS. For transient transfection assays, PAE cells, COS-7 cells, Stat5ab-/- cells, and SYF cells were seeded in phenol-red free DMEM (Life Technologies, Inc.) supplemented with 5% dextran-charcoal-stripped (DCS) FBS in 24-well microtiter plates, 24 h before transfection. Cells were transfected with 1 µg reporter plasmid, 250 ng cytomegalovirus-ß-galactosidase plasmid as an internal control, and various expression plasmids, as indicated in the figure legends, together with an empty expression vector to a total of 2 µg DNA per well, using a modified calcium phosphate coprecipitation method (68). The transfection medium was changed after 24 h to a phenol red-free DMEM supplemented with 0,05% DCS FBS. The hormones were added as indicated in the figures. After 24 h, the cells were harvested in lysis buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; 150 mM NaCl; and 0.65% Nonidet P-40). Extracts were assayed for luciferase and ß-galactosidase activity in a microplate reader (Lucy-1; Anthos, Salzburg, Austria). For immunofluorescence studies, COS-7 cells were seeded onto poly-L-lysine precoated coverslips placed in six-well tissue culture plates in phenol red-free DMEM supplemented with 5% DCS FBS. Cells were transfected as described above and then processed for histological studies.
Indirect Immunofluorescence
For histological studies, cells were washed with PBS and fixed in 3% paraformaldehyde solution [3% (wt/vol) paraformaldehyde, 0.1 mM CaCl2 and 0.1 mM MgCl2, pH 7.4, in PBS] for 20 min. The cells were then washed with PBS, permeabilized in 0.2% Triton X-100/PBS for 4 min, and then washed with 10 mg/ml BSA in PBS. ER and -ß proteins were detected using the ER polyclonal antibody H-184 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and an ERß polyclonal antibody (Upstate Biotechnology, Inc., Lake Placid, NY), respectively. The antibodies were diluted 1:100 in 10 mg/ml BSA in PBS, and 40 µl samples were placed on parafilm in a box containing moist Whatman 3MM (Clifton, NJ). Coverslips were inverted onto the antibody and incubated for 1 h at room temperature. They were then washed with 10 mg/ml BSA in PBS, and the above procedure was repeated with an appropriate fluorescein isothiocyanate-conjugated secondary antibody (DAKO A/S, Glostrop, Denmark). Cell nuclei were stained with 4,6-diamidino2-phenylindole. The coverslips were subsequently mounted on slides with Vectashield Mounting Medium (Vector Laboratories, Inc., Burlingame, CA). In all cases, untransfected or transfected cells incubated without a primary antibody were included as controls. Slides were examined using a DMRXA microscope (Leica Corp., Deerfield, IL), and digitally imaged using a Hamamatsu C4880–40 CCD camera (Hamamatsu Photonics Norden AB, Solna, Sweden), the Openlab software package (Improvision, Sollentuna, Sweden), and Adobe Photoshop software.
Western Blotting
PAE cells were seeded in six-well tissue culture plates in phenol red-free DMEM supplemented with 5% DCS FBS. After 48 h, cells were washed three times with PBS before incubation for 24 h in serum-free medium. Cells were then washed twice before addition of hormone and various inhibitors for the times indicated in the figure legends. Cells were lysed in hot Laemmelli lysis buffer directly in the petri dish, and proteins were separated by electrophoresis through a 10% polyacrylamide gel. Proteins were transferred to Hybond-C membranes (Amersham International, Buckinghamshire, UK). The membranes were probed using either phospho-Stat5A/B (Y694/699), phospho-Stat5A/B (S726/731) (Upstate Biotechnology, Inc.), Stat5a (L-20), Stat5b (C-17), Stat3 (F-2) (Santa Cruz Biotechnology, Inc.), phospho-Stat3 (Tyr705), phospho-Stat3 (Ser727), phospho-p44/42 MAPK (Thr202/Tyr204), or p44/p42 MAPK (Cell Signaling Technology) antibodies. Proteins were detected using the ECL chemiluminescence system (Amersham Pharmacia Biotech).
DNA Affinity Purification
Biotinylated oligonucleotide (5'-AGATTTCTAGGAATTCAAATC-3'), derived from the ß-casein promoter and including a Stat5 response element, was annealed to an antisense strand. An oligonucleotide with two point mutations in the consensus Stat5 response element (5'-AGATTTCTATTAATTCAAATC-3') and an oligonucleotide derived from the human CRP promoter and including a Stat3 response element (5'-CCTCTTCCCGAAGCTCT-3') were prepared in a similar manner. PAE cells were plated in phenol red-free DMEM supplemented with 5% DCS FBS. After 48 h, cells were washed three times with PBS before incubation for 24 h in serum-free medium. Cells were then washed twice before addition of hormone at various time points, as indicated in the figure legends, and nuclear extracts were prepared as previously described (60). Nuclear extracts (containing a total of 
300 µg protein) were mixed with 2 µg double-stranded biotinylated DNA oligo in 800 µl A-buffer (8 mM trisphosphate, pH 7.4; 120 mM KCl; 8% glycerol; 4 mM dithiothreitol; 100 µM Na orthovanadate; 1 mM phenylmethylsulfonylfluoride; 10,000 U/ml aprotinin) and incubated at 4 C for 1 h on a rotator. Streptavidin MagneSphere Paramagnetic Particles (Promega Corp.) resuspended in A-buffer were added to a final volume of 1 ml, and incubation was continued for 15 min. After washing, bound proteins were eluted in loading buffer and separated by electrophoresis through a 10% polyacrylamide gel. Proteins were transferred to Hybond-C membranes (Amersham International) and detected by Western blotting with the Stat5 (C-17) or Stat3 (F-2) antibody (Santa Cruz Biotechnology, Inc.). Proteins were detected using the ECL chemiluminescence system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AF-2, Activation function 2; CRP, C-reactive protein; DBD, DNA-binding domain; DCS, dextran-charcoal-stripped; EGF, epidermal growth factor; ER, estrogen receptor; ERß wt, wild-type ERß; ERß Mem, N terminus of ERß; ERE, estrogen response element; FBS, fetal bovine serum; Jak, Janus kinase; LBD, ligand-binding domain; LHRE, lactogenic hormone response element; MEK, MAPK kinase; NLSA, nuclear localization signals in the hinge domain; OHT, 4-hydroxytamoxifen; PAE, porcine aortic endothelial; PI3-kinase, phosphatidylinositol 3-kinase; PRL, prolactin; Stat, signal transducer and activator of transcription.
Received for publication February 18, 2002. Accepted for publication June 24, 2002.
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